Pharmaceutical preparations and their use

Abstract

The present invention provides a formulation of albumin having preferably high levels of free thiols and preferably low levels of albumin polymer. The present invention further provides a use of the formulation for stabilizing a virus, wherein the formulation comprises (i) albumin, which is substantially in an agglutinative form, and (ii) a plurality of negatively charged anions housed on a flexible skeleton. The present invention also provides a kit of formulations and components comprising (i) albumin, which is substantially in an agglutinative form, and (ii) a plurality of negatively charged anions housed on a flexible skeleton, and the use thereof for stabilizing a virus.

Description

[Technical Field]

[0001] This application includes a computer-readable sequence listing. The computer-readable sequence is incorporated herein by reference.

[0002] This specification consists of two parts, which are referred to herein as Part A and Part B.

[0003] Part A Sequence List Reference (Part A) This application includes a computer-readable sequence listing. The computer-readable sequence is incorporated herein by reference.

[0004] Technical field (Part A) The present invention relates to a formulation of albumin having a desirable high level of free thiols and a desirable low level of albumin polymer, and to the use thereof. [Background technology]

[0005] Background Technology (Part A) Human serum albumin (HSA, SEQ ID NO: 2) contains a free thiol at the cysteine ​​residue located at position 34 (Cys34). This thiol transmits many of albumin's antioxidant properties. The thiol provided by Cys34 is also available for binding to and conjugation of other molecules.

[0006] These other molecules include cysteine ​​and glutathione in vivo, but may also include molecules such as drugs (chemicals or peptides with therapeutic uses). However, molecules may have relatively short stability, either in the formulation or when administered to a patient. It is desirable to increase the efficacy and stability of molecules such as therapeutic agents, thereby reducing the dose and frequency required by the patient. Pharmaceutical companies are researching technologies to improve drug stability and extend efficacy. Technologies include different formulations, packaging, and storage conditions of the drug, or modifying the drug molecule itself by linking it to another molecule, for example, by PEGylation. However, not all of these approaches are feasible due to concerns such as cost, safety, reproducibility, and immunogenicity.

[0007] For example, conjugating drugs to larger molecules such as albumin via thiols provided by Cys34 has been shown to improve drug stability (e.g., Holmes et al. (2000) Bioconjug. Chem. 11, 439-444). The advantage of using wild-type albumin is that it is the same molecule as the naturally occurring one, with a proven record of safety and consistency. Theoretically, the free thiol level is one free thiol per albumin molecule due to the unpaired cysteine ​​at position 34. However, during long-term storage, the free thiols on wild-type albumin may substantially decrease due to oxidation, and polymer levels may increase due to aggregation.

[0008] Maintaining high levels of free thiols is desirable to maximize the efficiency of drug conjugation to albumin. Maintaining low levels of polymer is desirable to minimize the possibility of adverse immunogenic reactions in recipients of albumin or albumin-containing products. Polymer levels have been addressed, for example, by formulating albumin with fatty acids or amino acids (Anraku et al. (2004) Biochimica et Biophysica Acta. 1702, 9-17). However, when investigating the effect of fatty acids on albumin, the inventors observed that increasing the level of fatty acids resulted in a desirable decrease in polymer levels, but was accompanied by an undesirable decrease in the level of free thiols.

[0009] To date, no albumin formulations that retain both high levels of free thiols and low levels of polymer after storage are available. Such formulations would be desirable for use, for example, when conjugating albumin to conjugation partners such as drugs. [Overview of the project]

[0010] Summary of the Invention (Part A) The present invention provides a liquid formulation of albumin or its fragments or fusions, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin, after storage or incubation at at least 40°C for at least one month.

[0011] The present invention also provides a method for preparing a formulation of albumin or its fragments or fusions having high free thiol levels and low polymer levels, comprising formulation of albumin or its fragments or fusions in a buffer containing 2.5 to 7.5 mM fatty acids, at least 175 mM cations, and a pH of 5.5 to 6.5.

[0012] The present invention further provides the use of the buffer described herein for, for example, maintaining high free thiol levels and low polymer levels in albumin preparations after storage or incubation of albumin preparations.

[0013] The present invention also provides a method for producing a conjugate, which includes conjugating albumin or a fragment or fusion thereof with a conjugation partner.

[0014] The present invention provides a method for conjugating albumin or a fragment or fusion thereof to a partner with at least 75% efficiency, further comprising contacting the partner with a formulation of albumin or a fragment or fusion thereof, according to a first, second, third, or fourth aspect of the present invention.

[0015] The present invention also provides the use of albumin or its fragments or fusion formulations described herein for highly efficient conjugation to a conjugation partner.

[0016] The present invention further provides a conjugate comprising albumin and a conjugation partner.

[0017] The present invention also provides the use of formulations of albumin fragments or fusions thereof, produced according to the herein or by the methods described herein, for increasing the half-life of the molecule.

[0018] The present invention further provides conjugates described herein for the treatment of diseases, the treatment of illnesses, and / or diagnosis.

[0019] The present invention also provides conjugates described herein for the manufacture of agents for the treatment of diseases, the treatment of illnesses, and / or diagnosis. [Brief explanation of the drawing]

[0020] [Figure 1]Figure 1 (Part A) (A) shows the design space (unshaded) for sodium (Na, mM) and octanoate (Oct, mM) concentrations of albumin preparations having a free thiol level of at least 0.75 moles of free thiols and a maximum polymer level of 1% (w / w) per mole of albumin at a set pH of 6.0 after one incubation at 40°C for one month. The filled shaded area represents the design space with a free thiol level lower than 0.75 moles of free thiols per mole of albumin. (B) A three-dimensional representation of the relationship between octane, sodium, and thiol levels at a set pH of 6.0 after incubation at 40°C for one month, and (C) A three-dimensional representation of the relationship between octane, sodium, and polymer levels at a set pH of 6.0 after incubation at 40°C for one month. [Figure 2] Figure 2 (Part A) (A) Shows the design space (unshaded) for pH and octanoate (Oct, mM) concentration of albumin preparations having a free thiol level of at least 0.75 moles of free thiol per mole of albumin and a maximum polymer level of 1% (w / w) at a set sodium (Na) concentration of 250 mM after one incubation at 40°C for one month. The filled shaded area shows the design space with a free thiol level lower than 0.75 moles of free thiol per mole of albumin, and is shaded with diagonal lines. The region indicates a design space with polymer levels exceeding 1% (w / w), the intersection of the vertical and horizontal lines indicates a formulation with pH 6.0 and 5 mM octanoate, (B) a three-dimensional representation of the relationship between octanoate, pH, and thiol levels at a set sodium concentration of 250 mM after incubation at 40°C for one month, and (C) a three-dimensional representation of the relationship between octanoate, pH, and polymer levels at a set sodium concentration of 250 mM after incubation at 40°C for one month. [Figure 3] Figure 3 (Part A) (A) Shows the design space (unshaded) for sodium (Na, mM) concentration and pH of an albumin preparation having a free thiol level of at least 0.75 moles of free thiol per mole of albumin and a maximum polymer level of 1% (w / w) at a set octanoate concentration of 5 mM after one incubation at 40°C for one month. The filled shaded area shows the design space with a free thiol level lower than 0.75 moles of free thiol per mole of albumin, and is shaded with diagonal lines. The region indicates a design space with a polymer level greater than 1% (w / w), the intersection of the vertical and horizontal lines indicates a formulation with 250 mM Na and pH 6.0, (B) a three-dimensional representation of the relationship between pH, sodium, and thiol levels at a set octanoate concentration of 5 mM after incubation at 40°C for one month, and (C) a three-dimensional representation of the relationship between pH, sodium, and polymer levels at a set octanoate concentration of 5 mM after incubation at 40°C for one month. [Figure 4-1] Figure 1 (Part B) (A) Hydrodynamic radius over time during incubation at 25°C followed by incubation at 40°C (cumulant analysis) - solution in tris-citrate + Na2SO4 at high AAV2 concentration, (B) hydrodynamic radius at the start and end of incubation at 25°C and 40°C for high-concentration AAV2 in tris-citrate buffer and (C) tris-citrate buffer + Na2SO4. Radii were not reported for samples showing precipitates. [Figure 4-2] Figure 1 (Part B) (A) Hydrodynamic radius over time during incubation at 25°C followed by incubation at 40°C (cumulant analysis) - solution in tris-citrate + Na2SO4 at high AAV2 concentration, (B) hydrodynamic radius at the start and end of incubation at 25°C and 40°C for high-concentration AAV2 in tris-citrate buffer and (C) tris-citrate buffer + Na2SO4. Radii were not reported for samples showing precipitates. [Figure 5]Figure 2 (Part B) Hydrodynamic radii at the start and end of incubation at 25°C and 40°C. Larger error bars are due to sample aggregation but are plotted for comparison. Albumin at low concentrations AAV2 and 5 mg / mL. (A) Tris-citrate buffer and (B) Tris-citrate buffer + Na2SO4. [Figure 6-1] Figure 3 (Part B) Background membrane imaging - Images of wells containing (A) tris-citrate and (B) tris-citrate + Na2SO4. Solutions were stored at 4°C for 2 months. Scale bar is 1000 μm (microns). [Figure 6-2] Figure 3 (Part B) Background membrane imaging - Images of wells containing (A) tris-citrate and (B) tris-citrate + Na2SO4. Solutions were stored at 4°C for 2 months. Scale bar is 1000 μm (microns). [Figure 7] Figure 4 (Part B) (A) Number of particles with ECD ≥ 2 μm and (B) ThT fluorescent particles with ECD ≥ 2 μm in both tested formulations and in the presence of albumin from different sources. The solutions were stored at 4°C for 2 months. [Figure 8] Figure 5 (Part B) (Bright-field SIMI) Number of particles with ECD ≥ 2 μm in AAV2 solution after 4 cycles of freeze-thawing in (A) tris-citrate and (B) tris-citrate + Na2SO4. [Figure 9] Figure 6 (Part B) Number of ThT-positive particles with ECD ≥ 2 μm in AAV2 solution after 4 cycles of freezing in (A) tris-citrate and (B) tris-citrate + Na2SO4. [Figure 10-1] Figure 7 (Part B) Correlation between (A) polymer percentage measured by SEC-HPLC, (B) polydispersity index, and (C) hydrodynamic radius (measured by DLS), and (D) total lipid content (albumin at 100 mg / mL), and temperature incubation scoring for incubation experiments at T=25°C and 40°C. [Figure 10-2]Figure 7 (Part B) Correlation between (A) polymer percentage measured by SEC-HPLC, (B) polydispersity index, and (C) hydrodynamic radius (measured by DLS), and (D) total lipid content (albumin at 100 mg / mL), and temperature incubation scoring for incubation experiments at T=25°C and 40°C. [Figure 11-1] Figure 8 (Part B) Correlation between (A) polymer percentage measured by SEC-HPLC, (B) polydispersity index, and (C) hydrodynamic radius (measured by DLS) and the temperature incubation score of incubation experiments at T=40°C. [Figure 11-2] Figure 8 (Part B) Correlation between (A) polymer percentage measured by SEC-HPLC, (B) polydispersity index, and (C) hydrodynamic radius (measured by DLS) and the temperature incubation score of incubation experiments at T=40°C. [Figure 12] Figure 9 (Part B) Correlation between (A) total lipid content and (B) free cysteine ​​and temperature incubation scoring for incubation experiments at T=40°C. [Figure 13-1] Figure 10 (Part B) Correlation between particle coverage % measured by BMI and (A) polymer % (SEC-HPLC), (B) total lipids, (C) free cysteine, and (D) normalized free cysteine-normalized NTD (Elman assay and ESI-MS), respectively. Store at 4°C. [Figure 13-2] Figure 10 (Part B) Correlation between particle coverage % measured by BMI and (A) polymer % (SEC-HPLC), (B) total lipids, (C) free cysteine, and (D) normalized free cysteine-normalized NTD (Elman assay and ESI-MS), respectively. Store at 4°C. [Figure 14-1] Figure 11 (Part B) shows the correlation between the number of ThT-positive particles and the following, respectively: (A) free cysteine, (B) total lipids, (C) the ratio between two peaks in the fluorescence spectrum, and (D) zinc content. Store at 4°C. [Figure 14-2]Figure 11 (Part B) shows the correlation between the number of ThT-positive particles and the following, respectively: (A) free cysteine, (B) total lipids, (C) the ratio between two peaks in the fluorescence spectrum, and (D) zinc content. Store at 4°C. [Figure 15] Figure 12 (Part B) Correlation between particle number (BMI-SIMI) and the following, respectively: (A) amount of modification (excluding oxidation) measured by ESI-MS, (B) free Gibbs free energy, and (C) m-value calculated by chemical modification using guanidinium chloride (GdnHCl) as a denaturing agent. Freeze-thaw stress. [Figure 16-1] Figure 13 (Part B) Correlation between particle number (BMI-SIMI) and different metal groups. Freeze-thaw stress. [Figure 16-2] Figure 13 (Part B) Correlation between particle number (BMI-SIMI) and different metal groups. Freeze-thaw stress. [Figure 17-1] Figure 14 (Part B) Correlation between the number of ThT-positive particles and (A) ANS Kd, (B), and (C) various metals, respectively, under freeze-thaw stress. [Figure 17-2] Figure 14 (Part B) Correlation between the number of ThT-positive particles and (A) ANS Kd, (B), and (C) various metals, respectively, under freeze-thaw stress. [Figure 18-1] Figure 15 (Part B) Aggregation: (A) Area % of polymer peaks as determined by SEC-HPLC, (B) Hydrodynamic radius, and (C) Polydispersity index as determined by DLS. Note that since serum-derived albumin was polydispersible, radius or polydispersity values ​​for cumulant-derived albumin are not reported. The dashed line in the graph represents the proposed threshold for "good" albumin. [Figure 18-2] Figure 15 (Part B) Aggregation: (A) Area % of polymer peaks as determined by SEC-HPLC, (B) Hydrodynamic radius, and (C) Polydispersity index as determined by DLS. Note that since serum-derived albumin was polydispersible, radius or polydispersity values ​​for cumulant-derived albumin are not reported. The dashed line in the graph represents the proposed threshold for "good" albumin. [Figure 19] Figure 16 (Part B) shows the results of the Elman assay, expressed as the number of moles of Cys per mole of protein. The dotted line represents the threshold for "good" albumin. [Figure 20] Figure 17 (Part B) ESI-MS PTM: Percentage of PTM products measured by ESI-MS. 100% is the peak with the highest abundance. The dashed line is the proposed sum of percentages of the PTM threshold to define "good" albumin. [Figure 21] Figure 18 (Part B) ESI-MS PTM: Percentage of N-terminal degradation products measured by ESI-MS. 100% is the peak with the highest abundance. The dashed line is the proposed threshold for defining "good" albumin. [Figure 22] Figure 19 (Part B) Time course of change in hydrodynamic radius of albumin incubated at 40°C. All albumins were diluted to 5 mg / mL in DPBS. (A) Only "good" albumins. (B) Comparison of "poor" albumins with some "good" albumins. [Figure 23] Figure 20 (Part B) (A) Time course of change in the hydrodynamic radius of Rice 1 in Sacch.2.1 (black square) in DPBS, and in DPBS (white circle), Sacch.2 formulation buffer (high salt, hexagon with cross), and Sacch.1 formulation buffer (octanoate-containing buffer, black star), respectively, after incubation at 40°C. (B) reports the same data but uses a different scale on the Y axis to highlight the changes that occur even in the octanoate-containing buffer. These changes were not visible in (A) due to the high instability in one of the tested buffers (high salt). [Figure 24] Figure 21 (Part B) Stern-Volmer plot. Only one batch is shown for Sacch.1 and 2; there is no plot for Sacch.3, and all are equivalent. [Figure 25]Figure 22 (Part B) Stern-Volmer constants calculated from the first part of the Stern-Volmer plot. The dashed line represents the proposed threshold for KSV of "good" albumin. [Figure 26] Figure 23 (Part B) Total amount of lipids (excluding octanoates) present in various albumin raw materials. [Figure 27] Figure 24 (Part B) ANS Kd values. [Figure 28] Figure 25 (Part B) Differential scanning calorimetry traces of Sacch.2 at 1 mg / mL in citrate and phosphate buffer solutions. Effect of added NaCl. [Figure 29] Figure 26 (Part B) Differential scanning calorimetry traces of Sacch.2 at 1 mg / mL in various buffer solutions. [Figure 30] Figure 27 (Part B) Hydrodynamic radius measured after incubation of non-octanoate-containing albumin at 60°C for 2 hours. [Figure 31] Figure 28 (Part B) Stability study using Sacch.1 in different buffers. Total protein at (A) 6 months and (B) 36 months, as measured by SEC-HPLC. [Figure 32] Figure 29 (Part B) Monomer concentrations measured by SEC-HPLC at 36 months (normalized to a 0-1 scale to account for small variations between solutions). (A) Stored at 25°C and (B) Stored at 5°C. [Figure 33] Figure 30 (Part B) Comparison of residues in contact with citrate (1TF0) and residues involved in octanoate binding (Kawai et al, 2017). [Figure 34] Figure 31 (Part B): Compounds having the same structural characteristics as citrate.

[0021] Brief explanation of the drawing (Part A) Figure 1 (A) shows the design space (unshaded) for sodium (Na, mM) and octanoate (Oct, mM) concentrations of albumin preparations having a free thiol level of at least 0.75 moles of free thiols and a maximum polymer level of 1% (w / w) per mole of albumin at a set pH of 6.0 after one incubation at 40°C for one month. The filled shaded area represents the design space with a free thiol level lower than 0.75 moles of free thiols per mole of albumin. (B) A three-dimensional representation of the relationship between octane, sodium, and thiol levels at a set pH of 6.0 after incubation at 40°C for one month, and (C) A three-dimensional representation of the relationship between octane, sodium, and polymer levels at a set pH of 6.0 after incubation at 40°C for one month. Figure 2 (A) shows the design space (unshaded) for pH and octanoate (Oct, mM) concentration of albumin preparations having a free thiol level of at least 0.75 moles of free thiol per mole of albumin and a maximum polymer level of 1% (w / w) at a set sodium (Na) concentration of 250 mM after one incubation at 40°C for one month. The filled shaded area indicates the design space with a free thiol level lower than 0.75 moles of free thiol per mole of albumin, and is shaded with diagonal lines. The region indicates a design space with polymer levels exceeding 1% (w / w), the intersection of the vertical and horizontal lines indicates a formulation with pH 6.0 and 5 mM octanoate, (B) a three-dimensional representation of the relationship between octanoate, pH, and thiol levels at a set sodium concentration of 250 mM after incubation at 40°C for one month, and (C) a three-dimensional representation of the relationship between octanoate, pH, and polymer levels at a set sodium concentration of 250 mM after incubation at 40°C for one month. Figure 3 (A) shows the design space (unshaded) for sodium (Na, mM) concentration and pH of an albumin preparation having a free thiol level of at least 0.75 moles of free thiol per mole of albumin and a maximum polymer level of 1% (w / w) at a set octanoate concentration of 5 mM after one incubation at 40°C for one month. The filled shaded area indicates the design space with a free thiol level lower than 0.75 moles of free thiol per mole of albumin, and is shaded with diagonal lines. The region indicates a design space with a polymer level greater than 1% (w / w), the intersection of the vertical and horizontal lines indicates a formulation with 250 mM Na and pH 6.0, (B) a three-dimensional representation of the relationship between pH, sodium, and thiol levels at a set octanoate concentration of 5 mM after incubation at 40°C for one month, and (C) a three-dimensional representation of the relationship between pH, sodium, and polymer levels at a set octanoate concentration of 5 mM after incubation at 40°C for one month.

[0022] Definitions (Part A) The term "albumin" means a protein having the same and / or very similar tertiary structure as human serum albumin (HSA) or one or more (e.g., several) HSA domains, and possessing similar properties to HSA or related domains. Similar tertiary structures are, for example, the structures of albumin from the species described herein. Some of the main properties of albumin are: i) its ability to regulate plasma volume or osmotic pressure; ii) its long plasma half-life of approximately 19 ± 5 days; iii) ligand binding, such as binding to endogenous molecules including acidic lipophilic compounds such as bilirubin, fatty acids, hemin, and thyroxine (see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695, incorporated herein by reference); and iv) binding to small organic compounds with acidic or electronegative characteristics, such as drugs such as warfarin, diazepam, ibuprofen, and paclitaxel (see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695, incorporated herein by reference). Not all of these properties must be met for a protein or fragment to be characterized as albumin. For example, if a fragment does not contain a domain involved in the binding of a particular ligand or organic compound, then variants of such a fragment would not be expected to possess these properties. The term albumin includes variants, and / or derivatives such as fusions and / or fragments of albumin or albumin variants.

[0023] The term “albumin fusion” means a gene fusion of albumin (or a variant or fragment thereof) and a non-albumin protein or peptide. The non-albumin protein or peptide may be a therapeutic, prophylactic, or diagnostic protein or peptide. Examples of albumin fusions are provided in European Patent No. 624195, International Publication No. 2001 / 079271, International Publication No. 2003 / 059934, International Publication No. 2003 / 060071, International Publication No. 2011051489, International Publication No. 2011 / 124718, and European Patent Application Publication No. 11164955 (each incorporated herein in its entirety by reference).

[0024] The term “conjugate” means albumin (or its variants, fragments, or fusions) to which a non-albumin portion has been chemically conjugated. The non-albumin portion may be a therapeutic, prophylactic, or diagnostic protein. Examples of albumin conjugations are provided in International Publication 2011 / 124718 and European Patent Application Publication 11164955 (both incorporated herein by reference).

[0025] The term "fragment" means a polypeptide having one or more (several) amino acids deleted from the amino and / or carboxyl termini of a mature polypeptide, and / or from the internal region of albumin. A fragment may consist of one uninterrupted sequence derived from albumin, or it may contain two or more sequences derived from different parts of albumin. Fragments according to the present invention have a size of more than about 20 amino acid residues, preferably more than 30 amino acid residues, more preferably more than 40 amino acid residues, more preferably more than 50 amino acid residues, more preferably more than 75 amino acid residues, more preferably more than 100 amino acid residues, more preferably more than 200 amino acid residues, more preferably more than 300 amino acid residues, even more preferably more than 400 amino acid residues, and most preferably more than 500 amino acid residues. In a preferred embodiment, the fragment corresponds to one or more albumin domains. The preferred albumin domains of the present invention have at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5%, or 100% identity with HSA domain I, which consists of 1 to 194±1 to 15 amino acid residues of SEQ ID NO: 2, and at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5%, or This refers to a domain having 100% identity and at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5%, or 100% identity with HSA domain III, which consists of 381 to 585 ± 1 to 15 amino acid residues of SEQ ID NO: 2, or a combination of one or more of these domains, for example, fused domains I and II, domains II and III, or domains I and III.There is no generally accepted convention regarding the precise boundaries of albumin domains, and the allowance of overlaps within the above range, as well as the various lengths of ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at the N-terminus and / or C-terminus of each domain, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids, most preferably 1 to 5 amino acids, allowing for total dispersion of up to 30 amino acids for each domain, preferably up to 20 amino acids, more preferably up to 10 amino acids, reflects this fact, and there may be some differing opinions regarding which amino acid residues within the boundaries between domains belong to one or the other. For the same reasons, it may be possible to find references to amino acid residues of albumin domains different from the numbers above, but those skilled in the art will understand how to identify albumin domains based on the teachings in the literature and the teachings above. The corresponding domains of non-human albumin can be identified by alignment with the HSA using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is performed using the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 3.0.0 or later of the Needle program. Optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Alternative alignment tools, such as MUSCLE described herein, can also be used.Domains can also be defined according to Dockal or Kjeldsen: Dockal et al (The Journal of Biological Chemistry, 1999, Vol.274(41):29303-29310) define the domains of HSA as follows: Domain I: amino acids 1-197, Domain II: amino acids 189-385 of sequence 2, Domain III: amino acids 381-585 of sequence 2. Kjeldsen et al (Protein Expression and Purification, 1998, Vol 13:163-169) define the domains as follows: Domain I: amino acids 1-192, Domain II: amino acids 193-382, Domain III: amino acids 383-585.

[0026] Therefore, the following domain definitions are preferred in the present invention. The amino acid numbers correspond to those of Sequence ID No. 2 (HSA). However, these numbers can be used by those skilled in the art to identify corresponding domains in other albumin sequences. Domain I may or may not begin with amino acid 1, and may or may not end with any of amino acids 192, 193, 194, 195, 196, or 197, preferably any of amino acids 192, 194, or 197. Domain II may or may not begin with any of amino acids 189, 190, 191, 192, or 193, preferably any of amino acids 189, 192, or 193, and may or may not end with any of amino acids 382, ​​383, 384, 385, 386, or 387, preferably any of amino acids 382, ​​385, or 387. Domain III may or may not begin with amino acids 381, 382, ​​or 383, preferably 381 or 383, and may or may not end with amino acid 585. Domains in non-human albumin may have the same or different amino acid lengths and / or number of residues as HSA. For example, multiple alignments or paired alignments may be prepared using HSA and one or more other albumins, fragments, derivatives, variants, and / or fusions to identify domains corresponding to domains 1, 2, and / or 3 of HSA.

[0027] Each domain itself consists of two homologous subdomains, namely 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585, and flexible intersubdomain linker regions include residues Lys106-Glu119, Glu292-Val315, and Glu492-Ala511.

[0028] The term "free thiol" refers to a thiol group that is available to react with another group. For example, a free thiol is one that is not yet connected to another thiol group via a disulfide bond, or that has not been oxidized.

[0029] The terms “parent” or “parental albumin” mean albumin which modification is performed to produce the albumin variants of this disclosure. The parent may be a naturally occurring (wild-type) polypeptide or its allele. In preferred embodiments, the parental albumin is wild-type albumin, more preferably wild-type albumin from Homo sapiens (e.g., UNIPROT:P02768.2, SEQ ID NO: 10). Preferably, the wild-type albumin from Homo sapiens is a mature form of albumin, such as SEQ ID NO: 2. The mature form of the albumin sequence may be encoded by the nucleotide sequence SEQ ID NO: 1. Alternative wild-type albumin can be obtained from primate serum albumins such as orangutan serum albumin (UNIPROT:Q5NVH5.2), chimpanzee serum albumin (NCBI:XP_517233), and macaque serum albumin (UNIPROT:Q28522.1), or from rabbit serum albumin (UNIPROT:P49065.2), guinea pig serum albumin (UNIPROT:Q6WDN9), hamster serum albumin (UNIPROT:A6YF56), mouse serum albumin (UNIPROT:P07724.3), and rat serum albumin. The serum albumin can be selected from rodent serum albumins such as albumin (UNIPROT:P02770.2), or from serum albumins from ungulates such as bovine serum albumin (UNIPROT:P02769.4), horse serum albumin (UNIPROT:P35747.1), donkey serum albumin (UNIPROT:Q5XLE4.1), goat serum albumin (GENBANK:ACF1039.1), sheep serum albumin (UNIPROT:P14639.1), and pig serum albumin (UNIPROT:P08835.2), or from dog serum albumin (NCBI NP_001003026), or from chicken serum albumin (UNIPROT:P19121.2), or from any of the other albumin species shown in the table above. Preferably, the parent albumin is mature albumin.In another embodiment, the parent albumin is at least 70%, more preferably 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 99.8% identical to SEQ ID NO 2, and maintains at least one of the main properties of albumin or an albumin-like tertiary structure such as HSA. It is preferable that the parent albumin is a mature albumin, for example, one that does not contain a leader sequence.

[0030] In relation to "fusion partner," the term "partner" refers to a non-albuminic portion that can genetically fuse with albumin or its fragments or fusions; in relation to "conjugation partner," it refers to a non-albuminic portion that can chemically conjugate with albumin or its fragments or fusions. Conjugation partners may or may not be biologically active. Conjugation partners may or may not be therapeutic compounds. Conjugation partners may or may not be radiopharmaceuticals. Conjugation partners may or may not be imaging agents.

[0031] The term "polymer" refers to a gel permeation HPLC column that separates molecules within the molecular weight range of 10,000 to 500,000 Da, such as the TSK G3000SW. XL This refers to high molecular weight forms or aggregates of albumin that elute within the void volume. The polymer does not contain dimers or trimers.

[0032] The term "thiol" refers to a carbon-bonded sulfhydryl (-C-SH or R-SH, where R can be selected from the group consisting of alkanes, alkenes, or other carbon-containing groups).

[0033] The term "variant" refers to a polypeptide derived from a parent albumin that includes modifications, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid, deletion means removing an amino acid occupying a position, and insertion means adding one to three amino acids adjacent to an amino acid occupying a position. Modified polypeptides (variants) can be obtained through human intervention by modifying the polynucleotide sequence encoding the parent albumin. The variant albumin is preferably at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, and retains at least one (e.g., several) of the main properties of the tertiary structure similar to that of the parent albumin or HSA. For the purposes of this invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is performed using the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later of the Needle program. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the identity rate and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment).

[0034] Compared to the parent albumin, the variant may possess modified binding affinity and / or modified transcytosis rate to the neonatal Fc receptor (FcRn) across the endothelium, epithelium, and / or mesothelial single-cell layer. The variant albumin sequence is preferably not found in nature. The variant may be as described in International Publication Nos. 2010 / 092135, 2011 / 051489, 2011 / 124718, 2012 / 059486, 2012 / 150319, 2013 / 135896, 2014 / 072481, 2014 / 125082, or 2015 / 036579.

[0035] The term "wild-type" (WT) albumin refers to albumin that has the same amino acid sequence as albumin found naturally in animals or humans. Human serum albumin (HSA, sequence 1 for cDNA, or sequence number 2 for amino acid sequence) is an example of wild-type albumin and is derived from Homo sapiens. The wild-type (WT) human albumin (HSA) sequence is given by GenBank accession number AAA98797.1 (Minghetti, PP et al. "Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4", J. Biol. Chem. 261(15), 6747-6757 (1986)). [Modes for carrying out the invention]

[0036] Modes for carrying out the invention (Part A) A first aspect of the present invention provides a liquid formulation of albumin or its fragments or fusions, comprising at least 0.75 moles of free thiols and / or up to 1% (w / w) of albumin polymer per mole of albumin. The liquid formulation may contain at least 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, or 0.99 moles of free thiols per mole of albumin or its fragments or fusions. The liquid formulation may contain 1 mole of free thiols per mole of albumin or its fragments or fusions.

[0037] Albumin may be supplied from serum or recombinant sources. Advantageously, liquid formulations may contain recombinant albumin. That is, albumin may be supplied from recombinant organisms such as recombinant microorganisms, recombinant plants, or recombinant animals. Because some users prefer animal-free ingredients, it is more preferable that albumin be supplied from non-animal recombinant sources such as recombinant microorganisms or recombinant plants. Preferred organisms include, but are not limited to, prokaryotes, and more preferably eukaryotes such as animals, plants, fungi, or yeasts, and include, for example, the following species in which albumin has been successfully expressed as a recombinant protein: Fungi (including, but not limited to, Aspergillus (International Publication No. 06066595), Kluyveromyces (Fleer 1991, Bio / technology 9, 968-975), Pichia (Kobayashi 1998 Therapeutic Apheresis 2,257-262), and Saccharomyces (Sleep 1990, Bio / technology 8, 42-46)) Animals (Barash 1993, Transgenic Research 2, 266-276) Plants (including, but not limited to, potatoes and tobacco (Sijmons 1990, Bio / technology 8, 217, and Farran 2002, Transgenic Research 11, 337-346), as well as rice, e.g., Oryza sativa) Mammalian cells such as CHO and HEK Prokaryotes (Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285), for example, E. coli (European Patent No. 73,646).

[0038] Preferably, the albumin is a recombinant supplied from a fungal host, preferably Saccharomyces (e.g., Saccharomyces cerevisiae) or Pichia (e.g., Pichia pastoris), more preferably Saccharomyces, most preferably Saccharomyces cerevisiae. The albumin expressed by the host may include a leader sequence, for example, the albumin may include SEQ ID NO: 10 described herein or its variants, fragments, or fusions. The first 19 residues of SEQ ID NO: 10 correspond to the signal sequence, the following 5 residues correspond to the propeptide, and the following 585 residues correspond to the mature peptide according to SEQ ID NO: 2.

[0039] Albumin or its fragments may contain one or more (e.g., several) free thiol groups ("thiols") per molecule of albumin or its fragments, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 free thiol groups. For albumin or its fragments or fusions containing one free thiol group per molecule, such as human serum albumin containing free thiols provided by Cys34, the theoretical free thiol content is 1 mole of free thiols per mole of albumin or its fragments or fusions. For albumin or its fragments or fusions containing two free thiol groups per molecule, such as a variant of human serum albumin containing free thiols provided by Cys34 and additional free thiols provided by another Cys34, the theoretical free thiol content is 2 moles of free thiols per mole of albumin or its fragments. Preferably, the liquid formulation contains at least 75% theoretical free thiol levels, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% theoretical free thiol levels. Therefore, for albumin or its fragments or fusions containing 2 free thiols per molecule of albumin or its fragments, the free thiol level of at least 75% theoretical free thiol levels is 0.75 * 2. That is, the average (mean value) of at least 1.5 moles of free thiols per mole of albumin or its fragments or fusions. Liquid formulations may contain 100% theoretical free thiol levels.

[0040] Preferably, the desired free thiol level per mole of albumin or its fragment or fusion is observed after storage or incubation at 25°C for at least one month, more preferably at least two, three, four, five, six, nine, or twelve months. Preferably, the free thiol level per mole of albumin or its fragment is observed after storage or incubation at 40°C for at least one month, more preferably at least two, three, four, five, or six months. Preferably, the free thiol level per mole of albumin or its fragment is observed after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7-3, 4, 5, 6, 7, or 8°C, for example, about 4°C, over a duration of at least one month, more preferably at least two, three, four, five, or six months, or at least one, two, three, four, five, or six years.

[0041] Free thiol levels can be measured by free thiol assays, such as a colorimetric free thiol assay, for example, a DTNB assay or a DTDP assay. The DTDP assay is preferred.

[0042] A suitable DTDP assay comprises the following method, where the volume of components may be adjusted and the ratio of components should remain as described below: a) Using 0.65 mM glutathione and 950 μL (microliters) of buffer as controls, albumin (50 mg / mL in 50 μL (microliters)) was measured. -1 Prepare an albumin "sample" by mixing albumin and buffer (950 μL (microliters), for example, 0.1 M sodium phosphate, 1 mM EDTA, pH 7.4) (see, for example, Example 1). b) Measure the absorbance of the sample, control, and buffer "blank" at a wavelength of 324 nm. c) Add 50 μL (microliters) of 4 mM 4,4'-dithiodipyridine (DTDP) to the sample, control, and buffer "blank". d) Incubate at room temperature (15-25°C, 20°C is preferable) for 10 minutes. e) Measure the increase in absorbance at a wavelength of 324 nm for the albumin sample, control, and buffer "blank," and f) Determine the free thiol level in the sample.

[0043] A suitable DTNB assay comprises the following steps, where the volume of components may be adjusted and the ratio of components should remain as described below: a) Prepare an albumin "sample" by mixing albumin (80 μL of 50 mg / mL of albumin) and buffer (920 μL of buffer, for example, 0.1 M TRIS-HCl, 0.01 M EDTA, pH 8.0) using 80 μL (microliters) of 0.65 mM glutathione and 920 μL (microliters) of buffer as controls (see, for example, Example 1). b) Measure the absorbance of the sample, control, and buffer "blank" at a wavelength of 412 nm. c) Add 50 μL (microliters) of 0.01 M 5,5'-Dithiobis-(2-Nitrobenzoate) (DTNB) to the sample, control, and buffer "blank". d) Incubate at room temperature (15-25°C, 20°C is preferred) for 10 minutes, and e) Measure the increase in absorbance of the sample, control, and buffer "blank" at a wavelength of 412 nm, and f) Determine the free thiol level in the sample.

[0044] Free thiol levels observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation), preferably at least 70%, more preferably at least 72, 74, 76, 78, 80, 82, 84, 85, 86, 88, 90, 92, 94, 85, 96, 97, 98, 99%, are maintained after storage or incubation at 25°C for at least one month, more preferably two, three, four, five, or six months, or at least one, two, three, four, five, or six years. Free thiol levels observed at T0 (e.g., measured within 24 or 48 hours of formulation), preferably at least 70%, more preferably at least 72, 74, 76, 78, 80, 82, 84, 85, 86, 88, 90, 92, 94, 85, 96, 97, 98, 99%, are maintained after storage or incubation at 40°C for at least one month, more preferably two, three, four, five, or six months. Free thiol levels observed at T0 (e.g., measured within 24 or 48 hours of formulation), preferably at least 70%, more preferably at least 72, 74, 76, 78, 80, 82, 84, 85, 86, 88, 90, 92, 94, 85, 96, 97, 98, 99%, are maintained after storage or incubation at a temperature of 2-8°C, such as 2, 3, 4, 5, 6, or 7-3, 4, 5, 6, 7, or 8°C, for at least 1 month, more preferably 2, 3, 4, 5, or 6 months, or at least 1, 2, 3, 4, 5, or 6 years.

[0045] Preferably, the formulation contains up to 1% polymer, more preferably up to 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0% polymer. Preferably, polymer levels of up to 1%, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0% are observed at T0 (e.g., measured within 24 or 48 hours of formulation). Preferably, polymer levels of up to 1%, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0% are observed after storage or incubation at at least 25°C for at least one month, more preferably at at least 40°C for at least one month. Preferably, the polymer level after incubation at 25°C or 40°C for 2, 3, 4, 5, or 6 months is up to 5, 4, 3 times, or preferably up to 2 times, the level observed after incubation at the same temperature for 1 month. For example, the polymer level after incubation at 40°C for 3 months is up to 4 times, the level observed after incubation at the same temperature for 1 month. The polymer, preferably up to 1%, more preferably up to 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0%, observed at T0 (e.g., measured within 24 or 48 hours of formulation), is maintained after storage or incubation at a temperature of 2–8°C, such as 2, 3, 4, 5, 6, or 7°C, including 3, 4, 5, 6, 7, or 8°C, for a duration of at least 1 month, more preferably at least 2, 3, 4, 5, or 6 months, or at least 1, 2, 3, 4, 5, or 6 years. 1% polymer means that 1% albumin is present in polymer form. Polymer levels are, for example, 5 mg / mL. -1 Albumin can be used to measure, for example, gel permeation high-performance liquid chromatography (GP.HPLC).

[0046] A preferred liquid formulation according to the first aspect of the present invention is C6, C8, or C 10It contains 2.5 to 7.5 mM fatty acids, such as C6 fatty acids or larger, more preferably C7 fatty acids or larger, and most preferably C8 fatty acids (octanoates). The fatty acid concentration may be approximately 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, or 7.25 mM. A fatty acid concentration of approximately 3 mM to 7 mM, such as approximately 4 mM to 6 mM, is preferred, and particularly preferably approximately 5 mM.

[0047] A preferred liquid formulation according to a first aspect of the present invention contains a cation concentration of at least 175 mM, such as a cation concentration of about 175, 200, 225, 250, 275, or 300 mM to about 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mM. Preferred cation concentrations are about 200 to about 300 mM, particularly about 225, 230, 235, 240, 245, 250, 255, 260, 265, or 270 to about 230, 235, 240, 245, 250, 255, 260, 265, 270, or 275 mM, particularly about 250 mM.

[0048] The cations of the composition may be provided by any cation, and may be provided by one or more (e.g., several) classes or species described below. For example, the cation may be monovalent or divalent, monatomic or polyatomic, and may be provided by one or more (e.g., several) alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), or ammonium. The cation is preferably provided by sodium and / or potassium and / or magnesium, most preferably sodium or magnesium.

[0049] The cations can be provided by salts of inorganic acids (e.g., Group 1 or 2 metals or ammonium salts such as sodium chloride), salts of divalent acids (e.g., Group 1 or 2 metals or ammonium sulfate or ammonium phosphate such as sodium sulfate), or salts of organic acids (e.g., Group 1 or 2 metals or ammonium salts of acetates or citrates such as sodium acetate). For the liquid albumin formulation of the present invention, sodium chloride is a preferred cation source.

[0050] The cations and anions used to stabilize albumin may be provided by (i) salts and / or (ii) pH buffers as described herein. Thus, more than one (e.g., several) cations or anions may be present, such as two or three. More than one (e.g., several) sources of a single cation may be present, such as Na, which may be provided by both pH buffers (e.g., sodium phosphate) and salts (e.g., NaCl).

[0051] Anions useful in the present invention include inorganic anions such as phosphates, halides such as chlorides, and organic anions such as acetates and citrates. Anions can be monovalent or divalent, monatomic or polyatomic. Preferred anions include sulfates, acetate phosphates, and chlorides, in particular chlorides, sulfates, and acetates.

[0052] Therefore, the composition may contain one or more (for example, several) alkali metal phosphates or chlorides (such as sodium phosphate, potassium phosphate, sodium chloride, or potassium chloride), alkaline earth metal phosphates (such as calcium phosphate, magnesium phosphate, calcium chloride, or magnesium chloride), or ammonium phosphate or ammonium chloride.

[0053] A preferred liquid formulation according to a first aspect of the present invention has a pH of from about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, or 6.4 to about 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. A preferred pH is from about 5.7 to about 6.2, more preferably about 6.0.

[0054] A particularly preferred liquid formulation is given by the following formula: (a) Free thiol = 1.1786 - 0.05167 * pH - 0.0001544 * Cation - 0.01133 * FA + (pH - 6) * ((Cation - 198.3) * 0.0002659) + (pH - 6) * ((FA - 7.125) * 0.003160) + (Cation - 198.3) * ((FA - 7.125) * 0.000001935) + (pH - 6) * ((pH - 6) * - 0.0500) + (Cation - 198.3) * ((Cation - 198.3) * 0.000002583) + (FA - 7.125) * ((FA - 7.125) * 0.0001413) + (pH - 6) * ((Cation - 198.3) * ((FA - 7.125) * - 0.000009879)), and (b) Polymer = 9.1677 - 0.8417 * pH - 0.01044 * Cation - 0.2690 * FA + (pH - 6) * ((Cation - 198.3) * - 0.004796) + (pH - 6) * ((FA - 7.125) * 0.06523) + (Cation - 198.3) * ((FA - 7.125) *0.001494)+(pH-6) * ((pH-6) * 2.25) + (cation -198.3) * ((Cation-198.3) * 0.00006768)+(FA-7.125) * ((FA-7.125) * 0.02490)+(pH-6) * ((Cation-198.3) * ((FA-7.125) * Having a formulation that is in the design space defined by both of 0.001237), During the ceremony, "Free thiol" refers to the level of free thiol, where 1.0 means 1 mole of thiol per mole of albumin, and "cation" refers to the cation concentration (preferably sodium) in mM units. "FA" refers to fatty acid concentration (preferably octanoate) in mM units.

[0055] Albumin or a fragment thereof may or may not be genetically fused with a partner. The advantage of the albumin fusion according to the present invention is that a first partner can be genetically fused with albumin or a fragment thereof, and a second partner can be chemically conjugated with albumin or a fragment thereof or the fusion. The first and second partners may be different parts or copies of the same part. Examples of partners are disclosed under the ninth aspect of the present invention.

[0056] One or more free thiols in albumin or its fragments or fusions may be provided by one or more cysteine ​​residues at one or more (e.g., several) positions in albumin or its fragments or fusions. For example, free thiols are 34, 1, 2, 4, 38, 40, 48, 52, 55, 58, 60, 75, 76, 79, 80, 82, 83, 83, 86, 91, 104, 113, 115, 116, 121, 122, 124, 125, 129, 168, 169, 177, 229, 236, 266, 269, 270, 273, 283, 298, 300, 301, 303, 304, 308, 313, 314, 316, 318, 320, 321, 324, 325, 355, 360, 361, 364, 365, 368, 369, The free thiol may be provided by cysteine ​​at a position corresponding to one or more (e.g., several) of the positions selected from 371, 375, 379, 386, 390, 396, 397, 435, 439, 443, 471, 478, 479, 490, 496, 498, 501, 503, 504, 505, 506, 508, 512, 538, 541, 542, 546, 549, 550, 558, 560, 562, 564, 565, 566, 567, 573, 574, 577, 578, 580, 581, 582, 584, and 585. Preferably, the free thiol is provided by cysteine ​​at the position corresponding to position 34 of SEQ ID NO: 2.

[0057] Preferably, the albumin or its fragments or fusions have at least 70% sequence identity with mammalian albumin selected from the group consisting of human (SEQ ID NO: 2), mouse (SEQ ID NO: 3), rat (SEQ ID NO: 4), macaque (SEQ ID NO: 5), bovine (SEQ ID NO: 6), pig (SEQ ID NO: 7), horse (SEQ ID NO: 8), or rabbit (SEQ ID NO: 9) albumin. Preferably, albumin or its fragment has at least 70% sequence identity with HSA (SEQ ID NO: 2), more preferably 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, or 99.8 to 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, 99.8, or 100% identity with HSA (SEQ ID NO: 2). For example, a preferred albumin or its fragment or fusion has a maximum of 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation with respect to wtHSA (SEQ ID NO: 2) or its fragment or fusion.

[0058] A fusion of albumin or a fragment thereof comprises albumin or a fragment thereof genetically fused to a fusion partner, as described herein. The fusion partner may, in principle, be any polypeptide, but generally, it is preferable that the fusion partner is a polypeptide having biological activity, therapeutic, prophylactic (including vaccines), radiopharmaceutical, diagnostic, imaging, or other beneficial properties. Such properties may be referred to as “pharmaceutically beneficial properties.” Fusion polypeptides comprising albumin or a fragment thereof are known in the art. Such fusion polypeptides comprising albumin or a fragment thereof and a fusion partner polypeptide have been found to have a longer plasma half-life compared to the unfused fusion partner polypeptide alone. The fusion may be one or more (e.g., several) of the following: an N-terminal fusion (i.e., a fusion partner genetically fused to the N-terminus of albumin or a fragment thereof), a C-terminal fusion (i.e., a fusion partner genetically fused to the C-terminus of albumin or a fragment thereof), or an insertion into the sequence of albumin or a fragment thereof, such as an insertion into a loop.

[0059] Preferably, the albumin fragment or fusion thereof contains at least 175 consecutive amino acids from albumin having at least 70% sequence identity with HSA (SEQ ID NO: 2), such as 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, or 575-200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 580 amino acids. The fragment may contain, consist of, or substantially correspond to one or more (e.g., several) domains of albumin or its variants, such as HSA (SEQ ID NO: 2), including amino acids corresponding to domain I (residues 1-194±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), domain II (residues 192-387±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), or domain III (residues 381-585±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0060] The liquid formulation may contain approximately 5 to approximately 30% (w / v), for example, approximately 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, or 27.5%, most preferably approximately 10% or approximately 20%, of albumin or its fragments or fusions.

[0061] The liquid formulation preferably contains at least 50% albumin or its fragments or fusions, more preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% albumin or its fragments or fusions. Most preferably, the liquid formulation is substantially or completely free of proteins other than albumin, its fragments, or fusions.

[0062] Liquid formulations are preferably aqueous.

[0063] Liquid formulations of albumin or its fragments or fusions may or may not contain an inorganic buffer. The inorganic buffer may or may not be selected from organic buffers such as sodium phosphate, potassium phosphate, sodium acetate, or sodium citrate. The buffer may stabilize the pH. Preferably, the buffer is a pharmaceutically acceptable buffer. Liquid formulations of albumin or its fragments or fusions may or may not contain a surfactant such as polysorbate 80. For example, the liquid formulation may contain 0, 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10, or 15 mg / L of polysorbate 80 or similar. -1 Surfactants in concentrations of ~0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, or 20 mg / L -1 They may or may not contain surfactants. Liquid formulations may be substantially free of surfactants such as polysorbate 80, or completely free of them.

[0064] Liquid formulations of albumin or its fragments or fusions may or may not contain amino acids such as tryptophan, e.g., N-acetyltryptophan (i.e., “free” amino acids not present in the albumin fragment or fusion). For example, a liquid albumin formulation may or may not contain 0, 0.001, 0.005, 0.01, 0.1, 0.5, 1, 2, 3, or 4 mM free amino acids such as tryptophan to 0.001, 0.005, 0.01, 0.1, 0.5, 1, 2, 3, 4, or 5 mM free amino acids. A liquid albumin formulation may substantially or completely lack free amino acids such as tryptophan.

[0065] Liquid formulations of albumin or its fragments or fusions may contain substantially no, or may not contain at all, components other than albumin, a cation source, and fatty acids.

[0066] Liquid formulations may be supplied in rigid or flexible vials, bottles, or bags, such as BPCs or bioprocess containers. Suitable container volumes range from approximately 50 mL to approximately 10,000 mL, for example, 50 mL, 1,000 mL, 5,000 mL, and 10,000 mL. The containers preferably include one or more (e.g., several) inlets or outlets to allow for filling and / or dispensing from the containers. The albumin composition may be sterilized, for example, before or after filling the containers. Liquid formulations may or may not be supplied in unit dosage forms.

[0067] A remarkable advantage of the formulations of the first aspect of the present invention is that they provide high free thiol levels while also providing low polymer levels, which are desirable when conjugating a partner to albumin, as high free thiol levels enable good conjugation efficiency and low polymer levels minimize the possibility of adverse immunogenic responses when delivered to a target such as a patient. Therefore, the present invention provides a liquid formulation for maximizing free thiol levels and minimizing polymer levels of albumin.

[0068] A second aspect of the present invention provides a method for preparing a formulation of albumin or its fragments or fusions having a high free thiol level and a low polymer level, comprising: formulation of albumin or its fragments or fusions in a buffer containing 2.5 to 7.5 mM fatty acids, at least 175 mM cations, and a pH of 5.5 to 6.5; and optionally, filling the albumin or its fragments or fusions into a container. The formulation can maintain a high free thiol level and a low polymer level after storage or incubation as described herein.

[0069] A third aspect of the present invention provides the use of the buffer described herein for maintaining high free thiol levels and low polymer levels in an albumin preparation, for example, after storage or incubation of the albumin preparation.

[0070] The options and preferences of the first aspect of the present invention apply to the second and third aspects of the present invention.

[0071] A fourth aspect of the present invention provides a method for producing a conjugate, comprising conjugating albumin or a fragment or fusion thereof with a conjugation partner, wherein the albumin or a fragment or fusion thereof is a formulation produced by a method according to the first aspect of the present invention or by a method according to the second aspect of the present invention.

[0072] Optionally, conjugation is performed via a linker.

[0073] Conjugation may be performed by methods known in the art (for example, those provided by Pierce, Thermo Fisher Scientific, Rockford, IL, USA in the 2012 edition of the "Thermo Scientific Crosslinking Technical Handbook" (document identifier: 1602163 10 / 12) (available online at https: / / tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf)). These include, but are not limited to, incorporating or manipulating a thiol reactive group in or on the conjugation part by, for example, incorporating or manipulating another free thiol present on the conjugation partner, or incorporating or manipulating a pyridyl disulfide group on the conjugation partner, or incorporating or manipulating a haloacetyl group on the bioactive compound, or incorporating or manipulating a maleimide group on the conjugation partner, or incorporating or manipulating a thiosulfate base on the conjugation partner, or incorporating or manipulating a vinyl sulfone group on the conjugation partner.For example, but not limited to, N-ethylmaleimide (NEM, Pierce), 2-amino-2'-aminoethanethiol sulfonate (Pierce), N-beta-maleimidopropionic acid (BMPA, Pierce), and methyl methanethiosulfonate. These include thiosulfonate (MMTS, Pierce), fluorescein-5-maleimide (Pierce), 5-iodoacetamido-fluorescein (5-IAF, Pierce), or N-[6-7-amino-4-methylcoumarin-3-acetamido)hexyl]-3'-[2'-pyridyldithio]propionamide (AMCA-HPDP, Pierce).

[0074] If the conjugation partner contains at least one (e.g., several) thiols, the conjugation partner may be oxidized by methods known in the art, such as oxidation, or by 1,4-bis-maleimidibutane (BMB, Pierce), 1,4-bis-maleimidyl-2,3-dihydroxybutane (BMDB, Pierce), bis-maleimidohexane (BMH, Pierce), bis-maleimidoethane (BMOE, Pierce), 1,8-bis-maleimidotriethyleneglycolimide (BM[PEO]3, Pierce), 1,11-bis-maleimido Crosslinking reagents such as, but not limited to, tetraethylene glycol (1,11-Bis-Maleimidotetraethyleneglycol, BM[PEO]4, Pierce), 1,4-Di-[3'-(2'-pyridyldithio)-propionamido]butane (DPDPB, Pierce), dithio-bis-maleimidoethane (DTME, Pierce), 1,6-Hexane-bis-vinylsulfone (HBVS, Pierce), and tris-[2-maleimimidoethyl]amine (TMEA, Pierce) can be used to crosslink the albumin mutant protein of the present invention.

[0075] If the conjugation partner does not contain a thiol-reactive group, it may be modified to incorporate one or more (e.g., several) such groups by either chemical modification or genetic engineering by methods known in the art (Chapman, AP (2002) Adv. Drug Deliv. Rev., 54 531-545, Humphreys, D P et al. Protein Engineering, Design & Selection vol.20 no.5 pp.227-234, 2007, incorporated herein by reference). These two references describe methodologies for crosslinking PEG to engineered free thiols in antibodies or antibody fragments, and these techniques may be used to crosslink the conjugation partner to engineered free thiols in the albumin mutant protein of the present invention. Alternatively, the Drug Affinity Complex (DAC®) technology developed by ConjuChem Inc. (Montreal, Quebec, Canada, H2X 3Y8), as described in International Publication No. 2000 / 69902 (incorporated herein by reference), may be used. Each DAC® construct consists of three parts: 1) a drug component (the part involved in biological activity), 2) a linker bound to the drug component, and 3) a reactive chemical group at the opposite end of the linker, usually a soft electrophile selective for thiols, with maleimide being the most useful embodiment. Other applicable conjugation methods are described in International Publication No. 2007 / 071068 (incorporated herein by reference).

[0076] If the conjugation partner does not contain a thiol-reactive group but contains one or more (e.g., several) amino groups, it is one of the following: N-5-azido-2-nitrobenzoyloxysuccinimide (AMAS, Pierce), N-[beta-maleimidopropyloxy]succinimide ester (BMPS, Pierce), N-eta-maleimidocaproic acid (EMCA, Pierce), N-[eta-maleimidocaproyloxy]succinimide ester (EMCS, Pierce), N-[eta-maleimidocaproyloxy]sulfosuccinimide N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS, Pierce), N-[gamma-maleimidobutyryloxy]sulfosuccinimide ester (GMBS, Pierce), N-kappa-maleimidoundecanoic acid (KMUA, Pierce), N-[κ-maleimidoundecanoyloxy]sulfosuccinimide ester (GMBS, Pierce) m-maleimidobenzoyl-N-hydroxysuccinimide (MBS, Pierce), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (m-maleimidobenzoyl-N-hydroxysulfosuccinimideN-succinimidyl S-acetylthio-acetate (SATA, Pierce), N-succinimidyl S-acetylthiopropionate (SATP, Pierce), succinimidyl 3-[bromoacetamido]propionate (SBAP, Pierce), N-succinimidyl iodo acetate (N-succinimidyliodoacetate (SIA, Pierce), N-succinimidyl[4-iodoacetyl]aminobenzoate (SIAB, Pierce), sulfosuccinimidyl[4-iodoacetyl]aminobenzoate (sulfosuccinimidyl[4-iodoacetyl]aminobenzoate, sulfo-SIAB, Pierce), succinimidyl[4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC, Pierce), sulfosuccinimidyl[4-[N-maleimidomethyl]cyclohexane-1-carboxylate, sulfo-SMCC, Pierce) , succinimidyl-[4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate (LC-SMCC, Pierce), 4-succinimidyloxycarbonyl-methyl-α[2-pyridyldithio]toluene (SMPT, Pierce), sulfosuccinimidyl6-[alpha-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT, Pierce), succinimidyl4-[p-maleimidophenyl]-butyrate (succinimidyl 4-[p-maleimidophenyl]-butyrate (SMPB, Pierce), sulfosuccinimidyl 4-[p-maleimidophenyl]-butyrate4-[p-maleimidophenyl]-butyrate, sulfo-SMPB, Pierce), succinimidyl-6-[(beta-maleimidopropionamido)hexanoate], SMPH, Pierce), N-succinimidyl 3-[2-pyridyldithio]propionate These can be modified to incorporate one or more (e.g., several) thiol-reactive groups by chemical modification using methods known herein, such as the use of crosslinking reagents, including but not limited to 3-[2-pyridyldithio]propionate (SPDP, Pierce), succinimidyl[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP, Pierce), sulfosuccinimidyl[3'-(2-pyridyldithio)propionamido]hexanoate (sulfosuccinimidyl[3'-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP, Pierce) and N-succinimidyl-[4-vinylsulfonyl]benzoate (SVSB, Pierce). Blocking certain amine residues may be advantageous, as described by Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384 (incorporated herein by reference).

[0077] If the conjugation partner does not contain a thiol-reactive group but contains one or more (e.g., several) carbonyl (oxidized carbohydrate) groups, it is one of the following: N-β-maleimidopropionic acid hydrazide (BMPH, Pierce), N-[eta-maleimidocaproic acid]hydrazide (EMCH, Pierce), 4-[N-maleimidomethyl]cyclohexane-1carboxylhydrazide·HCl·1 / 2 dioxane (MMCCH, Pierce), 3-maleimidophenylboronic acid (MPBH, Pierce), N-[κ-maleimidoundecanoic acid]hydrazide (N-[κ-maleimidoundecanoic These can be modified to incorporate one or more (e.g., several) thiol-reactive groups by chemical modification using methods known in the art, such as the use of crosslinking reagents, including but not limited to acid]hydrazide (KMUH, Pierce) and 3-[2-pyridyldithio]propionyl hydrazide (PDPH, Pierce).

[0078] If the conjugation partner does not contain a thiol-reactive group but contains one or more (e.g., several) hydroxyl groups, it can be modified to incorporate one or more (e.g., several) thiol-reactive groups by chemical modification using methods known in the art, such as the use of a crosslinking agent, including but not limited to N-[p-maleimidophenyl]isocyanate (PMPI, Pierce).

[0079] Optionally, the method includes, for example, providing a conjugate in a container or filling a container with a conjugate, as described above. Optionally, the method also includes providing a conjugate in unit dosage form.

[0080] A fifth aspect of the present invention provides a method for conjugating albumin or its fragments or fusions to a partner with at least 75% efficiency, preferably at least 80, 85, 90, 95, 96, 97, 98, 99% efficiency, or 100% efficiency, comprising contacting a formulation of albumin or its fragments or fusions according to the first, second, third, or fourth aspect of the present invention. Optionally, the conjugation method includes a linker.

[0081] A sixth aspect of the present invention provides the use of albumin formulations prepared by the method of the first or second aspect of the present invention for high-efficiency conjugation to a conjugation partner. "High efficiency" includes conjugation with an efficiency of at least 75, 80, 85, 90, 95, 96, 97, 98, 99%, or 100% relative to the albumin component. For example, an efficiency of 75% means that, upon completion, for example, 75% of the free thiols in the albumin component are conjugated to the partner portion. High levels of free thiols in the formulation of albumin, its fragments, or fusions enable this high-efficiency conjugation.

[0082] A seventh aspect of the present invention provides a conjugate comprising albumin prepared according to a first aspect of the present invention or according to a second aspect of the present invention, and a conjugation partner. The seventh aspect of the present invention also provides a conjugate that can be obtained or obtained by the method of a fourth or fifth aspect of the present invention.

[0083] The conjugation partner may be a bioactive compound such as a peptide, polypeptide, protein, or small molecule drug, a radiopharmaceutical, or an imaging agent.

[0084] A conjugate may include two or more (several, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conjugation partners, each of which may be different from one another and / or may be multiple copies of the same compound.

[0085] Preferably, each conjugation partner is bound to albumin through a conjugable cysteine ​​residue of albumin, but the conjugation partners may be bound by other means, for example, by gene fusion or covalent bond to a non-cysteine ​​amino acid such as lysine, as described in a fourth aspect of the present invention.

[0086] An eighth aspect of the present invention provides the use of albumin preparations described herein or produced by the methods described herein for increasing the half-life of molecules such as bioactive agents, therapeutic agents, radiopharmaceuticals, imaging agents, diagnostic agents, contrast agents, or therapeutic compounds. The half-life can be increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% compared to molecules not conjugated with albumin.

[0087] A ninth aspect of the present invention provides a conjugate described herein for the treatment of a disease, the treatment of a disease, and / or the diagnosis of a disease.

[0088] A tenth aspect of the present invention provides a conjugate described herein for the manufacture of a pharmacopoeci for the treatment of a disease, the treatment of a disease, and / or the diagnosis of a disease.

[0089] Conjugation Partner The term "conjugation partner" includes therapeutic compounds such as bioactive agents, imaging agents, diagnostic agents, contrast agents, radiopharmaceuticals, and chemotherapeutic agents and radiopharmaceuticals.

[0090] Imaging agents, diagnostic compounds, contrast agents, and therapeutic compounds The use of diagnostic agents, imaging agents, and biological "contrast" agents is well known in the art. Diagnostic agents are any pharmaceutical products used as part of a diagnostic test (i.e., together with the instruments and procedures required to evaluate the test results). Diagnostic agents may be used in vivo, ex vivo, or in vitro.

[0091] The ability of albumin to accumulate in damaged muscle fibers of dystrophic muscles is well documented. For example, gadolinium-DTPA-albumin conjugates can be used as a combined diagnostic and therapeutic tool for visualizing and monitoring dystrophic muscles by magnetic resonance imaging (MRI) and for delivering albumin-bound putative therapeutic agents for effective targeting of dystrophic muscles (Amthor et al. (2004) Neuromuscular Disorders 14912:791-796). Malignant tumors often show increased albumin uptake and metabolism. The use of gadolinium-albumin conjugates has also been documented for improved imaging of malignant tumors and for determining tumors sensitive to treatment with drug-conjugated albumin by MRI (Kiessling et al. (2002) Investigative Radiology 37(4):93-198).

[0092] Current imaging agents often degrade rapidly, while longer-lasting agents are often toxic. The use of albumin conjugates can be particularly useful in increasing the half-life of imaging agents, thus enabling imaging over extended periods. International Publication 2005 / 082423 (incorporated herein by reference) describes the use of serum albumin conjugated with a fluorescent substance for imaging.

[0093] Albumin can be conjugated to two or more molecules (e.g., several) selected from bioactive agents, therapeutic agents, radiopharmaceuticals, imaging agents, diagnostic agents, therapeutic compounds, and contrast agents.

[0094] Tumors (and muscle degeneration) exhibit enhanced albumin uptake (EPR (Enhanced Permeation and Retention)). Albumin conjugates may be used for imaging enhancement and to assess whether tumors (or other tissues and organs) are suitable for albumin-conjugated drugs.

[0095] bioactive compounds Bioactive compounds may be therapeutic or diagnostic compounds. Therapeutic compounds may be chemotherapeutic agents for use in cancer chemotherapy. They may be cell proliferation inhibitors or cytotoxic agents, and may be tumor inhibitors.

[0096] Bioactive compounds may already contain free thiol groups, for example, polypeptides containing cysteine ​​residues along with free thiol groups. Alternatively, bioactive compounds may be modified to contain free thiol groups. Thus, the amino acid sequence of a polypeptide may be modified to contain cysteine ​​residues along with free thiol groups, or the bioactive compound may be chemically derivatized to contain free thiol groups.

[0097] Bioactive compounds can be polypeptides (proteins), particularly recombinant protein pharmaceuticals. They may be chemotherapeutic or radiotherapy agents used to treat cancer and other related diseases.

[0098] Albumin can be conjugated to at least one (e.g., several) bioactive compounds by methods known in the art, via free thiol groups, or, if the albumin mutant protein of the present invention contains more than one free thiol, via multiple free thiol groups. Bioactive compounds include peptides, polypeptides, or proteins (natural, recombinant, or synthetic) (Debinski, (2002) Cancer Investigation 20, 801-809, O'Keefe and Draper et al., (1985) JBC 260, 932-937, Xia et al., (2000) J. Pharmacology Experimental Therapeutics 295, 594-600, Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384, Humphries, et al., (1994) J. Tissue Culture Methods 16, 239-242, Wenning et al., (1998) Biotech. Bioeng. 57, 484-496, Yazdi and Murphy, (1994) Cancer Research 54,6387-6394, Weaver and Laske (2003) J. Neuro-Oncology 65,3-13, Widera et al., (2003) Pharmaceutical Research 20,1231-1238, Daniels, TR et al. (2006) Clinical Immunology 121,159-176, and references contained herein), therapeutic and diagnostic drugs or compounds (Mishra et al., (2006) J. Drug Targeting 14,45-53, Lim and Shen, (2004) Pharmaceutical Research 21,1985-1992, Fritzer et al., (1996) Biochemical Pharmacology 51,489-493, Lubgan and Jozwiak (2002) Cell.Mol.Biol.Lett.7,98, Daniels, TRet al. (2006) op.ci.High molecular weight complexes including but not limited to liposomes, viruses, and nanoparticles (Mishra et al., (2006) J. Drug Targeting 14, 45-53, Daniels, TR et al. (2006) op. cit., and references included therein), nucleic acids and radionuclides including DNA, RNA (including siRNA), and their analogues (Lee et al., (2005) Arch. Pharm. Res. 28, 722-729, Huang et al., (2007) FASEB J. 21, 1117-1125, Daniels, TR et al. (2006) op. cit., and references included therein), and apparatus (Humphries, et al., (1994) J. Tissue Culture Methods This includes, but is not limited to, pp. 16,239–242 (and the references contained therein). In addition, entities can be modified in themselves by methods known in the art.

[0099] therapeutic compounds Examples of therapeutic compounds include 4-1BB ligand, 5-helix, human CC chemokine, human L105 chemokine, human L105 chemokine designated as huL105_3, monokine induced by gamma-interferon (MIG), partial CXCR4B protein, platelet basic protein (PBP), α1-antitrypsin, ACRP-30 homolog, complement component C1q C, and adenoid-expressed chemokines. Chemokines (ADEC), aFGF, FGF-1, AGF, AGF protein, albumin, etoposide, angiostatin, anthrax vaccine, antibodies specific to collapsin, antistacin, anti-TGFβ family antibodies, antithrombin III, APM-1, ACRP-30, famoxin, apolipoprotein species, arylsulfatase B, b57 protein, BCMA, β-thromboglobulin protein Protein, β-TG, bFGF, FGF2, blood coagulation factors, BMP processing enzyme furin, BMP-10, BMP-12, BMP-15, BMP-17, BMP-18, BMP-2B, BMP-4, BMP-5, BMP-6, BMP-9, bone morphogenetic protein-2, calcitonin, calpain-10a, calpain-10b, calpain-10c, cancer vaccine, carboxypeptidase, CC chemokine, MCP2, CCR5 variant, CCR7, CD11a Mab, CD137, 4-1BB receptor protein, CD20 Mab, CD27, CD27L, CD30, CD30 ligand, CD33 immunotoxin, CD40, CD40L, CD52Mab, Cerebus protein, chemokine eotaxin, chemokine hIL-8, chemokine hMCP1, chemokine hMCP1a, chemokine hMCP1b, chemokine hMCP2, chemokine hMCP3, chemokine hSDF1b, chemokine MCP-4, chemokine TECK and TECK variants, full-length and mature chemokine-like protein IL-8M1, full-length and mature chemokine-like protein IL-8M10, chemokine-like protein IL-8M3, full-length and mature chemokine-like protein IL-8 M8, full-length and mature chemokine-like protein IL-8M9, full-length and mature chemokine-like protein PF4-414, full-length and mature chemokine-like protein PF4-426, full-length and mature chemokine-like protein PF4-M2, cholera vaccine, chondromodulin-like protein, c-kit ligand, SCF, mast cell growth factor, MGF, fibrosarcoma-derived stem cell factor, CNTF and its fragments (CNTFAx15' (Axokine®) etc.), coagulation factors in both precursor and active forms, collagen, complement C5 Mab, connective tissue activating protein-III, CTAA16.88Mab, CTAP-III, CTLA4-Ig, CTLA-8, CXC3, CXC3, CXCR3, CXC chemokine receptor 3, cyanobilin-N, darbepoetin, designated exodus, designated huL105_7, DIL-40, DNase, EDAR, EGF receptor Mab, ENA-78, endostatin, eotaxin, epithelial neutrophil-activating protein-78, EPO receptor, EPOR, erythropoietin (EPO) and EPO mimetic, Eutropin, Exodus protein, factor IX, factor VII, factor VIII, factor X and factor XIII, FAS ligand inhibitor protein (DcR3), FasL, FasL, FasL, FGF, FGF-12, fibroblast growth factor homologue-1, FGF-15, FGF-16, FGF -18, FGF-3, INT-2, FGF-4, Geronin, HST-1, HBGF-4, FGF-5, FGF-6, Heparin-binding secretory transforming factor-2, FGF-8, FGF-9, Glial activator, Fibrinogen, FLT-1, FLT-3 ligand, Follicle-stimulating hormone α subunit, Follicle-stimulating hormone β subunit, Follitropin, Fractalkine, Myofibrillar protein troponin I, FSH, Galactosidase, Galectin-4, G-CSF, GDF-1, Gene therapy, Glioma-derived growth factor, Glucagon, Glucagon-like peptide, Glucocerebrosidase, Glucose oxidase, Glucosidase, Glycoderin-A, Progesterone-related endometrial protein, GM-CSF, Gonadotropin, Granulocyte chemotactic protein-2 (Granulocyte Chemotactic protein-2 (GCP-2), granulocyte-macrophage colony-stimulating factor, growth hormone, growth-related oncogene-alpha (GRO-α), growth-related oncogene-beta (GRO-β), growth-related oncogene-gamma (GRO-γ), hAPO-4, TROY, hCG, hepatitis B surface antigen, hepatitis B vaccine, HER2 receptor Mab, hirudin, HIV gp120, HIVgp41, HIV inhibitor peptide, HIV inhibitor peptide, HIV inhibitor peptide, HIV protease inhibitor peptide, HIV-1 protease inhibitor, HPV vaccine, human 6CKine protein, human Act-2 protein, human lipid synthesis inhibitor, human B cell stimulator-2 receptor, human β-chemokine H1305 (MCP-2), human CC chemokine DGWCC, human CC chemokine ELC protein, human CC-type chemokine interleukin C, human CCC3 protein, human CCF18 chemokine, human CC-type chemokine protein designated as SLC (secondary lymphoid chemokine), human β-8 short type, human chemokine C10, human chemokine CC-2, human chemokine CC-3, human chemokine CCR-2, human chemokine Ckβ-7, human chemokine ENA-78, human chemokine eotaxin, human chemokine GROα, human chemokine GROα, human chemokine GROβ, human chemokine HCC-1, human chemokine HCC-1, human chemokine I-309, human chemokine IP-10, human chemokine L105_3, human chemokine L105_7, human chemokine MIG, human chemokine MIG-β protein, human chemokine MIP-1α, human chemokine MIP1β, human chemokine MIP-3α, human chemokine MI P-3β, human chemokine PF4, human chemokine protein 331D5, human chemokine MIP - human interleukin-1 receptor accessory protein, human chemokine protein 61164, human chemokine receptor CXCR3, human chemokine SDF1α, human chemokine SDF1β, human chemokine ZSIG-35, human Chr19Kine protein, human CKbeta-9, human CKβ-9, human CX3C 111 amino acids and chemokines, human DNAX interleukin-40, human DVic-1 CC chemokine, human DIRF I protein sequence, human EDIRF II protein sequence, human eosinophil CC-type chemokine eotaxin, human eosinophil-expressed chemokineChemokines (EEC), human fast-twitch skeletal muscle troponin C, human fast-twitch skeletal muscle troponin I, human fast-twitch skeletal muscle troponin subunit C, human fast-twitch skeletal muscle troponin subunit I protein, human fast-twitch skeletal muscle troponin subunit T, human fast-twitch skeletal muscle troponin T, human fetal spleen-expressed chemokines, FSEC, human GM-CSF receptor, human gro-α chemokine, human gro-β chemokine, human gro-γ chemokine, human IL-16 protein, human IL-1RD10 protein sequence, human IL-1RD9, human IL-5 receptor α chain, human IL-6 receptor, human IL-8 receptor protein hIL8RA, human IL-8 receptor protein hIL8RB, human IL-9 receptor protein Quality, Human IL-9 receptor protein variant #3, Human IL-9 receptor protein variant fragment, Human IL-9 receptor protein variant fragment #3, Human interleukin-1 delta, Human interleukin-10, Human interleukin-10, Human interleukin-18, Human interleukin-18 derivative, Human interleukin-1β precursor, Human interleukin-1β precursor, Human interleukin-1 receptor accessory protein, Human interleukin-1 receptor antagonist β, Human interleukin-1 type-3 receptor, Human interleukin-10 (precursor), Human interleukin-10 (precursor), Human interleukin-11 receptor, Human interleukin-12 40kD subunit, Human interleukin-12β-1 receptor, Human interleukin-12β-2 receptor, Human interleukin-12 p35 protein, Human interleukin-12p40 protein, human interleukin-12 receptor, human interleukin-13α receptor, human interleukin-13β receptor, human interleukin-15, human interleukin-15 receptor from clone P1, human interleukin-17 receptor, human interleukin-18 protein (IL-18), human interleukin-3, human interleukin-3 receptor, human interleukin-3 variant, human interleukin-4 receptor, human interleukin-5, human interleukin-6, human interleukin-7, human interleukin-7, human interleukin-8 (IL-8), human intracellular IL-1 receptor antagonist, human IP-10 and HIV-1 gp120 hypervariable region fusion protein, human IP-10 and human Muc-1 core epitope (VNT) fusion protein, human liver and activation-regulated chemokines Chemokine (LARC), full-length and mature human Lkn-1 protein, full-length and mature human mammary-associated chemokine (MACK) protein, human mature chemokine Ckbeta-7, human mature gro-α, human mature gro-γ polypeptide used to treat sepsis, human MCP-3 and human Muc-1 core epitope (VNT) fusion protein, human MI10 protein, human MI1A protein, human monocyte chemotactic factor hMCP-1, human monocyte chemotactic factor hMCP-3, human monocyte chemotactic proprotein (MCPP) sequence, human neurotactin chemokine-like domain, human non-ELR CXC chemokine H174, human non-ELR CXC chemokine IP10, human non-ELR CXC chemokine Mig, human PAI-1 mutant, human protein with IL-16 activity, human protein with IL-16 activity, human secondary lymphoid chemokine (SLC), human SISD protein, human STCP-1, human stromal cell-derived chemokine, SDF-1, human T cell mixed lymphocyte reaction-expressing chemokineChemokine (TMEC), human thymus and activation-regulated cytokine (TARC), human thymus-expressed, human TNF-α, human TNF-β (LT-α), human CC-type chemokine eotaxin 3 protein sequence, human type II interleukin-1 receptor, human wild-type interleukin-4 (hIL-4) protein, human ZCHEMO-8 protein, humanized anti-VEGF antibody and its fragments, humanized anti-VEGF antibody and its fragments, hyaluronidase, ICE 10kD subunit, ICE 20kD subunit, ICE 22kD subunit, iduronate-2-sulfatase, iduronidase, IL-1α, IL-1β, IL-1 inhibitor (IL-1i), mature IL-1, IL-10 receptor, IL-11, IL-11, IL-12 p40 subunit, IL-13, IL-14, IL-15, IL-15 receptor, IL-17, IL-17 receptor, IL-17 receptor, IL-19, IL-1i fragment, IL-1 receptor antagonist, IL-21 (TIF), IL-3-containing fusion protein, IL-3 mutant protein, IL-3 variant Ants, IL-3 variants, IL-4, IL-4 mutant protein, IL-4 mutant protein Y124G, IL-4 mutant protein Y124X, IL-4 mutant protein, IL-5 receptor, IL-6, IL-6 receptor, IL-7 receptor clone, IL-8 receptor, IL-9 mature protein variant (Met117 version), immunoglobulin or immunoglobulin system molecules or fragments of either (e.g., Small Modular ImmunoPharmaceutical (trademark) ("SMIP"), or Ab, Fab' fragment, F(ab')2, scAb, scFv or scFv fragment), plasminogen (but not limited to these), influenza vaccine, inhibin α, inhibin β, insulin, insulin-like growth factor, integrin Mab, interalphatrypsin inhibitor, interferon-γ-inducible protein (Ip-10), interferon (interferon α species and subspecies, interferon β species and subspecies, interferon γ species and subspecies), interleukin 6, interleukin 8 (Interleukin 8, IL-8) receptor, interleukin-8 receptor B, interleukin-1α, interleukin-2 receptor-related protein p43, interleukin-3, interleukin-4 mutant protein, interleukin-8 (IL-8) protein, interleukin-9, interleukin-9 (IL-9) mature protein (Thr117 version), interleukins (IL10, IL11, and IL2, etc.), Japanese encephalitis vaccine, kallikrein inhibitors, keratinocyte growth factor, Kunitz crude main protein (aprotinin with and without albumin fusion, amyloid precursor protein, and those described in International Publication No. 03 / 066824), LACI, lactoferrin, latent TGF-β binding protein II, leptin, liver-expressed chemokine-1 (LVEC-1), liver-expressed chemokine-1 (LVEC-1), liver-expressed chemokine-1chemokine-2, LVEC-2), LT-α, LT-β, ​​luteinizing hormone, lime vaccine, lymphotactin, macrophage-derived chemokine analog MDC(n+1), macrophage-derived chemokine analog MDC-eyfy, macrophage-derived chemokine analog MDC-yl, macrophage-derived chemokine, MDC, macrophage-derived chemokine (Macrophage-derived chemokine, MDC), Masupin, protease inhibitor 5, MCP-1 receptor, MCP-1a, MCP-1b, MCP-3, MCP-4 receptor, M-CSF, melanoma inhibitor protein, membrane-bound protein, Met117 human interleukin 9, MIP-3α, MIP-3β, MIP-γ, MIRAP, modified lantes, monoclonal antibody, MP52, mutant interleukin 6 S176R, myofibrillar contraction protein troponin I, natriuretic peptide, neurotrophin β, neurotrophin β2, neuropilin-1, neuropilin-2, neurotactin, neurotrophin-3, neurotrophin-4, neurotrophin-4a, neurotrophin-4b, neurotrophin-4c, neurotrophin-4d, neutrophil activating peptide-2 Peptide-2, NAP-2), NOGO-66 receptor, NOGO-A, NOGO-B, NOGO-C, PTEC, and other novel β-chemokines, N-terminally modified chemokines GroHEK / hSDF-1α, GroHEK / hSDF-1β, met-hSDF-1α, met-hSDF-1β, OPGL, osteomorphic protein-1, OP-1, BMP-7, osteomorphic protein-2, OX40, ACT-4, OX40L, oxytocin (neurophysin I), parathyroid hormone, Patched, Patched-2, PDGF-D, pertussis toxoid, and pituitary-expressed chemokines.Chemokines (PGEC), placental growth factor, placental growth factor-2, plasminogen activator inhibitor-1, PAI-1, plasminogen activator inhibitor-2, PAI-2, plasminogen activator inhibitor-2, PAI-2, platelet-derived growth factor, platelet-derived growth factor Bv-sis, platelet-derived growth factor precursor A, platelet-derived growth factor precursor B, platelet Mab, platelet-derived endothelial cell growth factor Factor (PD-ECGF), platelet-derived growth factor A chain, platelet-derived growth factor B chain, polypeptides used to treat sepsis, preproapolipoprotein "Milano" variant, preproapolipoprotein "Paris" variant, prethrombin, primate CC chemokine "ILINCK", primate CXC chemokine "IBICK", proinsulin, prolactin, prolactin 2, prosaptide, protease inhibitor peptide, protein C, protein S, prothrombin, prourokinase, RANTES, RANTES 8-68, RANTES 9-68, RANTES peptide, RANTES receptor, recombinant interleukin-16, resistin, restricosin, retroviral protease inhibitor, lysine, rotavirus vaccine, RSV Mab, saporin, salcin, secreted and transmembrane polypeptides, secreted and transmembrane polypeptides, serum cholinesterase, serum proteins (blood coagulation factors, etc.), soluble BMP receptor kinase protein-3, soluble VEGF receptor, stem cell inhibitors, staphylococcal vaccine, stromal cell-derived factor-1α, stromal cell-derived factor-1β, substance P (tachykinin), T1249 peptide, T20 peptide, T4 endonuclease, TACI, Tarc, TGF-β1, TGF-β2, Thr117 human interleukin-9, thrombin, thrombopoietin, thrombopoietin derivative 1, thrombopoietin derivative 2, thrombopoietin derivative 3, thrombopoietin derivative 4, thrombopoietin derivative 5, thrombopoietin derivative 6, thrombopoietin derivative 7, thymus-expressed chemokinesChemokines (TECK), thyroid-stimulating hormone, tick anticoagulant peptide, Tim-1 protein, TNF-α precursor, TNF-R, TNF-RII, TNF p75 receptor, cell death receptor, tPA, transferrin, transforming growth factor β, troponin peptide, truncated monocyte chemotactic protein 2 (6-76), truncated RANTES protein (3-68), tumor necrosis factor, uric acid oxidase, urokinase, vasopressin (neurophysin II), VEGF Examples include R-3, flt-4, VEGF receptor, KDR, flk-1, VEGF-110, VEGF-121, VEGF-138, VEGF-145, VEGF-162, VEGF-165, VEGF-182, VEGF-189, VEGF-206, VEGF-D, VEGF-E, VEGF-X, von Willebrand factor, wild-type monocyte chemotactic protein 2, wild-type monocyte chemotactic protein 2, and ZTGF-beta 9.

[0100] Chemotherapy drugs Examples of chemotherapy drugs include 13-cis-retinoic acid, 2-CdA, 2-chlorodeoxyadenosine, 5-azacitidine, 5-fluorouracil, 5-FU, 6-mercaptopurine, 6-MP, 6-TG, 6-thioguanine, Abraxane, Accutane®, Actinomycin D, Adriamycin®, Adrucil®, Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, and Alker an(registered trademark), all trans retinoic acid, alpha interferon, altretamine, ametopterin, amifostin, aminoglutethimide, anagrelide, Anandron(registered trademark), anastrozole, arabinosylcytosine, Ara-C, Aranesp(registered trademark), Aredia(registered trademark), Arimidex(registered trademark), Aromasin(registered trademark), Arranon(registered trademark), arsenic trioxide, asparaginase, ATRA, Avastin(registered trademark), azacitidine, BCG, BCNU, bevacizumab, bexarotene, B EXXAR(registered trademark), bicalutamide, BiCNU, Blenoxane(registered trademark), bleomycin, bortezomib, busulfan, Busulfex(registered trademark), C225, calcium leucovorin, Campath(registered trademark), Camptosar(registered trademark), camptothecin-11, capecitabine, Carac(trademark), carboplatin, carmustine, carmustine wafer, Casodex(registered trademark), CC-5013, CCNU, CDDP, CeeNU, Cerubidine(registered trademark), cetuximab, chlorambucil, Cisplatin, citroboram factor, cladribine, cortisone, Cosmegen®, CPT-11, cyclophosphamide, Cytadren®, cytarabine, cytarabine liposome, Cytosar-U®, Cytoxan®, dacarbazine, dacogen, dactinomycin, darbepoetin alfa, dasatinib, daunomycin, daunorubicin, daunorubicin hydrochloride, daunorubicin liposome, DaunoXome®, Decadron, decitabine, Delta-Cortef®,Deltazone (registered trademark), Deniloquin difutitox, DepoCyt (trademark), Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexazone, Dexrazoxane, DHAD, DIC, Geodex, Docetaxel, Doxil (registered trademark), Doxorubicin, Doxorubicin liposome, Droxia (trademark), DTIC, DTIC-Dome (registered trademark), Duralone (registered trademark), Efude x (registered trademark), Eligard (trademark), Ellence (trademark), Eloxatin (trademark), Elspar (registered trademark), Emcyt (registered trademark), Epirubicin, Epoetin alfa, Erbitux (trademark), Erlotinib, Erwinia L-asparaginase, Estramustine, Ethiol, Etopophos (registered trademark), Etoposide, Etoposide phosphate, Eulexin (registered trademark), Evista (registered trademark) (Registered Trademark), Exemestane, Fareston (Registered Trademark), Faslodex (Registered Trademark), Femara (Registered Trademark), Filgrastim, Fluxuridine, Fludara (Registered Trademark), Fludarabine, Fluoroplex (Registered Trademark), Fluorouracil, Fluoxymesterone, Flutamide, Folic Acid, FUDR (Registered Trademark), Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab Ozogamicin, Gemzar (Registered Trademark), Gleevec (Trademark), Gliadel (Registered Trademark) Wafer, GM-CSF, Goserelin, Granulocyte Colony-Stimulating Factor, Granulocyte Macrophage Colony-Stimulating Factor, Halotestin (Registered Trademark), Herceptin (Registered Trademark), Hexadol, Hexalen (Registered Trademark), Hexamethylmelamine, HMM, Hycamtin (Registered Trademark), Hydrea (Registered Trademark), Hydrocort Acetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortisone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®, IFN-Alpha, Ifosfamide, IL-11, IL-2, Imatinib Mesylate, Imidazole Carboxamide, Interferon α,Interferon α-2 b (PEG conjugate), Interleukin-2, Interleukin-11, Intron A (registered trademark) (Interferon α-2 b), Iressa (registered trademark), Irinotecan, Isotretinoin, Kidrolase (registered trademark), Lanacort (registered trademark), Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine (trademark), Leuprolide, Leulocristine, Leustatin (trademark), Liposome Ara-C, Liquid Pred (registered trademark), Lomustine, L-PAM, L-Sarcolicin, Lupron (registered trademark), Lupron Depot (registered trademark), Matulane (registered trademark), Maxidex, Mechloretamine, Mechloretamine hydrochloride, Medralone (registered trademark), Medrol (registered trademark), Megace (registered trademark), Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex (trademark), Methotrexate, Methotrexate sodium, Methylprednisolone, Meticorten (registered trademark), Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol (registered trademark), MTC, MTX, Mustargen (registered trademark), Mustine, Mutamycin (registered trademark), Myleran (registered trademark), Mylocel (trademark), Mylotarg (registered trademark), Navelbine (registered trademark), Nelarabine, Neosar (registered trademark), Neulasta (trademark), Neumega (registered trademark), Neupogen (registered trademark), Nexavar (registered trademark), Nilandron (registered trademark), Niltamide, Nipent (registered trademark), Nitrogen Mustard, Novaldex (registered trademark), Novantrone (registered trademark), Octreotide, Octreotide Acetate, Oncospar (registered trademark), Oncovin (registered trademark), Ontak (registered trademark), Onxal (trademark), Oplevkin, Orapred (registered trademark), Orasone (registered trademark), Oxaliplatin, Paclitaxel, Protein-bound Paclitaxel, Pamidronate, Panitumumab, Panretin (registered trademark), Paraplatin (registered trademark), Pediapred (registered trademark),PEG interferon, pegaspar gauze, pegfilgrastim, PEG-INTRON®, PEG-L-asparaginase, PEMETREXED, pentostatin, phenylalanine mustard, Platinol®, Platinol-AQ®, prednisolone, prednisone, Prelone®, procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 including carmustine implant, Purinethol®, raloxifene, Revlimid®, Rheumatrex®, Rituxan®, rituximab, Roferon-A® (interferon α-2a), Rubex®, rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR (registered trademark), Sarglamostim, Solu-Cortef (registered trademark), Solul-Medrol (registered trademark), Sorafenib, SPRYCEL (trademark), STI-571, Streptozocin, SU11248, Sunitinib, Sutent (registered trademark), Tamoxifen, Tarceva (registered trademark), Targretin (registered trademark), Taxol (registered trademark), Taxotere (registered trademark), Temodar (registered trademark), Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid (registered trademark), TheraCys (registered trademark), Thioguanine Tabloid (registered trademark), thiophosphoamide, Thioplex (registered trademark), thiotepa, TICE (registered trademark), Toposar (registered trademark), topotecan, toremifene, tositumomab, trastuzumab, tretinoin, Trexall (trademark), Trisenox (registered trademark) TSPA, TYKERB (registered trademark), VCR, Vectibix (trademark), Velban (registered trademark), Velcade (registered trademark), VePesid (registered trademark), Vesanoid (registered trademark), Viadur (trademark), Vidaza (registered trademark), vinblastine, vinblastine sulfate, Vincasar Pfs (registered trademark), vincristine, vinorelbine, vinorelbine tartrate, VLB, VM-26, vorinostat, VP-16,Examples include Vumon (registered trademark), Xeloda (registered trademark), Zanosar (registered trademark), Zevalin (trademark), Zinecard (registered trademark), Zoladex (registered trademark), Zoledronic acid, Zolinza, and Zometa (registered trademark).

[0101] Radiopharmaceuticals Examples of radiopharmaceuticals include carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gold-198, indium-111, indium-113m, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, nitrogen-13, oxygen-15, phosphorus-32, rhenium-186, rubidium-82, samarium-153, selenium-75, strontium-89, technetium-99m, thallium-201, tritium, xenon-127, xenon-133, and yttrium-90.

[0102] Imaging agent Examples of imaging agents include gadolinium, magnetite, manganese, technetium, I125, I131, P32, TI201, iopamidol, and PET-FDG.

[0103] The conjugation partners described herein may be peptides, organic chemicals, or small molecule pharmaceuticals. The conjugation partners may be bioactive, therapeutic, prophylactic (including vaccines), diagnostic, imaging, or radiopharmaceutical portions, or portions having other beneficial properties.

[0104] Preferably, the biological activity of the conjugation partner is maintained at at least 50, 60, 70, 80, 90, or 95% of the biological activity of the conjugation partner in the unconjugated state in the case of conjugation to albumin according to the present invention. Preferably, the biological activity of the conjugation partner is not reduced by conjugation to albumin according to the present invention.

[0105] Preferred Embodiment (Part A) 1. A liquid formulation of albumin or a fragment thereof, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or a fragment thereof, after storage or incubation at 40°C for at least 1, 2, 3, 4, 5, or 6 months.

[0106] 2. A liquid formulation of albumin or a fragment thereof, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or a fragment thereof, after storage or incubation at 25°C for at least 1, 2, 3, 4, 5, 6, 9, or 12 months.

[0107] 3. A liquid formulation of albumin or its fragments, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or its fragments, after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7°C, for a duration of at least 1, 2, 3, 4, 5, or 6 months or at least 1, 2, 3, 4, 5, or 6 years, such as 3, 4, 5, 6, or 8°C, for example, about 4°C.

[0108] A liquid formulation according to any one of Embodiments 1 to 3, comprising at least 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, or 0.99 moles of free thiols per 4.1 moles of albumin or a fragment thereof.

[0109] The liquid formulation according to Embodiment 4, comprising at least 1 mole of free thiol per 5.1 moles of albumin or a fragment thereof.

[0110] 6. A liquid formulation according to any one of Embodiments 1 to 5, comprising a theoretical free thiol level of at least 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0111] The liquid formulation according to Embodiment 6, comprising a theoretical free thiol level of 7,100%.

[0112] 8. A liquid formulation according to any one of Embodiments 1 to 7, containing approximately 2.5 to 7.5 mM of fatty acids.

[0113] 9. A liquid formulation according to Embodiment 8, comprising approximately 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 mM fatty acids to approximately 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5 mM fatty acids.

[0114] 10. A liquid formulation according to Embodiment 9, containing approximately 4 to approximately 6 mM fatty acids.

[0115] 11. A liquid formulation according to Embodiment 10, containing approximately 5 mM fatty acids.

[0116] 12. A liquid formulation according to any one of embodiments 1 to 11, having a cation concentration of at least about 175 mM.

[0117] 13. A liquid formulation according to Embodiment 12, having a cation concentration of approximately 200, 225, 250, 275, or 300 mM to approximately 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mM.

[0118] 14. A liquid formulation according to Embodiment 13, having a cation concentration of approximately 200 to approximately 300 mM.

[0119] 15. The liquid formulation according to Embodiment 14, having a cation concentration of approximately 225 to approximately 275 mM.

[0120] 16. A liquid formulation according to any one of Embodiments 1 to 15, having a pH of approximately 5.5 to approximately 6.5.

[0121] 17. A liquid formulation according to Embodiment 16, having a pH of approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 to approximately 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0122] 18. A liquid formulation according to Embodiment 17, having a pH of approximately 5.7 to approximately 6.2.

[0123] 19. A liquid formulation according to Embodiment 18, having a pH of approximately 6.

[0124] 20. A liquid formulation comprising albumin, fatty acids, and cations, wherein the formulation has the following formula: (a) Free thiols = 1.1786 - 0.05167 * pH-0.0001544 * Cation-0.01133 * FA+(pH-6) * ((Cation-198.3) * 0.0002659)+(pH-6) * ((FA-7.125) * 0.003160)+(cation-198.3) * ((FA-7.125) * 0.000001935)+(pH-6) * ((pH-6) * (-0.0500) + (cation -198.3) * ((Cation-198.3) * 0.000002583)+(FA-7.125) * ((FA-7.125) * 0.0001413)+(pH-6) * ((Cation-198.3) * ((FA-7.125) * -0.000009879)), and (b) Polymer = 9.1677 - 0.8417 * pH -0.01044 * Cation -0.2690 * FA+(pH-6) * ((Cation-198.3) *-0.004796)+(pH-6) * ((FA-7.125) * 0.06523)+(cation-198.3) * ((FA-7.125) * 0.001494)+(pH-6) * ((pH-6) * 2.25) + (cation -198.3) * ((Cation-198.3) * 0.00006768)+(FA-7.125) * ((FA-7.125) * 0.02490)+(pH-6) * ((Cation-198.3) * ((FA-7.125) * It is located within the design space defined by both of 0.001237)), During the ceremony, "Free thiol" refers to the level of free thiols. "Cation" refers to cation concentration in mM units. "FA" refers to a liquid formulation where the fatty acid concentration is measured in mM.

[0125] 21. For example, the formulation according to Embodiment 20, comprising at least 0.75 moles of free thiols per mole of albumin or its fragments, and up to 1% (w / w) of albumin polymer, after storage or incubation at 40°C for at least 1, 2, 3, 4, 5, or 6 months.

[0126] 22. The formulation is given by the following formula: (a) Free thiols = 1.1786 - 0.05167 * pH-0.0001544 * Cation-0.01133 * FA+(pH-6) * ((Cation-198.3) * 0.0002659)+(pH-6) * ((FA-7.125) * 0.003160)+(cation-198.3) * ((FA-7.125) *(0.000001935)+(pH - 6) * ((pH - 6) * -0.0500)+(Cation - 198.3) * ((Cation - 198.3) * 0.000002583)+(FA - 7.125) * ((FA - 7.125) * 0.0001413)+(pH - 6) * ((Cation - 198.3) * ((FA - 7.125) * -0.000009879)), and (b) Polymer = 9.1677 - 0.8417 * pH - 0.01044 * Cation - 0.2690 * FA+(pH - 6) * ((Cation - 198.3) * -0.004796)+(pH - 6) * ((FA - 7.125) * 0.06523)+(Cation - 198.3) * ((FA - 7.125) * 0.001494)+(pH - 6) * ((pH - 6) * 2.25)+(Cation - 198.3) * ((Cation - 198.3) * 0.00006768)+(FA - 7.125) * ((FA - 7.125) * 0.02490)+(pH - 6) * ((Cation - 198.3) * ((FA - 7.125) * within the design space defined by both of 0.001237)), wherein, "Free Thiol" means the level of free thiol, "Cation" means the cation concentration in mM units, "FA" means the fatty acid concentration in mM units, the liquid preparation according to any one of Embodiments 1 to 21.

[0127] 23. The fatty acid is C6, C8, or C10 A liquid formulation according to any one of Embodiments 8 to 22, wherein the fatty acid is selected from C6 fatty acids or larger fatty acids, preferably C7 fatty acids or larger fatty acids, most preferably C8 fatty acids (octanoate).

[0128] 24. A liquid formulation according to any one of Embodiments 12 to 23, wherein the cation is selected from sodium, potassium, calcium, magnesium, and ammonium, and preferably sodium.

[0129] 25. A liquid formulation according to any one of Embodiments 1 to 24, comprising about 225 to about 275 mM of cations, preferably sodium, and 4 to 6 mM of fatty acids, preferably octanoate, and having a pH of about 5.7 to about 6.2.

[0130] 26. The liquid formulation according to Embodiment 25, wherein the formulation comprises about 250 mM of cation, preferably sodium, about 5 mM of fatty acid, preferably octanoate, and about pH 6.0.

[0131] 27. A liquid formulation according to any one of embodiments 1 to 26, wherein albumin or a fragment thereof is fused to a partner.

[0132] 28. A liquid formulation according to any one of Embodiments 1 to 27, wherein albumin, a fragment or fusion thereof contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 free thiols.

[0133] 29. A liquid formulation according to any one of Embodiments 1 to 28, wherein one or more free thiols are provided by one or more cysteine ​​residues at one or more (e.g., several) positions in albumin or a fragment or fusion thereof.

[0134] 30. Free thiols are found in SEQ ID NO: 2, 34, 1, 2, 4, 38, 40, 48, 52, 55, 58, 60, 75, 76, 79, 80, 82, 83, 83, 86, 91, 104, 113, 115, 116, 121, 122, 124, 125, 129, 168, 169, 177, 229, 236, 266, 269, 270, 273, 283, 298, 300, 301, 303, 304, 308, 313, 314, 316, 318, 320, 321, 324, 325, 355, 360, 361, 364, 365, 368, 369, 371, 375 A liquid formulation according to Embodiment 29, provided by cysteine ​​at a position corresponding to one or more (e.g., several) of the positions selected from 379, 386, 390, 396, 397, 435, 439, 443, 471, 478, 479, 490, 496, 498, 501, 503, 504, 505, 506, 508, 512, 538, 541, 542, 546, 549, 550, 558, 560, 562, 564, 565, 566, 567, 573, 574, 577, 578, 580, 581, 582, 584, and 585.

[0135] 31. The liquid formulation according to Embodiment 30, wherein the free thiol is provided by cysteine ​​at the position corresponding to position 34 of SEQ ID NO: 2.

[0136] 32. A liquid formulation according to any one of Embodiments 1 to 31, wherein the albumin or fragment thereof is preferably selected from mammalian albumin or fragment thereof from the group consisting of HSA (SEQ ID NO: 2), mouse (SEQ ID NO: 3), rat (SEQ ID NO: 4), macaque (SEQ ID NO: 5), cattle (SEQ ID NO: 6), pig (SEQ ID NO: 7), horse (SEQ ID NO: 8), or rabbit (SEQ ID NO: 9).

[0137] 33. A liquid formulation according to any one of Embodiments 1 to 32, wherein albumin or a fragment or fusion thereof has at least 70% sequence identity with SEQ ID NO: 2.

[0138] 34. The liquid formulation according to Embodiment 33, wherein albumin or a fragment or fusion thereof has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 2.

[0139] 35. The liquid formulation according to Embodiment 33 or 34, wherein albumin or a fragment or fusion thereof has 1, 2, 3, 4, 5, 6, 7, 8, or 9 to 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences compared to SEQ ID NO: 2.

[0140] 36. The liquid formulation according to Embodiment 35, wherein albumin or a fragment or fusion thereof comprises SEQ ID NO: 2.

[0141] 37. A liquid formulation according to any one of embodiments 1 to 36, wherein the albumin fragment contains at least 175 amino acids.

[0142] 38. A liquid formulation according to any one of Embodiments 1 to 37, wherein the albumin fragment contains at least 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 500, or 575 amino acids.

[0143] 39. A liquid formulation according to any one of Embodiments 1 to 38, wherein the albumin fragment comprises at least one (e.g., several) of domain I, domain II, or domain III of HSA.

[0144] 40. A liquid formulation according to any one of Embodiments 1 to 39, wherein albumin or a fragment or fusion thereof is recombinant.

[0145] 41. A liquid formulation according to any one of Embodiments 1 to 40, wherein albumin or a fragment or fusion thereof is produced in a eukaryotic host or a prokaryotic host.

[0146] 42. The liquid formulation according to Embodiment 41, wherein the eukaryotic host is selected from fungi, plants, and mammals.

[0147] 43. The liquid formulation according to embodiment 42, wherein the fungus is selected from Aspergillus, Kluyveromyces, Pichia (e.g., Pichia pastoris), and Saccharomyces (e.g., Saccharomyces cerevisiae).

[0148] 44. The liquid formulation according to embodiment 43, wherein Saccharomyces is Saccharomyces cerevisiae.

[0149] 45. The liquid formulation according to embodiment 42, wherein the plant is selected from potato, tobacco, and rice.

[0150] 46. The liquid formulation according to embodiment 45, wherein the rice is Oryza sativa.

[0151] 47. The liquid formulation according to embodiment 41, wherein the mammal is mammalian cells.

[0152] 48. The liquid formulation according to embodiment 47, wherein the mammalian cells are CHO or HEK.

[0153] 49. The liquid formulation according to any one of embodiments 1 to 48, wherein the albumin is derived from human or animal serum.

[0154] 50. The liquid formulation according to any one of embodiments 1 to 49, wherein albumin or a fragment or fusion thereof is present at 5 - 30% (w / v), preferably 10 - 20% (w / v), most preferably about 20%.

[0155] 51. The liquid formulation according to any one of embodiments 1 to 50, wherein the protein content of the formulation is at least 50% albumin or a fragment or fusion thereof, more preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% albumin or a fragment or fusion thereof.

[0156] 52. A liquid formulation according to any of Embodiments 1 to 51, which is substantially or completely free of albumin, or proteins other than fragments or fusions thereof.

[0157] 53. A liquid formulation according to any one of Embodiments 1 to 52, wherein the formulation is aqueous.

[0158] 54. A liquid formulation according to any one of Embodiments 1 to 53, presented in a container.

[0159] 55. A method for preparing a formulation of albumin or its fragments or fusions having a high free thiol level and a low polymer level, comprising: formulation of albumin or its fragments or fusions in a buffer containing 2.5 to 7.5 mM fatty acids, at least 175 mM cations, and a pH of 5.5 to 6.5; and optionally, filling the albumin or its fragments or fusions into a container.

[0160] 56. Use of a buffer containing 2.5–7.5 mM fatty acids, at least 175 mM cations, and a pH of 5.5–6.5 to maintain high free thiol levels and low polymer levels in albumin preparations.

[0161] 57. Fatty acids are C6, C8, or C 10 The method or use described in Embodiment 55 or 56, wherein a fatty acid is selected from C6 fatty acids or larger, preferably C7 fatty acids or larger, most preferably C8 fatty acids (octanoate).

[0162] 58. The method or use according to any one of embodiments 55 to 57, wherein the cation is selected from sodium, potassium, calcium, magnesium, and ammonium, and preferably sodium.

[0163] 59. A method for producing a conjugate, comprising: optionally conjugating albumin or a fragment or fusion thereof having the formulation described in any of Embodiments 1 to 54 with a conjugation partner via a linker; optionally filling the conjugate into a container; and optionally providing the conjugate in unit dosage form.

[0164] 60. A method for conjugating albumin or a fragment or fusion thereof to a partner with at least 75% efficiency, comprising contacting a formulation of albumin or a fragment or fusion thereof according to any one of Embodiments 1 to 54 with a partner and optionally with a linker.

[0165] 61. Use of an albumin preparation according to any of Embodiments 1 to 54 for highly efficient conjugation to a conjugation partner.

[0166] 62. The use according to Embodiment 61, wherein the high-efficiency conjugation has an efficiency of at least 75%, more preferably at least 80, 85, 90, or 95%.

[0167] 63. The method or use according to any of Embodiments 55 to 62, wherein the conjugation partner is a peptide, an organic chemical, or a small molecule pharmaceutical.

[0168] 64. Use of albumin preparations according to any one of Embodiments 1 to 54 for increasing the half-life of molecules such as bioactive agents, imaging agents, diagnostic agents, contrast agents, or therapeutic compounds.

[0169] 65. Use according to Embodiment 63, wherein the half-life is increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% compared to the molecule not conjugated with albumin.

[0170] 66. A conjugate for the treatment of a disease, the treatment of an illness, and / or for diagnosis, comprising albumin according to any one of embodiments 1 to 54 or albumin produced by the method according to any one of embodiments 55 to 64.

[0171] 67. A conjugate for the manufacture of a medicament for the treatment of a disease, the treatment of an illness, and / or for diagnosis, comprising albumin according to any one of embodiments 1 to 53 or albumin produced by the method according to any one of embodiments 55 to 63.

[0172] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

Examples

[0173] Example (Part A)

[0174] Example 1: Free Thiol Assay The level of free thiol in the albumin sample was determined by the DTNB assay or by the DTDP assay.

[0175] DTNB Assay The Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoate) (DTNB), was used to detect free thiol groups such as cys-SH (Cys 34 in the case of HSA, SEQ ID NO: 2). The reaction between DTNB and the sulfhydryl group releases 5-thio-2-nitrobenzoate ion TNB 2- which has a maximum absorption at 412 nm. The free thiol content of albumin was calculated by measuring the increase in absorbance at 412 nm.

[0176] Buffer 1 (0.1 M Tris-HCl, 0.01 M EDTA, pH 8.0): Prepared to a final volume of 500 mL using laboratory-grade water, containing 4.44 g Tris hydrochloride (tris(hydroxymethyl)aminomethane hydrochloride, (Sigma)), 2.64 g Tris base (tris(hydroxymethyl)aminomethane, (Sigma)), and 1.86 g EDTA (ethylenediaminetetraacetic acid, disodium salt, (Sigma)).

[0177] Buffer solution 2 (0.05 M sodium phosphate pH 7.0): Prepared with laboratory-grade water to a final volume of 500 mL, containing 2.73 g of Na2HPO4·2H2O (disodium hydrogen orthophosphate dihydrate, Fisher Scientific) and 1.52 g of NaH2PO4·2H2O (sodium dihydrogen orthophosphate dihydrate, Fisher Scientific).

[0178] DTNB reagent (0.01 M DTNB in ​​phosphate buffer): 40 mg of DTNB (5,5'-dithiobis(2-nitrobenzoic acid), (Sigma)) in 10 mL of buffer 2.

[0179] Glutathione standard (6.5 mM glutathione stock solution): 2.0 g of glutathione (L-glutathione, reduced form, Sigma) prepared in 1000 mL of laboratory-grade water.

[0180] 0.65 mM glutathione test control: 1 mL of 6.5 mM glutathione standard, 9 mL of laboratory-grade water.

[0181] procedure 1. Dilute the albumin sample with 50 mg / mL of laboratory-grade water in a 1.5 ml microcentrifuge tube. -1 The "albumin test sample" was prepared by diluting it.

[0182] 2.1 mL reduced-volume plastic cuvettes were used in three different ways for each of the following solutions: blank, each albumin test sample, and glutathione test control.

[0183] 3.1.0 mL of buffer 1 was pipetted into the "blank" cuvette.

[0184] 4.80 μL (microliters) of diluted albumin test sample was pipetted into the test sample cuvette.

[0185] 5.80 μL (microliters) of 0.65 mM glutathione control was pipetted into a glutathione control cuvette.

[0186] 6.0.92 mL of buffer solution 1 was added to each test sample and glutathione control cuvette and gently mixed.

[0187] 7. The spectrophotometer was set to zero "relative to air," meaning that no cuvettes were in their designated positions.

[0188] 8. The initial absorbances of the blank, test samples, and glutathione control were measured at a wavelength of 412 nm (A1).

[0189] 9. Step 8 was repeated for all subsequent samples.

[0190] 10. After completing the initial absorbance readings for all cuvettes, 50 μL (microliters) of DTNB reagent was added to all cuvettes (i.e., blank, test sample, and glutathione control) and gently mixed. The samples were incubated at room temperature (approximately 15–25°C) for 10 minutes.

[0191] 11. Next, the final absorbance (A2) of each cuvette was measured.

[0192] 12. The free thiol levels of albumin test samples and glutathione test controls were calculated using the following formula.

[0193] Calculation of the molar ratio of free thiols to albumin (SH / albumin) 1. A for blanks and samples 412 The increase (A3) was calculated as follows: A3 = A2 - A1 In the formula, A1 is the initial absorbance, i.e., before DTNB. A2 represents the final absorbance, i.e., after DTNB.

[0194] 2. The true absorbance change for each sample was calculated by subtracting the average value A3 of three blank values ​​from each sample. ΔA 412 =A3 sample - average A3 blank

[0195] 3. The measured free thiols (SH, nmol) were recorded for each sample and test control replica.

[0196]

number

[0197] 4. The amount of albumin (nmol) assayed was calculated:

[0198]

number

[0199] 5. The amount of glutathione (nmol) in the test control solution was calculated:

[0200]

number

[0201] 6. The molar ratio of free thiols to albumin (SH / albumin) in each sample replication was calculated:

[0202]

number

[0203] 7. The molar ratio of free thiols to glutathione (SH / albumin) in each controlled replication was calculated:

[0204]

number

[0205] 8. The average value for each of the 3 molar ratios was calculated for each test sample and test control.

[0206] The average molar ratio for the test controls within the range of 9.0.95 to 1.04 indicated that the assay was effective.

[0207] DTDP assay Free thiol groups in albumin samples were also detected using 4,4'-dithiodipyridine (DTDP). The reaction between DTDP and sulfhydryl groups releases 4-thiopyridone ions (4-TP), which have a maximum absorption at 324 nm. The free thiol content of albumin was calculated by measuring the increase in absorbance at 324 nm.

[0208] Buffer solution (0.1 M sodium phosphate, 1 mM EDTA, pH 7.4): 1000 mL of laboratory-grade water containing 14.56 g disodium orthophosphate dihydrate (Fisher), 3.04 g disodium orthophosphate dihydrate (Fisher), and 0.37 g disodium EDTA (ethylenediaminetetraacetic acid) dihydrate (Sigma).

[0209] DTDP reagent (4 mM DTDP in dilute hydrochloric acid): 0.88 g of DTDP (Aldrithiol®-4, Sigma) was first dissolved in 50 mL of laboratory-grade water, then 1000 μL of concentrated hydrochloric acid was added, and finally, 1000 mL of laboratory-grade water was used to prepare a 1 mL volume. 1 mL aliquots were stored at -20°C for single use.

[0210] The glutathione standard (6.5 mM glutathione stock solution) was the same as that described for the DTNB assay (described above).

[0211] The 0.65 mM glutathione test control was the same as that described for the DTNB assay (above).

[0212] procedure The principle and general steps of the DTDP assay are the same as those of the DTNB assay; that is, a reagent that specifically reacts with free thiol groups is added to albumin, and the change in absorption is proportional to the amount of free thiols.

[0213] 1. Dilute the albumin sample with 50 mg / mL of laboratory-grade water in a 1.5 ml microcentrifuge tube. -1 The "albumin test sample" was prepared by diluting it.

[0214] A blank cuvette containing 2.1 mL of buffer and a control cuvette containing glutathione were prepared in the same manner as in the DTNB assay (described above).

[0215] 3. For the DTDP assay, 50 μL (microliters) of diluted albumin test sample (or 0.65 mM glutathione test control) was pipetteed into the test sample cuvette.

[0216] 4.0.95 mL of buffer solution was added to each test sample and glutathione control cuvette.

[0217] 5. The initial absorbances of the blank, test samples, and glutathione control were measured at 324 nm (A1).

[0218] 6. After completing the initial absorbance readings for all cuvettes, 50 μL (microliters) of DTDP reagent was added to all cuvettes (i.e., blank, test sample, and glutathione control). The samples were incubated at room temperature (approximately 15–25°C) for 10 minutes.

[0219] 7. Next, the final absorbance (A2) of each cuvette was measured.

[0220] 8. The free thiol levels of the albumin test sample and the glutathione test control were calculated using the following formula.

[0221] Calculation of the molar ratio of free thiols to albumin (SH / albumin) 1. A for blanks and samples 324 The increase (A3) was calculated as follows: (A3=A2-A1) In the formula, A1 is the initial absorbance, i.e., before DTDP. A2 represents the final absorbance, i.e., after DTDP.

[0222] 2. True absorbance change (ΔA 324 This was calculated by subtracting the average value A3 of three blank values ​​from each sample.

[0223] A 324 =A3 sample - average A3 blank

[0224] 3. The measured free thiols (nmol) were calculated for each sample and test control replica:

[0225]

number

[0226] 4. The amount of albumin (nmol) assayed was calculated:

[0227]

number

[0228] 5. The amount of glutathione (nmol) in the test control solution was calculated:

[0229]

number

[0230] 6. The molar ratio of free thiols to albumin (SH / albumin) in each sample replication was calculated:

[0231]

number

[0232] 7. The molar ratio of free thiols to glutathione (SH / albumin) in each controlled replication was calculated:

[0233]

number

[0234] 8. The average value for each of the 3 molar ratios was calculated for each test sample and test control.

[0235] The average molar ratio for the test controls within the range of 9.0.95 to 1.04 indicated that the assay was effective.

[0236] Example 2: Determination of polymer levels by gel permeation high-performance liquid chromatography (GPHPLC). GP.HPLC separates proteins according to their size. This method was used to separate monomers from polymers that may be present in the sample. Polymer levels were determined by measuring their peak area relative to the total peak area present in the sample.

[0237] GP.HPLC analysis was performed using a Shimadzu HPLC system (LC2010 system) including two LC 10AD pumps, an SPD-M10Avp UV / Vis detector set to 280 nm, and a CTO 10ACvp column oven set to 30°C.

[0238] TSK G3000 SW with 7.8mm inner diameter x 30cm length XL Analytical column, and TSK SW 6.0 mm diameter x 4 cm length. XL Separation was performed on a guard column.

[0239] 10x Starter GP.HPLC Buffer (0.25 M sodium phosphate, 1.0 M sodium sulfate, 0.5% (w / v) sodium azide, pH 6.5): Prepared in 5 L of laboratory-grade water and filtered through a 0.22 μm filter before use, containing 272.5 g (±1.36 g) disodium hydrogen orthophosphate dodecahydrate, 75.0 g (±0.375 g) sodium dihydrogen orthophosphate dihydrate, 710.0 g (±3.55 g) anhydrous sodium sulfate, and 125 mL of 20% (w / v) sodium azide. The pH of the solution was measured and acceptable if between 6.3 and 6.8.

[0240] Working GP.HPLC buffer (25 mM sodium phosphate, 0.1 M sodium sulfate, 0.05% sodium azide, pH 7.0): 10-fold starting GP.HPLC buffer, diluted 10-fold with laboratory-grade water and filtered through a 0.22 μm membrane. pH was measured and adjusted to 6.8–7.2 using 1 M NaOH or 1 M HCl if necessary.

[0241] 20% (w / v) sodium azide: 20g in 100mL of laboratory-grade water.

[0242] Auto-injector buffer: 1 mL of 30% BRIJ 35 solution (i.e., BRIJ 35 (polyoxyethylene (23) lauryl ether; C)) in 1 L of laboratory-grade water. 12 E 23 ), 1 mL of 20% sodium azide.

[0243] Albumin reference standard (10 mg / mL) -1 Recombinant albumin (SEQ ID NO: 2, in 0.9% NaCl solution).

[0244] procedure Shimadzu HPLC instrument, 1 mL / min. -1 The system was started at a flow rate of 10 mg / mL and equilibrated for 30 minutes. The system was calibrated by injecting 25 μL of recombinant albumin reference standard three times. -1 Less than (typically about 5 mg / mL) -1 As described above, the sample was diluted with laboratory-grade water, and 25 μL was injected into an HPLC instrument. The total protein concentration of the sample was quantified by peak height, and the percentages of monomers and polymers were calculated using peak area.

[0245] Example 3: Preparation of albumin preparations for Examples 4 and 5: As shown in Table 1, recombinant human albumin (100 g / L, 25 mM phosphate, 250 mM NaCl, pH 6.5, SEQ ID NO: 2) was reformulated into formulations at 200 mg / ml each at three different pH levels using four different combinations of NaCl and octanoate.

[0246] [Table 1]

[0247] In summary, 28 50 mL vials of 10% (w / v) raw material albumin sample were pooled, yielding approximately 1.4 L of material. This was concentrated by ultrafiltration using a Centramate Omega 10000 molecular weight cutoff membrane (Pall) until 750 mL of permeate and 680 mL of retention solution were produced. The resulting material was then dialysically ultrafiltered against 7 times its original volume of 50 mM NaCl buffer. The dialysically ultrafiltered retention solution, which reached a volume of approximately 750 mL, was further concentrated until a final volume of 500–550 mL was achieved. The apparatus was then rinsed with a final 100 mL of 50 mM NaCl to maximize albumin recovery. The final volume of albumin solution was approximately 650 mL.

[0248] The expected concentration of the albumin solution is 250 mg / mL. -1 The above is the result, which is 245 mg / mL measured by GP.HPLC. -1 It was quantified as follows.

[0249] Next, the albumin material was divided into three 200 mL aliquots for pH adjustment to 6.0, 6.5, and 7.0. The pH of the starting material was measured to be 6.45, and pH adjustment was performed by adding approximately 1–2 mL of 1 M NaOH or 1 M HCl. The resulting albumin samples were sterile filtered and stored at 5°C.

[0250] After confirming the precise pH, samples of each formulation were analyzed for their pre-formulation concentrations. Specifically, sodium was analyzed by flame emission spectroscopy and specific gravity, and octanoate was analyzed by gas chromatography. For all samples, the following concentrations were recorded: sodium at 63.2 mM, 64.6 mM, and 74.1 mM, and octanoate at 0.5555 mM, 0.5630 mM, and 0.5660 mM.

[0251] The required amounts of NaCl, octanoate, and water to be returned to the sample and added to produce each of the 12 target formulations (Table 1) were calculated. Each of the three samples was divided into four aliquots of approximately 45 mL each. Stock solutions of 5 M NaCl, 2 M octanoate, and laboratory-grade water were added to the aliquots in a laminar flow hood until the precise amounts required to achieve the target values ​​were obtained.

[0252] The protein content of the sample was determined by GP-HPLC to approximately 200-220 mg / mL. -1 The concentrations were quantified and finally readjusted using the same approach as above with 5M NaCl, 2M octanoate, and laboratory-grade water. All final concentrations were approximately 190 mg / mL. -1 These were proteins, and these were the final formulations used in stability testing.

[0253] The final material was calcined in a laminar flow cabinet, filled into 2 mL Type II glass vials (Adelphi), sealed, and transferred to controlled incubators at 25°C and 40°C.

[0254] The T0 vial was retained for analysis according to Examples 3, 4, and 5.

[0255] Example 4: Stability of albumin samples at 25°C and pH 6.0-7.0 The samples prepared according to Example 3 (Table 1) were analyzed for stability at 25°C at T0, T1m (1 month), T2m (2 months), and T3m (3 months). The changes in free thiol levels using the DTNB assay described in Example 1 and the polymer levels using Example 2 were compared throughout this time course. Stability testing included a colorimetric free thiol assay and a GP.HPLC assay for polymer levels (using the assay described in Example 2 above). The data are shown in Tables 2 and 3.

[0256] [Table 2]

[0257] [Table 3]

[0258] The data show that free thiol levels increase after incubation at 25°C by reducing the amount of octanoate present in the formulation. Albumin polymerization was not observed at 25°C during incubation periods of 0–3 months.

[0259] Example 5: Stability of albumin samples at 40°C and pH 6.0-7.0 The samples prepared according to Example 3 (Table 1) were analyzed for stability at T0, T1m, T2m, and T3m at 40°C, and the changes in free thiol levels and polymer levels were compared throughout this time course. The data are shown in Tables 4 and 5. The free thiol and polymer assays were the same as those used in Example 4.

[0260] [Table 4]

[0261] [Table 5]

[0262] The data shows that after incubation at 40°C, reducing the amount of octanoate present in the formulation increases free thiol levels. The data also shows that the addition of octanoate minimizes albumin polymerization. Finally, the data shows that reducing pH increases free thiol levels and reduces albumin polymerization.

[0263] Example 6: Preparation of albumin preparations for Examples 7 and 8 Samples prepared in the same manner as in Example 3 were analyzed in further studies for stability at 40°C at T0, T1m (1 month), T2m (2 months), and T3m (3 months). The changes in free thiol levels using the DTDP assay described in Example 1 and polymer levels using the assay in Example 2 were compared throughout this time course. The data are shown in Tables 6 and 7.

[0264] [Table 6]

[0265] [Table 7] NR: No results: Precipitation of the sample meant that the polymer content could not be accurately determined.

[0266] Example 7: Prediction of the optimal formulation Using the T1m and T3m data from Tables 6 and 7, a Design of Experiments (DoE) approach was employed to predict the optimal albumin formulation for maximizing free thiol stability while minimizing polymer formation.

[0267] The software used was JMP version 11.1.1 (32-bit) from SAS Institute Inc. The data design involved a full-factor DoE with pH set to 5.5, 6.0, and 6.5, sodium ("Na") set to 145, 200, and 250 mM, and octane ("Oct") set to 0.5, 4, 8, and 16 mM. The resulting data was then added, and statistical analysis was performed using a quadratic model employing the following script. Fit Model Effects pH, :Na, :Oct, pH * :Na, pH * :Oct, :Na * :Oct, pH * pH, :Na * :Na, :Oct * :Oct, pH * :Na * :Oct ), Y(:Thiol data), Y(Polymer data), PERSONALITY(Standard Least Squares) ) The thiol and polymer data pertain to the appropriate temperature and time of testing.

[0268] No weighting or data transformation was used. To select the optimal formulation, equal weighting was applied to maximize free thiols and minimize polymers. This script was used at both time points (T1m and T3m).

[0269] The fit of data for both thiol and polymer at both time points was very good, with significant model fit (predicted P<0.0001) for both. The results showed similar trends, with pH and octanoate primarily influencing thiol levels, and all parameters influencing polymer levels. Both time points also yielded similar optimal formulations with pH 5.9–6.0, 250 mM sodium, and 4–6 mM octanoate. Therefore, the preferred albumin formulation is approximately 20% albumin, approximately pH 6.0, approximately 250 mM cation (e.g., sodium), and approximately 5 mM fatty acid (e.g., octanoate).

[0270] Since the results at both time points (T1m, T3m) showed similar results and trends, the T1 month data at 40°C can be used to rapidly predict the stability of the proposed formulation. As a result, the model equation was derived from the T1 month data as follows.

[0271] Thiol level = 1.1786 - 0.05167 * :pH-0.0001544 * :Na-0.01133 * :Oct+(:pH-6) * ((:Na-198.3) * 0.0002659)+(:pH-6) * ((:Oct-7.125) * 0.003160)+(:Na-198.3) * ((:Oct-7.125) * 0.000001935)+(:pH-6) * ((:pH-6) * -0.0500)+(:Na-198.3) * ((:Na-198.3) * 0.000002583)+(:Oct-7.125) * ((:Oct-7.125) * 0.0001413)+(:pH-6) * ((:Na-198.3) * ((:Oct-7.125) * -0.000009879)) Polymer level = 9.1677 - 0.8417 * pH -0.01044 * :Na-0.2690 * :Oct+(:pH-6) * ((:Na-198.3) * -0.004796)+(:pH-6) * ((:Oct-7.125) * 0.06523)+(:Na-198.3) * ((:Oct-7.125) * 0.001494)+(:pH-6) * ((:pH-6) * 2.25)+(:Na-198.3) * ((:Na-198.3) * 0.00006768)+(:Oct-7.125) * ((:Oct-7.125) * 0.02490)+(:pH-6) * ((:Na-198.3) * ((:Oct-7.125) * 0.001237))

[0272] Based on these formulas, formulations that yield acceptable thiol and polymer levels after one month of storage or incubation at 40°C were defined. For example, by setting limits for thiol levels (e.g., at least 0.75 mol / mol) and polymers (e.g., less than 1%), the required limits for pH, cations (e.g., sodium), and fatty acids (e.g., octanoates) were identified. Since these limits cannot be easily represented in a single graph, they are shown in Figures 1, 2, and 3 as two-dimensional graphs with a third parameter set to the levels of preferred formulations, namely pH 6.0 (Figure 1), 250 mM sodium (Figure 2), and 5 mM octanoates (Figure 3).

[0273] In each case (see Graph A in Figures 1, 2, and 3), the filled shaded areas mark locations where the thiol level is outside the proposed limit, the diagonally shaded areas mark locations where the polymer is outside the proposed limit, and the unshaded white areas mark locations where both the polymer and thiol are within the proposed limit. The intersection of the black lines represents a preferred formulation, i.e., about 20% albumin, about pH 6.0, about 250 mM cation, e.g., sodium, and about 5 mM fatty acid, e.g., octanoate.

[0274] Statistical analysis showed that this is a very good mathematical model representing the relationship between (a) pH, cations, and fatty acids and (b) their effects on thiol levels and polymer levels. Based on this analysis and the stability tests of Examples 4, 5, and 6, the preferred formulation is approximately 20% albumin, approximately pH 6.0, approximately 250 mM sodium, and approximately 5 mM octanoate.

[0275] List of sequences described herein >Sequence 1 >Sequence 2 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >Sequence number 3 MKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFK ENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCD KPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGR VGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA >Sequence 4 MKWVTFLLLLFISGSAFSRGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQYLQKCPYEEHIKLVQEVTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNECFLQHKDDNPNLPPFQRPEAEAMCTSFQ ENPTSFLGHYLHEVARRHPYFYAPELLYYAEKYNEVLTQCCTESDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGERAFKAWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQATISSKLQACCD KPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKNLVKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGR VGTKCCTLPEAQRLPCVEDYLSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPDKEKQIKKQTALAELVKHKPKATEDQLKTVMGDFAQFVDKCCKAADKDNCFATEGPNLVARSKEALA >Sequence 5 MKWVTFISLLFFSSAYSRGVFRRDTHKSEVAHRFKDLGEEHFKGLVVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFH DNEATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYMCENQDSISSSKLKECCD KPLLEKSHCLAEVENDEMPADLPSLAADYVESKDVCNKNYAEAKDVFLGMFLYEYARRHPDYSVMLLLLRAKAYEATLEKCCAAADPHECYAKVFDEFQPLVEEPQNLVKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTTPTLVEVSRNLGK VGAKCCKLPEAKRMPCAEDYLSVVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALELDEAYVPKAFNAETFTFHADMCTLSEKEKQVKKQTALVELVKHKPKATKEQLKGVMDNFAAFVEKCCKADDKEACFAEEGPKFVAASQAALA > sequence no.6 MKWVTFISLLLLFSSAYSRGVFRRDTHKSEAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFK ADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCD KPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGK VGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA >Sequence ID 7 MKWVTFISLLFLFSSAYSRGVFRRDTYKSEEIAHRFKDLGEQYFKGLVLIAFSQHLQQCPYEEHVKLVREVTEFAKTCVADESAENCDKSIHTLFGDKLCAIPSLREHYGDLADCCEKEEPERNECFLQHKNDNPDIPKLKPDPVALCADFQ EDEQKFWGKYLYEIARRHPYFYAPELLYYAIIYKDVFSECCQAADKAACLLPKIEHLREKVLTSAAKQRLKCASIQKFGERAFKAWSLARLSQRFPKADFTEISKIVTDLAKVHKECCHGDLLECADDRADLAKYICENQDTISTKLKECCD KPLLEKSHCIAEAKRDELPADLNPLEHDFVEDKEVCKNYKEAKHVFLGTFLYEYSRRHPDYSVSLLLRIAKIYEATLEDCCAKEDPPACYATVFDKFQPLVDEPKNLIKQNCELFEKLGEYGFQNALIVRYTKKVPQVSTPTLVEVARKLGL VGSRCCKRPEEERLSCAEDYLSLVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYKPKEFVEGTFTFHADLCTLPEDEKQIKKQTALVELLKHKPHATEEQLRTVLGNFAAFVQKCCAAPDHEACFAVEGPKFVIEIRGILA >Sequence 8 MKWVTFVSLLFLFSSAYSRGVLRRDTHKSEIAHRFNDLGEKHFKGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKKCAADESAENCDKSLHTLFGDKLCTVATLRATYGELADCCEKQEPERNECFLTHKDDHPNLPKLKPEPDAQCAAFQ EDPDKFLGKYLYEVARRHPYFYGPELLFHAEEYKADFTECCPADDKLACLIPKLDALKERILLSSAKERLKCSSFQNFGERAVKAWSVARLSQKFPKADFAEVSKIVTDLTKVHKECCHGDLLECADDRADLAKYICEHQDSISGKLKACCD KPLLQKSHCIAEVKEDDLPSDLPALAADFAEDKEICKHYKDAKDVFLGTFLYEYSRRHPDYSVSLLLRIAKTYEATLEKCCAEADPPACYRTVFDQFTPLVEEPKSLVKKNCDLFEEVGEYDFQNALIVRYTKKAPQVSTPTLVEIGRTLGK VGSRCCKLPESERLPCSENHLALALNRLCVLHEKTPVSEKITKCCTDSLAERRPCFSALELDEGYVPKEFKAETFTFHADICTLPEDEKQIKKQSALAELVKHKPKATKEQLKTVLGNFSAFVAKCCGREDKEACFAEEGPKLVASSQLALA >Sequence 9 MKWVTFISLLFLFSSAYSRGVFRREAHKSEIAHRFNDVGEEHFIGLVLITFSQYLQKCPYEEHAKLVKEVTDLAKACVADESAANCDKSLHDIFGDKICALPSLRDTYGDVADCCEKKEPERNECFLHHKDDKPDLPPFARPEADVLCKAFH DDEKAFFGHYLYEVARRHPYFYAPELLYYAQKYKAILTECCEAADKGACLTPKLDALEGKSLISAAQERLRCASIQKFGDRAYKAWALVRLSQRFPKADFTDISKIVTDLTKVHKECCHGDLLECADDRADLAKYMCEHQETISSHLKECCD KPILEKAHCIYGLHNDETPAGLPAVAEEFVEDKDVCKNYEEAKDLFLGKFLYEYSRRHPDYSVVLLLRLGKAYEATLKKCCATDDPHACYAKVLDEFQPLVDEPKNLVKQNCELYEQLGDYNFQNALLVRYTKKVPQVSTPTLVEISRSLGK VGSKCCKHPEAERLPCVEDYLSVVLNRLCVLHEKTPVSEKVTKCCSESLVDRRPCFSALGPDETYVPKEFNAETFTFHADICTLPETERKIKKQTALVELVKHKPHATNDQLKTVVGEFTALLDKCCSAEDKEACFAVEGPKLVESSKATLG >Sequence code 10 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0276] Part B Sequence listing reference (Part B) This application includes a computer-readable sequence listing. The computer-readable sequence is incorporated herein by reference.

[0277] Technical field (Part B) This invention relates to a formulation of albumin in a substantially non-aggregated form and its use.

[0278] Background Technology (Part B) Virus production is an important application of cell culture systems for vaccine manufacturing, gene therapy, and cell therapy.

[0279] Gene and cell therapies offer novel solutions for treating diseases and disorders caused by genetic or epigenetic defects. Breakthroughs in these types of therapies mean further research into optimizing formulation and delivery methods. These therapies involve multiple steps, including the production, purification, storage, distribution, and administration of viruses.

[0280] Existing research indicates that the low stability of viruses poses a significant challenge when performing these steps. This is because viruses are typically unstable and lose their biological activity or become completely inactive when left for extended periods at storage temperatures above -80°C. Therefore, viruses must be stored and transported in frozen form and used immediately after thawing (Harrington et al, 2017, International Publication No. 2016100364).

[0281] Low stability can affect the quality and efficacy of the administered formulation. This is characterized by a significant decrease in viral titer over time. Therefore, it is desirable to increase the efficacy and stability of molecules such as therapeutic agents in order to reduce the dose and frequency of administration required by the patient.

[0282] Cost is a crucial factor when considering improvements to vaccination programs in developing countries. One way to reduce vaccine costs is to produce more stable vaccines, for example, vaccines that do not require a cold chain, as these should be cheaper to deliver and store. Furthermore, the loss of vaccine activity under suboptimal storage conditions is thought to significantly contribute to less effective vaccination programs (Brandau et al, 2003). The World Health Organization (WHO) requires a minimum titer of vaccine preparations after one week of storage at 37°C. However, many vaccines can lose more than 50% of their potency after being stored at room temperature for just one hour (Plotkin & Orenstein, 2004).

[0283] Existing techniques to increase viral stability include keeping formulations at very low temperatures (-90°C to -70°C). However, loss of viral viability and / or infectivity occurs after multiple freeze-thaw cycles or after very short thawing periods. This can lead to the waste of a significant amount of expensive drug or vaccine formulations.

[0284] Another approach involves using native viral variants obtained through selection or mutagenesis (Domingo et al, 2006), but this is not a uniform approach, as mutations that work to stabilize some viruses may not work to stabilize others.

[0285] Another approach involves drying the virus (e.g., by freeze-drying / lyophilization, spray drying, or foam drying) (Lovalenti et al, 2016). However, drying requires specialized equipment for sample preparation (e.g., freeze dryers, vacuum pumps, etc.) and exposes the virus to extreme temperature and / or pressure conditions (Ohtake et al, 2011).

[0286] Further approaches include the addition of stabilizing pharmaceutical excipients to the formulation (Brandau et al., 2003). Excipients are substances that exclude active pharmaceutical ingredients (APIs) (e.g., viruses) that are intentionally included in the drug delivery system. Various excipients can be used, such as carbohydrates (sucrose, lactose, maltose, and trehalose), sugar alcohols (sorbitol and mannitol, etc.) (Bovarnick et al., 1950, J. Bedu-Addo et al., 2004), chitosan, and glutamates (Kissmann et al., 2008).

[0287] The inventors have observed that an albumin formulation comprising substantially non-aggregated albumin and a plurality of negatively charged anions housed on a flexible skeleton possesses beneficial properties for stabilizing viruses, including preserving the integrity of the viral structure and viral infectivity during storage at low and ambient temperatures, and across multiple freeze-thaw cycles. Such formulations help reduce constraints during the manufacture, transport, storage, and use of viruses by providing flexibility without unacceptable loss of stability, structure, and / or infectivity.

[0288] Summary of the Invention (Part B) The present invention provides the use of a formulation for stabilizing a virus, wherein the formulation comprises (i) albumin, which is substantially in an agglutinative form, and (ii) a plurality of negatively charged anions housed on a flexible skeleton.

[0289] The present invention also provides a formulation comprising (i) albumin, which is substantially in an agglutinated form, and (ii) a plurality of negatively charged anions housed on a flexible skeleton.

[0290] The present invention also provides pharmaceutical compositions comprising the formulations described herein and their pharmaceutically active ingredients, carriers, or diluents.

[0291] The present invention also provides formulations described herein for use in pharmaceuticals.

[0292] The present invention also provides formulations described herein for use in gene therapy.

[0293] The present invention further provides formulations described herein for use in cell therapy.

[0294] The present invention further provides formulations described herein for use in vaccination and / or immunization.

[0295] The present invention further provides formulations described herein for use in immunotherapy, wherein the immunotherapy is optionally oncolytic virus therapy.

[0296] The present invention further provides a method for stabilizing a virus, comprising combining the virus with a formulation or pharmaceutical composition described herein.

[0297] The present invention further provides a method for selecting a suitable albumin for use in stabilizing a virus, the method comprising determining whether the albumin is substantially in an agglutinating form.

[0298] Modes for carrying out the invention (Part B) A first aspect of the present invention provides the use of a formulation for stabilizing a virus, wherein the formulation comprises (i) albumin, which is substantially in an agglutinative form, and (ii) a plurality of negatively charged anions housed on a flexible skeleton.

[0299] As used herein, the term "albumin" includes human serum albumin (HSA, in particular SEQ ID NO: 2) or a protein having the same and / or very similar tertiary structure as the HSA domain and possessing similar characteristics to HSA or a related domain. Similar tertiary structures include, for example, albumins from non-human species, e.g., non-human primate albumins (chimpanzee albumin (e.g., predicted sequence GenBank XP_517233.2), gorilla albumin or macaque albumin (e.g., GenBank NP_001182578)), rodent albumins (hamster albumin (e.g., GenBank A6YF56), guinea pig albumin (e.g., UniProt Q6WDN9-1), mouse albumin (e.g., GenBank AAH49971 or UniProt P07724-1 version 3), and rat albumin (e.g., GenBank AAH85359 or UniProt P02770-1 version 2)), bovine albumin (e.g., bovine albumin (e.g., UniProt This includes structures such as P02769-1), equine albumin (e.g., UniProt P35747-1, or donkey albumin (e.g., UniProt Q5XLE4-1), rabbit albumin (e.g., UniProt P49065-1 version 2), goat albumin (e.g., GenBank ACF10391), sheep albumin (e.g., UniProt P14639-1), canine albumin (e.g., NCBI NP_001003026), chicken albumin (e.g., UniProt P19121-1 version 2), and porcine albumin (e.g., UniProt P08835-1 version 2), or any one of the structures of Sequence IDs 4-19 of International Publication No. 2013 / 006675 incorporated herein by reference. All of these albumins are included within the scope of the present invention.The mature form of albumin (e.g., the form in which all post-translational modifications and / or processing steps have been completed) is particularly preferred, and those skilled in the art can identify the mature form using publicly available information such as protein data banks and / or by using signal peptide recognition software such as SignalP (e.g., SignalP (Nielsen et al, 1997, Protein Engineering 10(1):1-6)). SignalP version 4.0 is preferred (Petersen et al, 2011, Nat Methods 8(10):785-786). Albumin formulations for use in the methods and compositions of the present invention may comprise one or more (several) albumins. To avoid misunderstanding, the sequence numbers provided in Part B of this application correspond to the sequence numbers provided in Part A.

[0300] Some of the main properties of albumin are: i) its ability to regulate plasma volume, ii) its long plasma half-life of approximately 19 ± 5 days, iii) ligand binding, such as binding to endogenous molecules including acidic lipophilic compounds such as bilirubin, fatty acids, hemin, and thyroxine (see also Table 1 of Kragh-Hansen et al, 2002, Biol Pharm Bull 25(6):695-704, incorporated herein by reference), and iv) binding to small organic compounds with acidic or electronegative characteristics, such as drugs such as warfarin, diazepam, ibuprofen, and paclitaxel (see also Table 1 of Kragh-Hansen et al, 2002, Biol Pharm Bull 25(6):695-704, incorporated herein by reference). Not all of these properties must be met for a protein or fragment to be characterized as albumin. For example, if a fragment does not contain a domain involved in binding a particular ligand or organic compound, then variants of such a fragment are not expected to possess these properties.

[0301] The term albumin includes derivatives such as fragments, variants, and / or fusions and / or conjugates of albumin or albumin variants. The term "fusion" includes the meaning of a genetic fusion of albumin (or its variants or fragments) and a non-albumin protein or peptide. The non-albumin protein or peptide may be a therapeutic, prophylactic, or diagnostic protein or peptide. Examples of albumin fusions are provided in European Patent No. 624195, International Publication No. 2001 / 079271, International Publication No. 2003 / 059934, International Publication No. 2003 / 060071, International Publication No. 2011051489, International Publication No. 2011 / 124718, and European Patent Application Publication No. 11164955 (each incorporated herein in its entirety by reference).

[0302] The term "variant" includes the meaning of a polypeptide derived from a parent albumin that includes modifications, i.e., substitutions, insertions, and / or deletions, at one or more (several) positions. It is preferable that the parent albumin is a mature albumin, e.g., one that does not contain a leader sequence. Substitution includes replacing an amino acid occupying a position with a different amino acid, deletion includes removing an amino acid occupying a position, and insertion includes adding an amino acid (e.g., 1 to 3 amino acids) adjacent to an amino acid occupying a position. For example, a modified polypeptide (variant) can be obtained through human intervention by modifying the polynucleotide sequence encoding the parent albumin. The mature form of the albumin sequence may be encoded by SEQ ID NO: 1 of the nucleotide sequence. The variant albumin is preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or 100% identical to SEQ ID NO: 2.

[0303] Sequence ID No. 2 - Wild-type human albumin: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDMPADLPSLAADFVESKDVCNKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0304] Preferably, the albumin is derived from human (SEQ ID NO: 2), mouse (GenBank AAH49971 or UniProt P07724-1 version 3), rat (GenBank AAH85359 or UniProt P02770-1 version 2), macaque (GenBank NP_001182578), cattle (bovine albumin (e.g., UniProt P02769-1)), pig (UniProt P08835-1 version 2), horse (UniProt P35747-1), rabbit (UniProt P49065-1 version 2), dog (Canis lupus familiaris, residues 1-18 are the signal peptide, 19-24 are the propeptide, and residues 25-608 are the mature sequence, accession number NP_001003026), or guinea pig (Cavia It has at least 70% sequence identity with mammalian albumin selected from the group consisting of porcellus, UniProt Q6WDN9-1) albumin, where residues 1-18 are the signal peptide and residues 25-608 are the mature sequence.Preferably, albumin is HSA (the immature sequence provided in SEQ ID NO: 2, with residues 1-18 being the signal peptide, 19-24 being the propeptide, and residues 25-609 being the mature sequence, as provided in SEQ ID NO: 10), mouse serum albumin (residues 1-18 being the signal peptide, 19-24 being the propeptide, and residues 25-608 being the mature sequence, as provided in SEQ ID NO: 3), rat serum albumin (residues 1-18 being the signal peptide, 19-24 being the propeptide, and residues 25-608 being the mature sequence, as provided in SEQ ID NO: 4), macaque serum albumin (residues 1-18 being the signal peptide, 19-24 being the propeptide, and residues 25-608 being the mature sequence, as provided in SEQ ID NO: 5), The albumin variant has at least 70% sequence identity with mammalian albumin selected from any of the following: porcine serum albumin (residues 1-18 are signal peptides, 19-24 are propeptides, and residues 25-607 are the mature sequence, SEQ ID NO: 6), porcine serum albumin (residues 1-18 are signal peptides, 19-24 are propeptides, and residues 25-607 are the mature sequence, SEQ ID NO: 7), horse serum albumin (residues 1-18 are signal peptides, 19-24 are propeptides, and residues 25-607 are the mature sequence, SEQ ID NO: 8), or rabbit serum albumin (residues 1-18 are signal peptides, 19-24 are propeptides, and residues 25-608 are the mature sequence, SEQ ID NO: 9). It is preferable that the albumin variant is a variant of the mature form of albumin. For example, if the parent albumin is mouse serum albumin, preferably the variant has at least 70% sequence identity with residues 25-608 of SEQ ID NO: 3, and similarly for other mammalian sequences disclosed herein. Preferably the albumin has at least 70% sequence identity with HSA (SEQ ID NO: 2), more preferably 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, or 99.8 to 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, 99.8, or 100% identity with HSA (SEQ ID NO: 2).For example, preferred albumin has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation compared to wild-type HSA (SEQ ID NO: 2). To avoid misunderstanding, the SEQ ID NOs provided in Part B of this application correspond to the SEQ ID NOs provided in Part A.

[0305] Preferably, the albumin contains at least 175 consecutive amino acids from albumin having at least 70% sequence identity with HSA (SEQ ID NO: 2), such as 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, or 575-200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 580 amino acids. The fragment may contain, consist of, or substantially correspond to one or more (e.g., several) domains of albumin or its variants, such as HSA (SEQ ID NO: 2), including amino acids corresponding to domain I (residues 1-194±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), domain II (residues 192-387±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), or domain III (residues 381-585±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0306] Typically, variant albumins retain at least one of the key tertiary structural characteristics similar to those of the parent albumin or HSA. For the purposes of this invention, sequence identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (Needleman & Wunsch, 1970, J Mol Biol 48(3):443-453), which is implemented using the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet 16(6):276-277), preferably version 5.0.0 or later of the Needle program. Typical parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "Longest Identity" (obtained using the -nobrief option) can be used as the identity rate and can be calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment).

[0307] Examples of albumin variants include International Publication Nos. 2011 / 051489, 2011 / 124718, 2012 / 059486, 2012 / 150319, 2014 / 072481, 2013 / 135896, 2015 / 036579, 2010 / 092135, 2013 / 075066, and 2014 Examples include those described in International Publication No. / 179657, International Publication No. 2009 / 126920, International Publication No. 2010 / 059315, International Publication No. 2011 / 103076, International Publication No. 2012 / 112188, International Publication No. 2015 / 063611, and International Publication No. 2017 / 029407 (the contents of which are incorporated herein by reference, in particular by reference to albumin variants, in their entirety).

[0308] As used herein, the terms “parent” or “parental albumin” include albumins from humans or other animals (e.g., mammals). Preferably, other mammalian albumins are albumins from clinically relevant animals such as mice, rats, rabbits, dogs, or guinea pigs. The parent may be a naturally occurring (wild-type) polypeptide or its allele, or a variant as described above.

[0309] As used herein, the term “wild-type” (WT) albumin includes albumin having the same amino acid sequence as the dominant allele variant of albumin found naturally in animals or humans. Sequence ID No. 2 is an example of wild-type albumin, which is wild-type albumin from Homo sapiens.

[0310] The term “substantially non-aggregated albumin” implies that the majority of albumin molecules are present in the formulation in a non-aggregated form and therefore, for example, are not aggregated with other albumin molecules and / or non-albumin molecules. It will be understood that proteins such as albumin can form covalent or non-covalent aggregates, which may lead to the formation of high molecular weight aggregates. Examples of covalent aggregation may or may not include aggregates formed by cysteine ​​or lysine crosslinking. Examples of non-covalent aggregation may or may not include aggregates formed by electrostatic interactions (e.g., ionic, hydrogen, halogen bonds), van der Waals forces, the π effect, and electromagnetic interactions such as hydrophobic interactions. In the formulations of the present invention, it will be understood that the majority of albumin molecules are not present in such high molecular weight aggregates, but rather in a non-aggregated form. For example, at least 80% of the albumin molecules in the formulation, such as at least 85%, 90%, 95%, or 99%, or all of the albumin molecules, may be present in a non-aggregated form. The term “non-aggregated” includes monomers, dimers, and trimers of albumin, and especially monomers. The term “aggregated” includes tetramers, pentamers, hexamers, and higher-order polymers of albumin. Examples of higher-order polymers may or may not include fibrils such as amyloid fibrils or protein fibrils. Preferably, the albumin of the present invention is substantially non-aggregated (e.g., existing in the form of monomers and / or dimers and / or trimers), and / or preferably, any aggregation of albumin referred to herein is non-covalent aggregation.

[0311] Various methods are known in the art and can be used to assess whether albumin exists in a substantially non-aggregated form, as described below. Any preferred method can be used.

[0312] For example, the percentage of albumin in polymer form may be used to indicate whether albumin is present in a substantially non-aggregated form. Therefore, in a preferred embodiment, albumin present in a substantially non-aggregated form is defined as less than 0.025% (w / w) of albumin in polymer form in the formulation. The term "polymer" includes the meaning of high molecular weight forms or aggregates of albumin that elute within the void volume of a gel permeation HPLC column separating molecules in the molecular weight range of 10,000 to 500,000 Da, such as a TSK G3000SWXL. To avoid misunderstanding, the term "polymer" does not include dimers or trimers, but rather includes tetramers and higher-order aggregates / multimers, such as tetramers, pentamers, and hexamers. Preferably, less than 0.025% (w / w) of albumin in the formulation, more preferably 0.024%, 0.02%, 0.015%, 0.010%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less than 0% (w / w) of albumin is present in polymer form. Preferably, polymer levels of less than 0.025%, 0.02%, 0.015%, 0.010%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less than 0% are observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation and / or filling into containers such as vials, bottles, or bags). However, it will be understood that albumin is preferably substantially non-aggregated throughout its shelf life at the recommended storage temperature, and therefore, for example, less than 0.025% (w / w) of albumin in a formulation is preferably in polymer form throughout its shelf life. Typical shelf life ranges from 6 months to 5 years, and therefore, it is preferable that less than 0.025% (w / w) of albumin is in polymer form for at least 1 month, 2 months, 3 months, 4 months, 5 months, more preferably at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years after T0. The recommended storage temperature for liquid formulations is typically in the range of 2 to 8°C, such as about 2, 3, 4, 5 to about 6, 7, 8°C.The recommended storage temperature for lyophilized formulations is typically either 2–8°C (e.g., approximately 2°C, 3°C, 4°C, 5°C to approximately 6°C, 7°C, 8°C) or -24–-16°C (e.g., approximately -24°C, -23°C, -22°C, -21°C, -20°C to approximately -19°C, -18°C, -17°C, -16°C).

[0313] Albumin polymer levels can be measured by chromatography, for example, high-performance liquid size exclusion chromatography (SEC-HPLC), or any other suitable technique in the art, using albumin provided at a concentration within the dynamic range of the assay, such as less than 100 mg / mL, preferably 20–40 mg / mL, or diluted to that concentration. In a preferred embodiment, albumin present in a substantially non-aggregated form is less than 0.025% (w / w) of albumin in the formulation, which is a polymer form measured by high-performance liquid size exclusion chromatography (SEC-HPLC) using an injection sample of albumin provided at less than 100 mg / mL, preferably 20–40 mg / mL, or diluted to that concentration. A further description of a method for evaluating albumin aggregation by evaluating polymer levels using chromatography is provided in the examples, in particular by the method of Example 3.

[0314] In another example, dynamic light scattering (DLS) may be used to indicate whether albumin in a formulation exists in a substantially non-aggregated form.

[0315] Therefore, in another embodiment, albumin substantially present in a non-aggregated form has a hydrodynamic radius (R) of albumin when evaluated by DLS, when the albumin is incubated at 40°C for a set period. H) remains substantially unchanged. Preferably, the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of at least 3 days, such as 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.25, or 0% or less. Preferably, a change of 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.25, or 0% or less is observed on the 3rd day. The hydrodynamic radius of albumin (for example, at a concentration of about 5 mg / mL) is about 3.5 nm, and it will be understood that albumin existing in a substantially non-aggregated form may maintain a hydrodynamic radius of about 3.5 ± 0.2 nanometers (nm), preferably ± 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, or 0 nm when incubated at 40°C for a duration of at least 3 days. Measuring the hydrodynamic radius of albumin by dynamic light scattering (DLS) is a standard technique in the art, which can be performed, for example, using albumin at 5 mg / mL. Further descriptions of methods using DLS to assess the hydrodynamic radius, including DLS cumulant analysis, are provided in the examples. It will be understood that the measurement of the hydrodynamic radius of albumin by DLS may be performed in or out of the presence of octanoate. In the presence of octanoate, albumin aggregation is expected to take longer.

[0316] In yet another embodiment, albumin substantially present in a non-aggregated form is one in which the polydispersity index (PD index) of the albumin, as assessed by DLS when the albumin is in solution (e.g., in PBS) in the absence of the virus, is 0.05 or less. Preferably, the PD index is less than 0.05, more preferably 0.04, 0.03, 0.02, 0.01, or less than 0. Preferably, 0.05, 0.04, 0.03, 0.02, 0.01, or less than 0 is observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation) and / or throughout its shelf life at the recommended storage temperature. Preferred duration of shelf life and preferred storage temperature include those described above. Preferably, the PD is determined for albumin provided at about 5 mg / mL or diluted to about 5 mg / mL. The PD index is a number calculated from a simple two-parameter fit (cumulant analysis) to correlated data. The PD index is dimensionless and is incremented or decremented so that values ​​less than 0.05 are rarely seen except when using a highly monovariance standard. Values ​​greater than 0.7 indicate that the sample has a very broad size distribution. The calculation of these parameters is defined in ISO standard documents 13321:1996 E and ISO 22412:2008 (https: / / www.malvernpanalytical.com / en / learn / knowledge-center / whitepapers / WP111214DLSTermsDefined). Measuring the PD index of albumin by dynamic light scattering (DLS) is a standard technique in the art, which can be performed, for example, using albumin at 5 mg / mL. Further descriptions of methods using DLS to assess the PD index, including DLS cumulant analysis, are provided in the examples.

[0317] As further described in the examples, the inventors have identified the following parameters: Less than 0.025% (w / w) of albumin in the formulation is in polymer form. The polydispersity index of albumin is 0.05 or less, and The hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days. We have found that albumin in a substantially non-aggregating form is particularly good at stabilizing viruses. Therefore, we have designated such albumin as “good albumin” in this specification. Accordingly, the albumin in the formulation possesses one or more of the following parameters (e.g., two or more, or all three): Less than 0.025% (w / w) of albumin in the formulation is in polymer form. The polydispersity index of albumin is 0.05 or less, and It is particularly preferable that the hydrodynamic radius of the albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days.

[0318] Further desirable properties of albumin identified by the present inventors are described below.

[0319] In one embodiment, the total amount of lipids in the formulation (excluding octanoates) is less than or equal to 50 μg (micrograms) of lipids per mL of 100 mg / mL of albumin, such as 40 μg of lipids / mL, 30 μg of lipids / mL, 20 μg of lipids / mL, or less than or equal to 10 μg of lipids / mL. The total amount of lipids (excluding octanoates) can be measured by any suitable technique in the art, including gas chromatography-mass spectrometry (GC-MS). Different lipids can be characterized by their peaks and identified using standard calibration curves known in the art. Throughout this document, the term “lipids” includes “fatty acids.”

[0320] In another embodiment, the albumin is one in which the N-terminal degradation of albumin is less than 12.5%, such as less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less than 4%, or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 12.5%. Albumin having less than 8% N-terminal degradation is particularly preferred. The phrase "less than 12.5% ​​N-terminal degradation of albumin" includes the meaning of less than 12.5% ​​of albumin molecules exhibiting a certain level of N-terminal degradation.

[0321] Preferably, the N-terminal degradation of albumin is 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.75, 1.5, 1.25, 1, 0.75, 0.50, 0.25, or less than 0%, for example, 0, 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 1.96, 1.97, 1.98, 1.99, 2, 2.5, 3, 3.5, 4, 4.5 , 5, 5.5, 6, 6.5, 7, or 7.5% ~ 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 1.96, 1.97, 1.98, 1.99, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, or 12.5% ​​are observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation) and / or during the shelf life. Preferred shelf life duration and preferred storage temperature include those described above. The percentage of N-terminal degradation of albumin can be assessed using any suitable technique in the art, including mass spectrometry such as electrospray ionization mass spectrometry (ESI-MS). For example, when ESI-MS is used, the peak with the highest abundance (attributed to non-N-terminal degradation albumin) may be normalized to 100%, and all peaks attributable to N-terminal degradation albumin should be less than 12.5% ​​of the "100% peak". A preferred ESI-MS protocol for assessing the intact mass of albumin is provided in Example 5.

[0322] Albumin may or may not be modified, such as by cysteinization. "Cysteinization" includes the addition of a cysteine ​​residue via a disulfide bond to an existing cysteine ​​residue, such as Cys34 in wild-type HSA (SEQ ID NO: 2), or the corresponding Cys in albumin or albumin variants from other species. Such cysteinization may generate "branching" from the albumin sequence. The calculation of the relative intensity of N-terminal degradation may preferably include both the amount of intact unmodified (e.g., uncysteined) albumin and intact modified (e.g., cysteined) albumin, as well as the amount of unmodified (e.g., uncysteined) albumin exhibiting N-terminal degradation and the amount of modified (e.g., cysteined) albumin exhibiting N-terminal degradation. Alternatively, the relative intensity of N-terminal degradation can be calculated based on the amount of intact, unmodified (e.g., non-cysteine) albumin and the amount of unmodified (e.g., non-cysteine) albumin exhibiting N-terminal degradation. Preferably, the calculation of the relative intensity of N-terminal degradation does not include the amount of intact, modified (e.g., cysteine) albumin and the amount of modified (e.g., cysteine) albumin exhibiting N-terminal degradation.

[0323] In yet another embodiment, the albumin is one in which all oxidative species are excluded, but the total percentage of all post-translational modifications is less than 75%. "Total percentage of all post-translational modifications, excluding all oxidative species, is less than 75%" means that less than 75% of the albumin molecule is post-translationally modified by means other than oxidation. Preferably, the total percentage of all post-translational modifications other than oxidation of albumin is less than 75%, such as 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, and less than 9.9, such as 9.8, 9.75, 9.5, 9.25, 9, 8, 7, 6, 5, 4, 3, 2, 1, or about 0%. Preferably, the total percentage of all post-translational modifications other than oxidation is less than 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 10, 9.9, 9.75, 9.5, 9.25, 9, 8, 7, 6, 5, 4, 3, 2, less than 1%, or about 0%, and is observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation) and / or during the shelf life. Particularly preferred albumins have a total percentage of all post-translational modifications other than oxidation of less than 30%, more preferably less than 25%, and even more preferably less than 10%. Preferred shelf life durations and preferred storage temperatures include those described above. The percentage of post-translational modifications may be evaluated using any preferred technique in the art, including mass spectrometry such as electrospray ionization mass spectrometry (ESI-MS). Preferably, ESI-MS is performed using a Bruker MicroTOF II with injection via a Waters® Acquity UPLC, which has a sensitivity greater than 25 pmol (picomoles) and a resolution of approximately 3500. Thus, it will be understood that albumin may exclude oxidized species, but the total percentage of all post-translational modifications may be less than 75% when determined using such instrumentation and parameters.

[0324] For example, when using ESI-MS, the peak with the highest abundance (e.g., the peak attributable to untranslated albumin) is normalized and set to 100%. All peaks attributable to posttranslational modification of albumin (e.g., glycation and cysteine) are summed up to produce a total that should be less than 75% of the "100% peak". The measurement is a measure of intensity, and "75%" corresponds to "75% of the intensity of the highest peak".

[0325] In yet another embodiment, the albumin is one in which the amount of copper (Cu) in the formulation is less than 0.15 μg / g (micrograms / gram) albumin. Preferably, the amount of copper in the formulation is less than 0.14 μg / g albumin, such as 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01, or about 0 μg / g albumin. Preferably, the amount of copper in the formulation, which is less than 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or about 0 μg / g albumin, is observed at T0 (e.g., time zero measured within 24 or 48 hours of formulation) and / or during the shelf life. Preferred shelf life durations and preferred storage temperatures include those described above. The amount of copper can be evaluated by any suitable technique in the art, including mass spectrometry such as inductively coupled plasma mass spectrometry (ICP-MS).

[0326] The inventors have found that albumin having a certain level of free thiol groups is particularly good at stabilizing viruses, and therefore, in one embodiment, the formulation contains at least 0.5 moles of free thiol per mole of albumin. Thus, the formulation may contain at least 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, or 0.99 moles of free thiol per mole of albumin. The formulation may contain at least 1 mole or 1 mole of free thiol per mole of albumin. The term "free thiol" includes the meaning of a thiol group that is available for reaction with another group. For example, a free thiol is one that is not yet connected to another thiol group via a disulfide bond or has not been oxidized. Free thiols may be provided by cysteine ​​amino acids such as Cys34 of HSA (SEQ ID NO: 2), or equivalent Cys in albumin variants or albumins from other species.

[0327] Therefore, in one embodiment, it will be understood that albumin may contain one or more (e.g., several) free thiol groups ("thiols") per molecule of albumin, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 free thiol groups. For albumins containing one free thiol group per molecule of albumin, such as human serum albumin containing free thiols provided by Cys34, the theoretical free thiol content is 1 mole of free thiols per mole of albumin or its fragment or fusion. For albumins containing two free thiol groups per molecule of albumin or its fragment, such as a variant of human serum albumin containing free thiols provided by Cys34 and additional free thiols provided by another Cys34, the theoretical free thiol content is 2 moles of free thiols per mole of albumin, and so on. Albumin containing two or more cysteine ​​residues that provide free thiols includes mouse albumin and albumin variants.

[0328] Preferably, the desired free thiol level per mole of albumin is observed after storage or incubation at 25°C for at least one month, more preferably at least two, three, four, five, six, nine, or twelve months. Preferably, the free thiol level per mole of albumin is observed after storage or incubation at 40°C for at least one month, more preferably at least two, three, four, five, or six months. Preferably, the free thiol level per mole of albumin is observed after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7-3, 4, 5, 6, 7, or 8°C, for example, about 4°C, for a duration of at least one month, more preferably at least two, three, four, five, or six months, or at least one, two, three, four, five, or six years.

[0329] Free thiol levels can be measured by any suitable free thiol assay known in the art, such as a colorimetric free thiol assay, for example, a DTNB assay or a DTDP assay. The DTNB assay is preferred.

[0330] A particularly preferred DTNB assay comprises the following method, where the volume of components may be adjusted and the ratio of components should remain as described below: a) On the day of measurement, prepare a stock solution of DTNB at 4 mg / mL in assay buffer (0.1 M phosphate buffer, pH 8.0) and protect it from light. For the standard curve, prepare a 1.5 mM stock solution of L-cysteine ​​in water (Sigma #30089, MW = 121.165 g / mol) on the same day as the assay. b) Prepare cysteine ​​standard solutions from this stock solution by diluting it in assay buffer at the following concentrations: 1.5, 1.25, 1.0, 0.75, 0.5, 0.25, and 0 mM. c) Prepare the assay solution by mixing 500 μL (microliters) of assay buffer with 50 μL of standard or sample. d) Add 10 μL (microliters) of 0.01 M 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) to the sample, control, and buffer "blank". e) Incubate in a dark place at room temperature (15-25°C, 20°C is preferable) for 10 minutes, and f) Measure the increase in absorbance of the sample, control, and buffer "blank" at a wavelength of 412 nm, and g) Determine the free thiol level in the sample.

[0331] In one embodiment, the albumin in the formulation has one or more (preferably all three) of the following parameters: Less than 0.025% (w / w) of albumin in the formulation is in polymer form. The polydispersity index of albumin must be 0.05 or less. The hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, and one or more of the following parameters are met: i) The total amount of lipids in the preparation (excluding octanoates) is 50 μg (micrograms) of lipids / mL albumin or less. ii) The albumin has less than 12.5% ​​N-terminal degradation. iii) Albumin has a total percentage of less than 75% of all post-translational modifications. iv) The albumin contains less than 0.15 μg / g (micrograms / gram) of copper (Cu) in the preparation, and v) The formulation satisfies the requirement that it contains at least 0.5 moles of free thiols per mole of albumin.

[0332] Albumin may be supplied from serum or recombinant sources. Advantageously, the formulation may contain recombinant albumin. That is, albumin may be supplied from recombinant organisms such as recombinant microorganisms, recombinant plants, or recombinant animals. Since some users prefer animal-free ingredients, it is more preferable that albumin be supplied from non-animal recombinant sources such as recombinant microorganisms or recombinant plants. Preferred organisms include, but are not limited to, prokaryotes, and more preferably animals, plants, fungi, or eukaryotes such as yeast, and include, for example, the following species in which albumin has been successfully expressed as a recombinant protein: Fungi (including, but not limited to, Aspergillus (International Publication No. 06066595), Kluyveromyces (Fleer 1991, Bio / technology 9, 968-975), Pichia (Kobayashi 1998 Therapeutic Apheresis 2,257-262), and Saccharomyces (Sleep 1990, Bio / technology 8, 42-46)) Animals (Barash 1993, Transgenic Research 2, 266-276) Plants (including, but not limited to, potatoes and tobacco (Sijmons 1990, Bio / technology 8, 217, and Farran 2002, Transgenic Research 11, 337-346), as well as rice, e.g., Oryza sativa) Mammalian cells such as CHO and HEK Prokaryotes (Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285), for example, E. coli (European Patent No. 73,646).

[0333] Preferably, albumin is a recombinant supplied from a fungal host (e.g., a yeast host) from a genera selected from preferably Saccharomyces (e.g., Saccharomyces cerevisiae), or Pichia (e.g., Pichia pastoris), or Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, and Arxula adeninivorans, Candida (e.g., Candida utilis), Zygosaccharomyces bailii, more preferably Saccharomyces, most preferably Saccharomyces cerevisiae (https: / / doi.org / 10.1016 / j.biotechadv.2011.09.011, i.e., Celik & Calik, 2012, https: / / doi.org / 10.1007 / 978-1-4939-9024-5_1, i.e. Gunduz Ergun et al, 2019).

[0334] In embodiments, the formulation contains 0.4 to 300 mg / mL of albumin, such as 0.5 to 300 mg / mL. For example, the formulation may contain approximately 75, 100, 125, 150, 175, 200, 225, 250, or 275 mg / mL of albumin. In specific examples, the formulation may contain approximately 50 to 300 mg / mL of albumin, such as approximately 100 to 200 mg / mL.

[0335] Preferably, the formulation used in the first embodiment of the present invention contains 0.4 to 50 mg / mL of albumin, such as 0.5 to 50 mg / mL. For example, the formulation may contain about 0.4, 0.5, 1, 2.5, 5, 10, 15, 20, 25, 30, 35, or 40 to about 25, 30, 35, 40, 45, or 50 mg / mL of albumin. Albumin concentrations of about 1 to 20 mg / mL are preferred, and albumin concentrations of about 1 mg / mL, about 2.5 mg / mL, and about 5 mg / mL are particularly preferred.

[0336] It will be understood that albumin in a formulation used to stabilize a virus according to a first aspect of the present invention may itself be provided by a pre-prepared formulation (referred to herein as an “albumin composition”). “Albumin in a formulation provided by an albumin composition” means that albumin in a formulation is provided by re-formulating a pre-prepared albumin composition, for example, by (i) combining the albumin composition with a plurality of negatively charged anions housed on a flexible skeleton and optionally one or more other components such as a buffer, as described herein with respect to a first aspect of the present invention, and optionally (ii) diluting the albumin composition to obtain a desired albumin concentration.A preferred example of a suitable albumin composition is (a) containing 130-160 mM, preferably 145 mM, sodium, 29-35 mM, preferably 32 mM, polysorbate 80, 10-20 mg / L, preferably 10-15 mg / L, albumin (w / v) of 190-210 mg / mL, preferably 200 mg / mL, with a pH of 6.7-7.3, preferably 7, and a percentage (w / w) of polymerized albumin of 0-1%, more preferably The composition (b) contains 120-160 mM, preferably 145 mM, sodium; 4-12 mM, preferably 8 mM, octanoate; 0-50 mg / L, preferably 25-45 mg / L, polysorbate 80; and 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v), with a pH of 6.4-7.4, preferably 7, and a percentage (w / w) of polymerized albumin of 0-1%, more preferably 0. The composition is less than 0.25%, and contains (c) 120-160 mM, preferably 145 mM, sodium, 8-24 mM, preferably 16 mM, octanoate, 0-100 mg / L, preferably 50-70 mg / L, polysorbate 80, and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v), with a pH of 6.4-7.4, preferably 7, and a percentage (w / w) of polymerized albumin of 0-1%, more preferably 0.0 (d) comprising any composition selected from the group consisting of less than 25%, (d) containing 200-300 mM, preferably 230-260 mM, sodium, 0-3 mM, preferably 0-1 mM, octanoate, and 95-105 mg / mL, preferably 100 mg / mL of albumin (w / v), with a pH of 6-7, preferably 6.5, and a percentage (w / w) of polymerized albumin of 0-1%, preferably less than 0.025%. It will be understood that the albumin in these albumin compositions is considered “good” albumin.

[0337] Particularly preferred examples of substantially non-aggregated albumins (e.g., recombinant human albumin) include those produced in yeast, and in particular, the following known commercially available preparations of recombinant yeast-derived albumins, such as Recombumin® Prime (formerly Recombumin®), Recombumin® Elite (formerly AlblX®), Recombumin® Alpha (formerly Albucult®) (all supplied by Albumedix Limited), or any similar preparations, manufactured in Saccharomyces cerevisiae. It will be understood that all of these albumins are considered “good” albumins.

[0338] It will be further understood that a formulation used to stabilize a virus according to a first aspect of the present invention may be conveniently prepared from a stock solution of albumin containing a higher concentration of albumin than the concentration of albumin in the formulation ready for use to stabilize the virus. For example, the stock solution of albumin may contain 50 to 300 mg / mL of albumin (e.g., about 100 or about 200 mg / mL of albumin), such as 100 to 200 mg / mL, and may be diluted so that the concentration of albumin in the formulation ready for use to stabilize the virus is 0.4 to 50 mg / mL of albumin (e.g., about 1 to 20 mg / mL, such as 1 mg / mL, 2.5 mg / mL, or 5 mg / mL). It will be understood that any of the already prepared albumin compositions (a) to (d) or any of the commercially available albumin offerings described above may constitute such a stock solution.

[0339] It will be understood that the formulations described herein may or may not contain octanoates, and / or the formulations described herein may or may not contain phosphates.

[0340] In a preferred embodiment, the formulation used to stabilize the virus is a liquid formulation.

[0341] In the embodiments, the formulations used to stabilize the virus are substantially free of sugars (such as sucrose, lactose, maltose, or trehalose) and / or sugar alcohols (such as sorbitol or mannitol). The inventors have found that mannitol alone generally does not stabilize the virus, and that mannitol-containing samples result in oligomerization and aggregate formation. However, mannitol may be included when used in combination with other excipients and / or salts.

[0342] The term “anion having multiple negative charges housed on a flexible framework” includes any flexible framework that houses two or more negative charges, such as three or more, four or more, or five or more negative charges. Thus, the anion can be multivalent, such as divalent, trivalent, or tetravalent. In one embodiment, the multiple negative charges housed on the flexible framework are discontinuous. In another embodiment, the multiple negative charges housed on the flexible framework are continuous. “Flexible framework” includes a framework of single covalent bonds connecting atoms having negatively charged groups such that rotational degrees of freedom exist between the negative charges. In yet another embodiment, the multiple negative charges housed on the flexible framework are located at least at both ends of the framework.

[0343] Suitable examples of anions having multiple (two, three, four, five, etc.) negative charges housed on a flexible skeleton include polyvalent organic anions such as organic carboxylic acids. For example, the anion may be a divalent or trivalent carboxylic acid. The anion may have carboxylic acid groups at both ends of a flexible skeleton. The anion may have two, three, or four carboxylic acid groups and no one or more amino groups bonded to the skeleton. The anion may have at least two carboxylic acid groups (e.g., at least two, three, four, or five carboxylic acid groups) and no one or more amide groups. In one embodiment, the anion is a citric acid cycle intermediate such as citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, or oxaloacetate. In further embodiments, the anion is tartaric acid, maleic acid, succinic acid, aconitate, adenosine triphosphate, or sodium tripolyphosphate. Inorganic anions such as EDTA are also included. In preferred embodiments, the anion is an intermediate of the tricarboxylic acid cycle, preferably a citrate or oxaloacetate, more preferably a citrate.

[0344] In one embodiment, the multiple negatively charged anions housed on a flexible skeleton are not one or more of EDTA (ethylenediaminetetraacetic acid), glutamates, glutamic acid, alkali metal glutamates, glycosaminoglycans, negatively charged polysaccharide branches, or polysaccharides.

[0345] In one embodiment, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or neither one nor both.

[0346] Preferably, multiple negatively charged anions housed on a flexible skeleton thermally stabilize albumin.

[0347] The statement "anions thermally stabilize albumin" implies that the anions increase the thermal stability of albumin compared to the thermal stability of albumin in the absence of the anions.

[0348] Thermal stability can be evaluated by measuring stability as a function of time at a given temperature. It can be readily determined, for example, by measuring binding to a binding partner, or by using spectroscopic methods such as fluorescence, circular dichroism (CD), light scattering, or differential scanning calorimetry (DSC) at a specific temperature, using, for example, albumin at 5 mg / mL. For instance, the ability of albumin to retain binding to a binding partner after incubation over a set period at a specific temperature can be used to determine the thermal stability of albumin. Alternatively, the tertiary structure of albumin after incubation over a set period at a specific temperature can be evaluated, for example, by spectroscopic methods. It will be understood that the tertiary structure can be evaluated directly (e.g., by spectroscopic methods) or indirectly (e.g., by evaluating one or more of the structure-dependent activities of albumin). Since conformational stability can be related to stabilization against thermal stress, it can be understood that anions that increase the thermal stability of albumin may also increase the conformational stability of albumin.

[0349] In one embodiment, multiple negatively charged anions housed on a flexible framework do not cause a change in the hydrodynamic radius of albumin in formulations of more than 5% when incubated at 40°C for at least 20 hours. In another embodiment, multiple negatively charged anions housed on a flexible framework maintain the hydrodynamic radius of albumin at approximately 3.5 ± 0.2 nanometers (nm) when incubated at 40°C for at least 20 hours.

[0350] While we do not wish to be constrained by theory, the inventors believe that anions bind to albumin and thermally stabilize the albumin by “stapling” the helix. “Stapleting” means that the helix is ​​constrained by binding to albumin. In other words, anions may bind to albumin and thermally stabilize the albumin by “coordinating” the helix. “Coordinating” means that the helix is ​​constrained by intramolecular binding to albumin, which can be driven by direct interactions or indirect charge / solvent effects. By fixing the albumin conformation to a more stable one, anions can preserve their stabilizing ability even under conditions where possible structural modifications could impair it (e.g., stress the formulated product). Binding can be evaluated directly (e.g., by isothermal titration calorimetry, ITC) or indirectly (e.g., by increased thermal stability) by any suitable technique known in the art.

[0351] In further embodiments, multiple negatively charged anions housed on a flexible skeleton may increase the conformational and / or chemical stability of albumin.

[0352] Preferably, multiple negatively charged anions housed on a flexible backbone lead to higher protein recovery in the formulation after storage at 25°C for 1 to 36 months (e.g., 2 to 36 months). Preferably, multiple negatively charged anions housed on a flexible backbone lead to higher monomer recovery in the formulation after 36 months at 5°C or 25°C. "Higher protein or monomer recovery" means that the recovery of protein or monomers is higher in the presence of anions compared to the recovery of protein or monomers in the absence of anions. Protein and monomer recovery can be measured using any suitable technique in the art, such as mass spectrometry. For example, the amount of total protein or monomeric protein can be measured at time zero using any suitable technique in the art, such as SEC-HPLC. This allows for the determination of the starting protein or monomer concentration (mg / mL) before any aggregation occurs during storage. Next, the amount of protein or monomers can be measured at subsequent time intervals during storage, and these values ​​can be normalized to the total amount of protein or monomeric protein at time zero, thereby allowing for comparison between formulations. The starting amount of protein or monomers can be set to 1, and the recovery rate can be expressed on a scale of 0 to 1. For example, if the total protein concentration at time zero is 9.8 mg / mL, the recovery of 0.985 of the total protein is 0.985 × 9.8 mg / mL = 9.653 mg / mL, and so on. When measured using SEC-HPLC, any decrease in the amount of total protein indicates lost material on the pre-column and the presence of aggregates filtered in the pre-column, and any decrease in the amount of monomeric protein correlates with the appearance of other peaks (i.e., dimers, trimers, and unfiltered polymers).

[0353] In one embodiment, a plurality of negatively charged anions housed on a flexible skeleton enable the recovery of at least 0.985 normalized total protein at 25°C after at least one month, for example, after at least two, three, four, five, or six months, and / or enable the recovery of 0.99 normalized total protein at 5°C after at least one month, for example, after at least two, three, four, five, or six months.

[0354] In another embodiment, a plurality of negatively charged anions housed on a flexible skeleton enable a normalized monomer amount of at least 0.97 at 25°C and / or at least 0.98 at 5°C after at least 36 months.

[0355] It will be understood that albumin tends to undergo deamidation, oxidation, N-terminal degradation, and / or fragmentation over time.

[0356] In one embodiment, multiple negatively charged anions housed on a flexible skeleton prevent albumin deamidation. For convenience, deamidation is measured by analyzing the ammonia concentration using an enzyme assay (e.g., Ammonia Assay Kit AA0100 (sigmaaldrich.com)). The decrease in absorbance at 340 nm due to the oxidation of NADPH (e.g., measured with a Shimadzu UV-2101PC UV-VIS spectrophotometer) is proportional to the ammonia concentration in the sample. For convenience, a positive ammonia control (e.g., an inorganic ammonium salt) may be used to demonstrate the validity of the assay performance. The statement "anions prevent albumin deamidation" means that any anion, by its presence, reduces deamidation (partially or substantially entirely) compared to the level of deamidation in the absence of the anion. Preferably, multiple negatively charged anions housed on a flexible skeleton prevent deamidation so that the ammonia concentration remains below 100 μM (micromoles) for 36 months in a sample containing 10 mg / mL albumin incubated at 25°C.

[0357] In yet another embodiment, multiple negatively charged anions housed on a flexible skeleton prevent N-terminal degradation of albumin. The statement "anions prevent N-terminal degradation of albumin" means that any anion, by its presence, reduces (partially or substantially all) N-terminal degradation of albumin compared to the level of N-terminal degradation in the absence of the anion. N-terminal degradation has been identified and characterized as autolysis that occurs during storage at temperatures above 30°C (Chan et al., 1995). This degradation is specific to human-derived albumin and is attributed to a particular N-terminal sequence. N-terminal degradation can be detected by ESI-MS from the presence of less abundant species, which is calculated by subtracting the first two amino acids from the intact albumin mass (i.e., the total molecular weight determined by mass spectrometry without pre-digestion or fragmentation) (Δ=-186Da, corresponding to Des-Asp'-Ala'-HSA (HSA lacking the first two N-terminal amino acid residues)). Preferably, N-terminal degradation of albumin is measured by mass spectrometry of albumin by electrospray mass spectrometry (ESI-MS) of the intact mass after desalting, or by any preferred technique known in the art and further described in the examples. Typically, the sample analyzed has an albumin concentration of 0.3 mg / mL or less. When measured by ESI-MS, the level of N-terminal degradation is typically expressed as a percentage of the most abundant peak (resulting in no N-terminal degradation), which is considered 100%.Preferably, in the presence of anions, the N-terminal degradation of albumin does not exceed 12.5% ​​when albumin is incubated at 25°C, and more preferably 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.75, 1.5, 1.25, 1, 0.75, 0.50, 0.25, or 0% or less, for example, 0, 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 , 1.8, 1.9, 1.95, 1.96, 1.97, 1.98, 1.99, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5% ~ 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 1.96, 1.97, 1.98, 1.99, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, or 12.5%. For example, if albumin is incubated at 25°C for a desired duration in the presence of anions, and ESI-MS is used to measure N-terminal degradation, the peak with the highest abundance (attributed to non-N-terminally degraded albumin) should be set to 100%, and all peaks attributable to N-terminally degraded albumin should be less than 12.5% ​​of the "100% peak". The measurement is a measure of intensity height, and "12.5%" corresponds to "12.5% ​​of the intensity of the highest peak".

[0358] A preferred formulation using the first aspect of the present invention contains 2 to 500 mM anions having multiple negative charges housed on a flexible skeleton, such as 10 to 500 mM anions having multiple negative charges housed on a flexible skeleton. The anion concentration may be about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to about 50, 55, 60, 65, 70, 75, 100, 200, 300, 400, or 500 mM. Anion concentrations of about 10 mM to about 100 mM or about 25 mM to about 100 mM are preferred, such as about 2 mM to about 60 mM, about 10 mM to about 60 mM, about 40 mM to about 60 mM, and particularly about 2 mM, about 10 mM, or about 50 mM. In one embodiment of this preferred formulation, the multiple negatively charged anions housed on the flexible skeleton are not one or both glutamic acid and ATP.

[0359] A formulation according to the first aspect of the present invention may or may not include a buffer in addition to non-aggregated albumin and a plurality of negatively charged anions housed on a flexible skeleton. Preferably, the buffer maintains or increases and / or does not decrease the stability of albumin in the presence of the anions. "Does not decrease the stability of albumin in the presence of the anions" means that, in the presence of the buffer, the stability of albumin in the presence of the anions is substantially less than the stability of albumin in the presence of the anions in the absence of the buffer. "Maintains or increases the stability of albumin in the presence of the anions" means that, in the presence of the buffer, the stability of albumin in the presence of the anions is the same as or greater than the stability of albumin in the presence of the anions in the absence of the buffer. The stability of albumin can be measured by any suitable technique known in the art, including those described above, such as techniques for evaluating aggregation and thermal stability. Typically, in the presence of a buffer, the stability of albumin in the formulation is 95% or more of the stability of albumin in the formulation in the absence of a buffer, and preferably, it is the same as or greater than the stability of albumin in the formulation in the absence of a buffer.

[0360] Buffer solutions may or may not be charged. As used herein, the term “charged” includes the standard chemical definition of the total charge of a molecule, i.e., the sum of the charges of all charged groups, e.g., carboxyl anions (-1) and protonated amines (+1) as measured at pH 7.4. The charge of each potentially charged group can be calculated at a given pH by referring to the pKa of each potentially charged group, according to methods well known in the art. The pKa of each potentially charged group can also be determined according to methods well known in the art.

[0361] In one embodiment, the buffer is positively charged. In an alternative embodiment, the buffer is negatively charged. In yet another embodiment, the buffer is uncharged.

[0362] It will be understood that the final pH of the formulation is affected by the presence of all components of the formulation, including albumin, multiple negatively charged anions housed on a flexible skeleton, buffer, and one or more other excipients described herein.

[0363] Considering that multiple negatively charged anions (e.g., citrates) housed on a flexible skeleton are already negatively charged, it may be desirable to have a positively charged buffer so that the final pH of the formulation is between 6 and 8, for example, about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9 to about 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0364] As described in the examples, the formulations according to the present invention were found to have a stabilizing effect on viruses over a pH range of at least 4 to 8. Therefore, in the embodiments, the buffer was used so that the final pH of the formulation was 4 to 8, for example, about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, The pH values ​​are selected to be approximately 7.7, 7.8, 7.9 to 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[0365] In one embodiment, the buffer has a pKa higher than the pH of the formulation, for example, so that the buffer is positively charged.

[0366] Particularly preferred forms of the buffer include tris, bis-tris, and / or bis-trispropane. Other buffers may or may not contain phosphate-buffered saline (PBS) and / or PBS + 0.001% poloxamer (Vigene Biosciences / Charles River Laboratories). Other buffers may or may not contain bis-trisphosphate (BTP) and / or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-sulfonic acid (HEPES). HEPES and BTP are preferred buffers that may be used. In one embodiment, if such a buffer is present, preferably the multiple negatively charged anions housed on a flexible backbone are not one or both glutamic acid and ATP.

[0367] If a buffer is present, a preferred formulation contains a buffer of 0.1 mM to 250 mM, such as 1 to 250 mM. The buffer concentration may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM to about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 250 mM. A buffer concentration of about 10 mM to about 200 mM, particularly about 150 mM, is preferred, and in particular, the buffer is Tris. If the buffer is Tris, the buffer may be present at a concentration of about 0.1 to about 50 mM, preferably 50 mM, such as about 30 to about 50 mM. If the buffer is BTP or HEPES, the buffer may be present at a concentration of about 0.1 to about 50 mM, preferably about 30 mM, such as about 30 to about 50 mM. In one embodiment, when such a buffer is present, the multiple negatively charged anions housed on the flexible skeleton are not one or both of glutamic acid and ATP.

[0368] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 50 mM magnesium chloride. For example, the formulation may contain about 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 mM to about 20, 25, 35, 40, 45, or 50 mM magnesium chloride. The formulation may contain about 0 mM to about 10 mM magnesium chloride, such as about 1 mM to about 10 mM, or about 2 mM to about 10 mM magnesium chloride, such as about 5 mM to about 10 mM. Magnesium chloride concentrations of about 5 to about 10 mM or about 15 mM to about 35 mM are preferred, and magnesium chloride concentrations of about 5 mM, about 10 mM, or about 25 mM are particularly preferred. In one embodiment, when magnesium chloride is present, the multiple negatively charged anions housed on the flexible skeleton are not one or both glutamic acid and ATP.

[0369] In embodiments, the formulations used in the first aspect of the present invention contain sulfates ranging from 0 to 500 mM, such as 0 to 200 mM, including sulfates selected from sodium sulfate, sodium bisulfate, sodium thiosulfate, ammonium sulfate, ammonium bisulfate, magnesium sulfate, magnesium bisulfate, potassium sulfate, and potassium bisulfate. For example, the formulations may contain sulfates ranging from about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 475 to about 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 475, and 500 mM. A sulfate concentration of approximately 50 mM is preferred, or a sulfate concentration of approximately 200 mM is preferred. Particularly preferred concentrations of sulfate include sodium sulfate in concentrations of approximately 25 to 500 mM, such as approximately 50 to 200 mM (e.g., approximately 200 mM or approximately 50 mM); ammonium sulfate in concentrations of approximately 25 to 500 mM, such as approximately 50 to 200 mM (e.g., approximately 200 mM or approximately 50 mM); sodium thiosulfate in concentrations of approximately 25 to 500 mM, such as approximately 50 to 200 mM (e.g., approximately 200 mM or approximately 50 mM); sodium bicarbonate in concentrations of approximately 25 to 500 mM, such as approximately 50 to 200 mM (e.g., approximately 200 mM or approximately 50 mM); and magnesium sulfate in concentrations of approximately 25 to 500 mM, such as approximately 50 to 200 mM (e.g., approximately 200 mM or approximately 50 mM). In one embodiment, when sulfate is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or neither.

[0370] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 500 mM disodium phosphate (Na2HPO4), such as 0 to 200 mM. For example, the formulation may contain approximately 0, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 to approximately 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 500 mM disodium phosphate. Particularly preferred concentrations of disodium phosphate include about 10 to 500 mM, such as about 10 to 250 mM (e.g., about 10 mM or about 200 mM). A disodium phosphate concentration of about 10 mM or about 200 mM is preferred. In one embodiment, when disodium phosphate is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0371] As further discussed in the examples, albumin has been found to confer stability to the formulation even in the presence of EDTA. Therefore, in embodiments, the formulation in use of the first aspect of the present invention contains 0 to 50 mM EDTA. For example, the formulation may contain about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 mM EDTA. An EDTA concentration of about 1 mM is preferred. In one embodiment, when EDTA is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0372] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 200 mM glutamic acid. For example, the formulation may contain about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 to about 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mM glutamic acid. A glutamic acid concentration of about 50 mM is preferred. If glutamic acid is present in the formulation, it is preferable that the formulation includes one or more additional components in addition to albumin, a plurality of negatively charged anions housed on a flexible skeleton, and optionally a buffer. In one embodiment, if glutamic acid is present, the plurality of negatively charged anions housed on a flexible skeleton are not ATP.

[0373] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 500 mM mannitol. For example, the formulation may contain mannitol in concentrations of about 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 to about 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mM. A mannitol concentration of about 300 mM is preferred. If mannitol is present in the formulation, the formulation preferably contains albumin, a plurality of negatively charged anions housed on a flexible skeleton, and optionally a buffer, plus one or more additional components. In one embodiment, if mannitol is present, the plurality of negatively charged anions housed on a flexible skeleton are not glutamic acid and ATP, or both.

[0374] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 500 mM sucrose. For example, the formulation may contain sucrose in concentrations of about 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 to about 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mM. A sucrose concentration of about 300 mM is preferred. If sucrose is present in the formulation, the formulation preferably contains albumin, a plurality of negatively charged anions housed on a flexible skeleton, and optionally a buffer, plus one or more additional components. In one embodiment, if sucrose is present, the plurality of negatively charged anions housed on a flexible skeleton are not glutamic acid and ATP, or both.

[0375] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 500 mM arginine, such as 0 to 200 mM. For example, the formulation may contain approximately 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 to approximately 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 It may contain arginine in concentrations of 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500 mM. Arginine concentrations of about 25 mM, about 50 mM, or about 200 mM are preferred. In one embodiment, when arginine is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or neither.

[0376] In embodiments, the formulation in use of the first aspect of the present invention contains chloride salts in concentrations of 0 to 500 mM, such as 0 to 200 mM. For example, the formulation may contain chloride salts in concentrations of about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 to about 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 mM. Preferred concentrations of chloride salts include approximately 25 to 500 mM, such as approximately 50 to 200 mM. Chloride salt concentrations of approximately 50 mM or approximately 200 mM are particularly preferred. Therefore, in embodiments, the formulation used in the first aspect of the present invention contains 0 to 500 mM sodium chloride, such as 0 to 200 mM. For example, the formulation may contain sodium chloride in concentrations of approximately 0, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 to approximately 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500 mM. Preferred concentrations of sodium chloride include approximately 25 to 500 mM, such as approximately 25 to 250 mM, such as approximately 50 to 200 mM. A sodium chloride concentration of approximately 50 to 200 mM is particularly preferred, and more preferably about 200 mM. In one embodiment, when a chloride salt is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or both.

[0377] In embodiments, the formulation used in the first aspect of the present invention contains 0 to 200 mM histidine. For example, the formulation may contain histidine in concentrations of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 to about 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mM. If present, a histidine concentration of about 50 mM is preferred. However, in another preferred embodiment, the formulation does not contain histidine. In one embodiment, if histidine is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0378] In some embodiments, the formulations used in the first aspect of the present invention contain 0 to 200 mM glycine. For example, the formulations may contain glycine in concentrations of about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 to about 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mM. If present, a glycine concentration of about 50 mM or about 200 mM is preferred. However, in another preferred embodiment, the formulation does not contain glycine. In one embodiment, if glycine is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0379] In some embodiments, the formulation in use of the first aspect of the present invention contains 0 to 200 mM glutamate. For example, the formulation may contain about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 to about 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mM glutamate. If present, a glutamate concentration of about 50 mM is preferred. However, in another preferred embodiment, the formulation does not contain glutamate. In one embodiment, if glutamate is present, the multiple negatively charged anions housed on a flexible skeleton are not ATP.

[0380] In embodiments, the formulation in use of the first aspect of the present invention contains 0 to 200 mM aspartate. For example, the formulation may contain about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 to about 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mM aspartate. An aspartate concentration of about 50 mM is preferred. In one embodiment, when aspartate is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0381] In the embodiments, the formulation used in the first aspect of the present invention contains 0 to 0.01% (w / v) of a nonionic surfactant, such as a nonionic surfactant selected from poloxamers (Poloxamer 188, P188, MST-188) such as Pluronic® F-68 (https: / / www.selleckchem.com / products / pluronic-f-68.html, Guler et al, 2017) or polysorbates. Preferably, the polysorbate is polysorbate 20 or polysorbate 80. For example, the formulation may contain a nonionic surfactant in concentrations of approximately 0, 0.00025, 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, or 0.007 to approximately 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, or 0.01% (w / v). A nonionic surfactant concentration of approximately 0.001% or approximately 0.01% (w / v) is preferred. In one embodiment, when a nonionic surfactant is present, the multiple negatively charged anions housed on the flexible skeleton are not glutamic acid and ATP, or either one or both.

[0382] In the embodiments, the formulation used in the first embodiment of the present invention has a pH of approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 to approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. A pH of approximately 6.0 to approximately 8.0 is preferred, preferably approximately 5.0 to approximately 7.5, such as approximately 5.5 to approximately 6.5, or preferably approximately 6.0 or approximately 6.5. In another preferred embodiment, the pH of the formulation is 6.8. In an alternative preferred embodiment, a pH of approximately 4.0 to approximately 5.0 is preferred. It will be understood that one advantage of adding or including albumin is that it allows for stability in a more commercially acceptable or physiologically relevant pH range. In embodiments in which the formulation has such a pH, the multiple negatively charged anions housed on the flexible skeleton are not one or both of glutamic acid and ATP.

[0383] In preferred embodiments, the formulation for use in the first aspect of the present invention contains "good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 150 mM), Citrate (for example, 2-100 mM or 25-100 mM, preferably 40-60 mM, more preferably about 50 mM), and It contains magnesium chloride (for example, 1 to 10 mM or 15 to 35 mM, preferably 25 mM), It has a pH of 5 to 7, preferably about 6 to 6.5.

[0384] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 150 mM), Citrate (for example, 2-100 mM or 25-100 mM, preferably 40-60 mM, more preferably about 50 mM), Magnesium chloride (for example, 1-10 mM or 15-35 mM, preferably 25 mM), It contains sodium sulfate (for example, 25-75 mM or 25-74 mM, preferably about 50 mM), It has a pH of 5 to 7, preferably about 6 to 6.5.

[0385] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, or about 5 mg / mL), Citrate (for example, 2-100 mM or 25-100 mM, preferably 40-60 mM, more preferably about 50 mM), It contains magnesium chloride (for example, 1 to 10 mM or 15 to 35 mM, preferably 25 mM), It has a pH of 5 to 7, preferably about 6 to 6.5.

[0386] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 30-50 mM, more preferably about 50 mM), Citrate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0387] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 30-50 mM, more preferably about 30 mM), Oxaloacetate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0388] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 30-50 mM, more preferably about 30 mM), Fumarate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0389] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 30-50 mM, more preferably about 30 mM), succinate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0390] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), Tris (for example, 10-200 mM, preferably about 30-50 mM, more preferably about 30 mM), Maleic acid (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0391] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), BTP (for example, 10-200 mM, preferably about 30-50 mM, more preferably 30 mM), Citrate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0392] In another preferred embodiment, a formulation for use in the first aspect of the present invention is: "Good" albumin (e.g., 0.4 to 300 mg / mL, preferably 0.5 to 50 mg / mL, more preferably 1 to 20 mg / mL, even more preferably about 1 mg / mL, about 2.5 mg / mL, about 5 mg / mL), HEPES (for example, 10-200 mM, preferably about 30-50 mM, more preferably 30 mM), Citrate (for example, 2 to 100 mM, preferably about 10 mM), and It contains magnesium chloride (for example, 1 to 10 mM, preferably 5 mM, such as 2 to 10 mM), (Having a pH of 4 to 8, preferably about 6.5 to 7, more preferably about 6.8, such as 5 to 7.5).

[0393] "Stabilizing the virus" means that when the virus comes into contact with the formulations described herein, the stability of the virus increases compared to the stability of the virus in the absence of the formulations (e.g., a formulation that does not contain albumin and / or does not contain multiple negatively charged anions housed on a flexible skeleton, but is otherwise identical or substantially identical). In other words, if the stability of the virus is evaluated under specific conditions, contact with the formulations described herein should increase the stability of the virus when evaluated under the same specific conditions. The conditions selected may be any of the "stress conditions" described in the Examples, e.g., a freeze-thaw cycle, temperature incubation (e.g., at a set temperature (e.g., 40°C or 60°C) over a set period (e.g., 2 weeks, 1 month, or 2 months), and storage or incubation for 2 months at a temperature of 2 to 8°C, e.g., about 4°C, such as 2, 3, 4, 5, 6, or 7°C). The freeze-thaw cycle may be at a set period (e.g., The freeze-thaw stress applied to the virus may involve storing it at a temperature of approximately -20°C for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, followed by thawing (for example, at room temperature (15-25°C, preferably 20°C) for a set period such as at least 3, 4, 5, 6, 7, or 8 hours).

[0394] Typically, increased viral stability is manifested by a reduction in viral aggregation over a given period. Therefore, upon contact with the formulations described herein, the virus exhibits a reduction in aggregation compared to the level of viral aggregation in the absence of the formulation, but under otherwise identical conditions. The level of viral aggregation can be evaluated using any suitable technique within the art, as described below and in the examples.

[0395] In one embodiment, the increased stability of the virus is revealed, for example, by evaluating the hydrodynamic radius of the virus-containing formulation (e.g., by DLS) when the formulation is incubated at 25°C or 40°C, preferably 40°C, for a duration of at least 20 hours, or when the formulation undergoes multiple freeze-thaw cycles (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 freeze-thaw cycles). The specific hydrodynamic radius depends on the virus present, but the more stable the virus, the less variation there will be in the hydrodynamic radius (e.g., measured by DLS) over the duration of the study. Therefore, in further embodiments, the increased stability of the virus is evident by the fact that the hydrodynamic radius of the virus-containing formulation is no more than 6 times (preferably 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.5 times or less) of the hydrodynamic radius of the virus in monomer form after, for example, freeze-thaw stress (e.g., subjecting the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storing at a temperature of approximately -20°C for a set period (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) and then thawing (e.g., subjecting the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storing at a temperature of approximately -20°C for a set period (e.g., at least 6, 7, 8, 9, 10, 10, 10, 10, 10, 10, 10, 15, 5, 6, 7, or 10, 15 to 25°C, preferably 20°C)). A preferred free-thaw cycle involves storage at approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours. The monomeric form of the virus includes the meaning of a solution of the virus that has not undergone any of the “stress conditions” described herein, e.g., a freeze-thaw cycle, temperature incubation (e.g., at a set temperature (e.g., 40°C or 60°C) for a set period (e.g., 2 weeks, 1 month, or 2 months), and storage or incubation at a temperature of 2 to 8°C, such as 4°C for 2 months, e.g., 3, 4, 5, 6, or 7°C).The monomeric form of the virus can be prepared by providing a stock solution of the virus that can be diluted to evaluate its hydrodynamic radius. It will be understood that the monomeric form of the virus represents a monodisperse system.

[0396] Variations in the hydrodynamic radius can be evaluated by determining the coefficient of variation. For example, a virus can be stabilized such that the coefficient of variation of the hydrodynamic radius of a virus-containing formulation is 5% or less when the formulation is incubated at 40°C for a duration of at least 20 hours. For formulations containing multiple components, it will be understood that additional analysis, such as the method described in Example 14, which can be used to provide the mass recovery rate of the virus under stability test conditions, may be required to calculate the hydrodynamic radius. Therefore, in one embodiment, the increased stability is evident over the duration of the study, for example, by a mass recovery rate of at least 50% of the virus, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, after freeze-thaw stress (e.g., subjecting the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storing at a temperature of approximately -20°C for a set period (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) and then thawing (e.g., subjecting it to 1, 2, 3, 4, 5, 6, 7, or 8 hours at room temperature (15-25°C, 20°C is preferred)). Preferably, the increased stability is evident by a mass recovery rate of at least 50% of the virus, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, after 10 freeze-thaw cycles (for example, the formulation is frozen for a set period of about 6 hours at about -20°C, then frozen again at about -20°C, and thawed for a set period of about 3 hours at room temperature before repeating the cycle).

[0397] Similarly, in another embodiment, the increased stability is evident over the duration of the study by a higher viral mass recovery rate than the viral mass recovery rate in the absence of the formulation described herein (e.g., in the absence of the formulation used to increase viral stability according to the first aspect of the present invention) after freeze-thaw stress (e.g., subjecting the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storing at a temperature of approximately -20°C for a set period (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) followed by thawing (e.g., subjecting the formulation to 1, 2, 3, 4, 5, 6, 7, or 8 hours at room temperature (15-25°C, 20°C preferred)). For example, the mass recovery rate of the virus may increase or improve by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 percentage points compared to the mass recovery rate of the virus in the absence of the formulation after at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percentage points over the duration of the study, for example, after at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percentage points compared to the mass recovery rate of the virus in the absence of the formulation. The mass recovery rate of the virus in the absence of the formulation described herein will be understood to be typically the mass recovery rate of the virus in a formulation that does not contain albumin and / or does not contain multiple negatively charged anions housed on a flexible skeleton, but is otherwise identical or substantially identical. The mass recovery rate may be measured by the DLS described herein.

[0398] Another method for evaluating viral aggregation is by particle coverage. Thus, in another embodiment, increased stability is evident by particle coverage %, which is less than 15%, more preferably 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 0.5%, preferably about 5% or less. Preferably, particle coverage of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 0.5% is observed during storage or incubation at temperatures of 2 to 8°C, such as about 4°C, including 2, 3, 4, 5, 6, or 7°C, over a duration of at least 1 month, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1 year. Alternatively, particle coverage rates of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% are observed at a maximum storage temperature of 25°C over a duration of at least 2 weeks to 2 months, preferably 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, or 2 months. Alternatively, particle coverage rates of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% are observed when the formulation has undergone multiple freeze-thaw cycles (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 freeze-thaw cycles). Particle coverage rates may be measured by background membrane imaging (BMI) using bright-field microscopy to image a 50 μL (microliter) formulation containing 5 mg / mL and approximately E+13 viral particles (vp) / mL. One technique that may be used is the Aura system from Halo Labs, as described in Examples 1 and 15. "Particle coverage" refers to the percentage of membrane coverage in BMI measurements and is related to the number of aggregates.

[0399] For example, it will be understood that it may be desirable to express the "particle count" data as an improvement factor or value relative to the "particle count" of a control solution or stock solution of the virus (e.g., a solution of the virus in the absence of albumin and / or multiple negatively charged anions contained on a flexible skeleton), using the calculation "number of raw material virus particles" / "number of sample particles". Therefore, in one embodiment, the increased stability is evident from the result of the calculation "number of raw material virus particles" / "number of sample particles" greater than 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or at least 150, 200, 250, or 300. In other words, the increased stability may be evident from the number of particles in the virus-containing formulation being less than the number of particles in the control solution of the virus (e.g., a solution of the virus in the absence of albumin and / or multiple negatively charged anions housed on a flexible skeleton) after freeze-thaw stress (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles of storage at a temperature of approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours).

[0400] In yet another embodiment, the increased stability is evident from the particle count of fewer than 60,000 particles, preferably fewer than 60,000, 55,000, 50,000, 45,000, 40,000, 35,000, 30,000, or 25,000 particles, for storage or incubation at temperatures of 2 to 8°C, such as 3, 4, 5, 6, 7, or 8°C, for example, about 4°C, and the estimated circular diameter (ECD) of the particle size is 2 μm (microns) or less. Preferably, the storage time is at least 1.5, 2, 3, 4, or 5 months to at least 6, 6.5, 7, 8, 9, 10, 11, or 12 months. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. "Particle count" refers to the number of particles per 1 mL. The particle count can be calculated by counting the number of particles with an ECD of ≥ 2 μm, which are measured by background membrane imaging (BMI) using bright-field microscopy.

[0401] Alternatively, the particle count is less than 200,000 particles after four freeze-thaw cycles, preferably less than 200,000, 150,000, 100,000, or 50,000 particles, and the estimated circular diameter (ECD) of the particle size is 2 μm or less. A "freeze-thaw cycle" includes a cycle in which the formulation is frozen at approximately -20°C for a set period (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours, or at least 19 hours, or at least 20, 21, 23, or 24 hours), frozen again at approximately -20°C, and thawed at room temperature for a set period (e.g., at least 3 or 4 hours, or at least 5 hours) before repeating the cycle. The number of particles can be calculated by counting the number of particles with an ECD of ≥ 2 μm measured in bright-field light and subtracting the side illumination membrane intensity (SIMI) for particles within the same size range measured by bright-field SIMI.

[0402] In another embodiment, the increased stability is evident by the number of particles less than 15,000, preferably less than 15,000, 12,500, 10,000, 7,500, 5,000, 2,500, or 1,000, for storage or incubation at temperatures of 2 to 8°C, such as 3, 4, 5, 6, 7, or 8°C, for at least 1 month to at least 12 months, with an estimated circle diameter (ECD) of 2 μm or less, and the particles being thioflavin T positive. Preferably, the storage time is at least 1.5, 2, 3, 4, or 5 months to at least 6, 6.5, 7, 8, 9, 10, 11, or 12 months. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The number of particles can be calculated by counting the number of particles having an ECD of ≥ 2 μm, i.e., particles that fluoresce when stained with the protein stain thioflavin T (ThT), which are measured by background membrane imaging (BMI) using bright-field and fluorescence microscopy.

[0403] As will be further discussed in the examples, particularly Example 16, it will be understood that (i) the mass recovery rate of the virus measured by DLS, for example, and (ii) the number of particles measured by Halo analysis relative to the number of particles in the undiluted virus solution can be combined to provide an overall rank of the stabilizing effect of any given formulation against the virus. Formulations with a high mass recovery rate and a high normalized particle count may be considered very stable, while formulations with a low mass recovery rate and a low normalized particle count may be considered less stable. Formulations with a high mass recovery rate and a low normalized particle count may still be considered to provide a stabilizing effect against the virus. Formulations with a low mass recovery rate and a high normalized particle count require judgment by considering the overall results of the hydrodynamic radius, the mass fraction recovered, and the aggregation behavior.

[0404] In another embodiment, the increased stability is evident in the preservation of viral integrity.

[0405] In another embodiment, the increased stability is manifested by a virus that retains the ability to bind to a specific binding partner (e.g., a monoclonal antibody) in the presence of the formulation. Binding can be evaluated using an enzyme-linked immunosorbent assay (ELISA). “A virus that retains the ability to bind to a specific binding partner in the presence of the formulation” means that, in the presence of the formulation, the virus is more likely to retain its binding ability to a specific binding partner compared to its binding ability in the absence of the formulation. If the virus contains a capsid protein, it will be understood that the increased stability may be manifested by a capsid protein that retains the ability to bind to a specific binding partner, such as an antibody that selectively binds to an epitope on the capsid. Such retention in binding ability can be evaluated using commercially available ELISA kits. For example, Progen manufactures a commercially available ELISA kit (https: / / www.progen.com / AAV / AAV-ELISA / ) that can be used to evaluate antibody binding to AAV capsids, in which monoclonal antibodies specific to conformational epitopes on aggregated AAV capsids are coated onto microtiter strips and used to capture AAV particles from a sample.

[0406] In another embodiment, the increased stability is manifested by a virus that retains its ability to infect cells in the presence of the formulation. Cell infection can be assessed using an infectivity assay (TU assay) or any other technique used in the art. "A virus that retains its ability to infect cells in the presence of the formulation" means that, in the presence of the formulation, the virus is more likely to retain its infectivity than its infectivity in the absence of the formulation.

[0407] As used herein, the term “virus” includes viruses, viral particles, viral fragments, virus-like particles (VLPs), viral vectors, or vector viruses. As used herein, the terms “viral particle” and “virus-like particle” refer to a non-replicating viral shell derived from any of several viruses further discussed below. Viral particles and VLPs generally consist of one or more viral proteins, or particle-forming polypeptides derived from these proteins, including but not limited to proteins referred to as capsid, coat, shell, surface, and / or envelope proteins. In addition, they may or may not contain additional genetic material. The term “viral particle” refers to an entire virus that is infectious unless attenuated or inactivated. VLPs are non-infectious particles. As used herein, the term “viral fragment” refers to a part or portion of a virus or viral particle, preferably non-replicating. As used herein, the terms “viral vector” and “vector virus” refer to a carrier (e.g., a modified virus) used to deliver genetic material into a cell. Viral vectors utilize mechanisms evolved by viruses to efficiently transport their genomes into the cells they infect. Viral vectors and vector viruses are commonly used in basic research, gene therapy, and vaccine development.

[0408] In one embodiment, the virus may be a live virus, a weakened virus, a live weakened virus, or an inactivated virus.

[0409] In another embodiment, the virus may be a vector virus or a virus-like particle.

[0410] In another embodiment, the virus is in the form of a vaccine, and preferably the vaccine is a virus-based vaccine.

[0411] In another embodiment, the virus is a hybrid virus or a chimeric virus. As used herein, the terms “chimeric virus” or “hybrid virus” refer to a virus in which functional nucleic acids or amino acid sequences, preferably amino acid sequences, from two or more distinctly different viruses are combined to form a viable organism.

[0412] In preferred embodiments, the virus, preferably Simian virus, may be a parvovirus (e.g., adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV)), adenoviridae (e.g., adenovirus), herpesviridae (e.g., herpes simplex virus), rhabdoviridae (e.g., vesicular stomatitis virus or maraba virus), poxviridae (e.g., vaccinia virus), paramyxoviridae (e.g., measles virus or Newcastle disease virus), reoviridae (e.g., rotavirus or reovirus), picornavirus (e.g., poliovirus type I, coxsackievirus A21, or Seneca Valley virus), flavivirus (e.g., dengue virus, yellow fever virus, West Nile virus, or Zika virus), togaviridae (e.g., alphavirus), or coronavirusidae (e.g., coronavirus). The term "Simian" includes monkeys, apes, and humans.

[0413] In one embodiment, AAV or rAAV may be AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, AAV-PHP.S, preferably AAV2, AAV9, AAV8, or AAV5.

[0414] In a preferred embodiment, the virus is 1 × 10 6 ~1 × 1024 It can be found at concentrations of vg / ml. The viral concentration is approximately 1 × 10⁻⁶. 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 or 1 × 10 12 ~Approx. 1×10 18 , 1 x 10 19 , 1 x 10 20 , 1 x 10 21 , 1 x 10 22 , 1 x 10 23 , 1 x 10 24 It is possible. Approximately 1 × 10 11 ~Approx. 1×10 13 A certain virus concentration is preferred. For example, it will be understood that it may be desirable to vary the concentration depending on the purpose of the formulation, its delivery route to the target, and the properties of the virus.

[0415] A second aspect of the present invention provides a formulation comprising albumin, which is substantially in an agglutinated form, and a plurality of negatively charged anions housed on a flexible skeleton.

[0416] In this embodiment, the formulation is a liquid formulation.

[0417] The preference for albumin and anions includes those described above with respect to the first aspect of the present invention. The preference for the pH of the formulation includes those described above with respect to the first aspect of the present invention.

[0418] For example, in an embodiment, a formulation according to a second aspect of the present invention contains 0.4 to 300 mg / mL of albumin, such as 0.5 to 300 mg / mL. For example, a formulation may contain about 75, 100, 125, 150, 175, 200, 225, 250, or 275 mg / mL of albumin. In a particular example, a formulation may contain about 50 to 300 mg / mL of albumin, such as about 100 to 200 mg / mL. If a formulation according to a second aspect of the present invention has an albumin concentration of about 50 to 300 mg / mL, it will be understood that it may constitute a stock solution. Before using the formulation, it may be desirable to dilute the stock solution to have an albumin concentration of approximately 0.4 to 50 mg / mL, such as 0.5 to 50 mg / mL, to stabilize the virus according to the first aspect of the present invention.

[0419] In another embodiment, a formulation according to a second aspect of the present invention contains 0.4 to 50 mg / mL of albumin, such as 0.5 to 50 mg / mL. For example, the formulation may contain about 0.4, 0.5, 1, 2, 5, 5, 10, 15, 20, 25, 30, 35, or 40 to about 25, 30, 35, 40, 45, or 50 mg / mL of albumin. An albumin concentration of about 1 to 20 mg / mL is preferred, and albumin concentrations of about 1 mg / mL, about 2.5 mg / mL, or about 5 mg / mL are particularly preferred.

[0420] The formulation may be supplied in a rigid or flexible vial, bottle, or bag, such as a bioprocess container (BPC). Suitable container volumes range from approximately 50 mL to approximately 10,000 mL, for example, approximately 50, 100, 250, 500, 750, 1000, 2500, 5000 mL to approximately 100, 250, 500, 750, 1000, 2500, 5000, 10000 mL, for example, 50 mL, 1000 mL, 5000 mL, and 10000 mL. The container preferably includes one or more (e.g., several) inlets or outlets to allow for filling and / or dispensing from the container. The formulation may be sterilized, for example, before or after filling the container. The formulation may or may not be supplied in unit dosage forms.

[0421] A formulation according to a second aspect of the present invention may or may not contain a buffer in addition to non-aggregated albumin and a plurality of negatively charged anions housed on a flexible skeleton. Suitable buffers and their concentrations are those described above with respect to the first aspect of the present invention. Particularly preferred forms of the buffer include Tris, bis-Tris, and / or bis-Trispropane. Other preferred buffers include BTP and / or HEPES.

[0422] Preferably, the buffer has a pKa higher than the pH of the formulation, such that the buffer is positively charged, for example, net positively charged. In this way, the binding between the anion (e.g., citrate) and albumin is not weakened.

[0423] It will be understood that a formulation according to a second aspect of the present invention may further contain one or more of the following components described in relation to the first aspect of the present invention: sulfate, sodium chloride, disodium phosphate, EDTA, glutamic acid, mannitol, sucrose, arginine, chloride salt, histidine, glycine, glutamate, aspartate, and a nonionic surfactant. Preferred examples and appropriate concentrations of these components are those described above.

[0424] Although not constrained by theory, the constituent components may act as osmolites for aqueous preparations of viral vectors, and are expected to provide stability by inducing protein folding and reducing denaturation. Furthermore, the use of stabilizing salts may enhance hydrophobic interactions by increasing surface tension at the water-protein interface, keeping hydrophobic groups away from water molecules, and inducing preferential hydration of the protein.

[0425] In one embodiment, two or more components are provided in combination. Preferred combinations include sodium sulfate and arginine, sodium sulfate and histidine, sodium sulfate and EDTA, sodium sulfate and pluronic acid, disodium phosphate and glutamic acid, disodium phosphate and mannitol, sodium chloride and arginine, sodium chloride and histidine, sodium chloride and EDTA, sodium sulfate, arginine, and pluronic acid.

[0426] A formulation according to a second aspect of the present invention may or may not contain a virus in addition to non-aggregated albumin and a plurality of negatively charged anions housed on a flexible skeleton. Suitable viruses include any of those described above with respect to the first aspect of the present invention. If the formulation contains a virus (e.g., one to be stabilized), it is preferable that albumin be present at a concentration of 0.4 to 50 mg / mL, such as 0.5 to 50 mg / mL, such as 1 to 20 mg / mL, more preferably about 1 mg / mL, about 2.5 mg / mL, or about 5 mg / mL.

[0427] A third aspect of the present invention is: (i) Albumin, which is substantially in an unaggregated form, (ii) A kit of components comprising a plurality of negatively charged anions housed on a flexible skeleton.

[0428] The preference for albumin and anions includes those described above with respect to the first aspect of the present invention.

[0429] It will be understood that components (i) and (ii) of the kit may be provided in separate containers to allow for further processing of one or both of the components before they are mixed and used for any purpose (e.g., to stabilize a virus). For example, it may be desirable that one or both components be provided in a concentrated form (e.g., as a stock solution) that can be diluted before or when they are added to the other component, before use in any purpose. Additionally or alternatively, it may be desirable that one or more other components be added to one or both components before they are mixed and ready for use.

[0430] Preferably, the albumin component (i) of the kit may be provided in a concentrated form (e.g., as a stock solution) that can be diluted before or at the time of addition to the multiple negatively charged anions housed on a flexible skeleton. It will be understood that any of the albumin compositions (a) to (d) described above may constitute such a stock solution. In one example, the stock solution of the albumin component (i) of the kit may be a 20- to 40-fold stock solution. Typically, the stock solution contains 50 to 300 mg / mL of albumin. For example, the stock solution may contain about 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275 mg / mL of albumin to about 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg / mL of albumin. In certain cases, the stock solution may contain approximately 100–200 mg / mL of albumin. It will then be understood that the stock solution may be diluted to have an albumin concentration of approximately 0.4–50 mg / mL, for example, 0.5–50 mg / mL, and mixed with the anionic component (ii) of the kit before use in any application (e.g., to stabilize a virus).

[0431] Preferably, the anionic component (ii) of the kit may be provided in a concentrated form (e.g., as a stock solution) that can be diluted before or at the time of addition to albumin. In one example, the stock solution of the anionic component (ii) of the kit may be approximately 2 to 10 times stock solution, such as approximately 5 times stock solution.

[0432] A 2x concentrate means a solution that is twice as concentrated as the working concentration for any application (e.g., to stabilize a virus), a 10x concentrate means a solution that is ten times as concentrated as the working concentration for any application (e.g., to stabilize a virus), and the same applies to 20x and 40x concentrates. If the solution contains more than one component, for example, a 2x concentrate is preferably twice as concentrated as the working concentration for each of these components, and the same applies to 10x, 20x, and 40x concentrates. "Working concentration" means a concentration that is suitable for any application in which the solution is used. For example, if the application is to stabilize a virus, the working concentrations of various components (e.g., buffers from substantially non-aggregated albumin, multiple negatively charged anions housed on a flexible skeleton, sulfates, sodium chloride, disodium phosphate, EDTA, glutamic acid, mannitol, sucrose, arginine, chloride, histidine, glycine, glutamate, aspartate, and nonionic surfactants) include those described above with respect to the first aspect of the present invention.

[0433] It will be understood that the components of a kit according to a third aspect of the present invention may be mixed together to provide a formulation to be used according to a first aspect of the present invention. Preferably, when the components of the kit are mixed, the resulting formulation has a pH of approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 to approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. A pH of approximately 6.0 to approximately 8.0 is preferred, such as greater than 5.0 to approximately 7.5, approximately 5.5 to approximately 6.5, approximately 5.5 to approximately 7.0, or preferably approximately 6.0 or approximately 6.5. In an alternative embodiment, a pH of approximately 4.0 to approximately 5.0 is preferred.

[0434] It will be understood that a kit according to a third aspect of the present invention may further include a buffer in addition to albumin and a plurality of negatively charged anions housed on a flexible skeleton. Preferred buffers and their concentrations are any of those described above with respect to the first aspect of the present invention. Particularly preferred forms of the buffer include Tris, bis-Tris, and / or bis-Trispropane. Other buffers may or may not include phosphate-buffered saline (PBS) and / or PBS + 0.001% poloxamer (Vigene biosciences). Other buffers may or may not include BTP and / or HEPES. Preferred buffers include BTP and HEPES.

[0435] Preferably, the buffer solution has a pKa higher than the pH of the albumin component (i) and / or anionic component (ii) of the kit. When components (i) and (ii) of the kit are mixed, the resulting formulation is as described above, i.e., approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6. It will be understood that selections may be made to ensure that the pH is 3, 6.4, 6.5 to approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0436] For convenience, the buffer may be used to dilute the albumin component (i) and / or anionic component (ii) of the kit (when the kit components are provided as stock solutions) before use for any purpose (e.g., to stabilize the virus).

[0437] The buffer solution may be provided in a separate container from the respective containers containing components (i) and (ii) of the kit, or it may be in the same container as one or both of the components.

[0438] In a preferred embodiment, the anionic component (ii) of the kit also contains a buffer. For example, the kit may include: (i) Albumin, which is substantially in a non-aggregated form and preferably in the form of the stock solution (for example, a stock solution diluted about 20 to 40 times), containing, for example, 50 to 300 mg / mL, more preferably 100 to 200 mg / mL, (ii) Preferably, the solution comprises a plurality of negatively charged anions housed on a flexible skeleton and a buffer (e.g., one or more of Tris, bis-Tris, bis-Trispropane, BTP, and / or HEPES) in the form of a stock solution (e.g., a stock solution approximately 2 to 10 times dilution, such as a 5-fold stock solution).

[0439] It will be further understood that a kit according to a third aspect of the present invention may further comprise one or more of the following components described with respect to the formulations of the first and second aspects of the present invention: sulfate, sodium chloride, disodium phosphate, EDTA, glutamic acid, mannitol, sucrose, arginine, chloride salt, histidine, glycine, glutamate, aspartate, and nonionic surfactant. Preferred examples and appropriate concentrations of these components are those described above.

[0440] It will be understood that one or more such components may be provided in one or more containers separate from the respective containers that contain components (i) and (ii) of the kit, or they may be in the same container that contains one of these components (i) and (ii) of the kit.

[0441] If the kit further includes a buffer solution, and the buffer solution is provided in a separate container from the kit components (i) and (ii), it will be understood that one or more such components may be provided in one or more containers separate from the respective containers containing the kit components (i) and (ii), and the container containing the buffer solution, or they may be in the same container as the one containing one of the kit components (i) and (ii), or the same container as the one containing the buffer solution.

[0442] If the kit further includes a buffer, and the buffer is provided in the same container as one or both of components (i) and (ii) of the kit, it will be understood that one or more such components may be provided in one or more containers separate from the respective containers containing components (i) and (ii) of the kit, or in the same container as the one containing one or both of components (i) and (ii) of the kit.

[0443] Therefore, in a preferred embodiment, if the anionic component (ii) of the kit also contains a buffer solution, the kit is (i) Albumin, which is substantially in a non-aggregated form and preferably in the form of the stock solution (for example, a stock solution diluted about 20 to 40 times), containing, for example, 50 to 300 mg / mL, more preferably 100 to 200 mg / mL, (ii) Preferably, the solution may include, in stock form (e.g., a 2x to 10x solution such as a 5x stock), a plurality of negatively charged anions housed on a flexible skeleton, a buffer (e.g., one or more of Tris, bis-Tris, bis-Trispropane, BTP, and / or HEPES), and one or more components selected from sulfates, glutamic acid, mannitol, sucrose, arginine, chloride salts (e.g., sodium chloride), histidine, glycine, glutamate, aspartate, and nonionic surfactants, glutamic acid, mannitol, and sucrose. A 2x stock of component (ii) means that the solution is 2x concentrated with respect to the anions, buffer, and one or more components than the working concentrations of the anions, buffer, and one or more components for any application (e.g., for stabilizing viruses), and the same applies to 5x and 10x.

[0444] A fourth aspect of the present invention provides a pharmaceutical composition comprising a formulation as defined in the first or second aspect of the present invention and a pharmaceutically active ingredient, carrier, or diluent thereof.

[0445] The formulations, buffers, and virus preferences include those described above with respect to the first and second aspects of the present invention.

[0446] A fifth aspect of the present invention provides a formulation for use in a pharmaceutical, the formulation being as defined in the first or second aspect of the present invention. This aspect also includes a kit of components according to the third aspect of the present invention for use in a pharmaceutical.

[0447] The preference for formulations and viruses includes those described above in relation to the first or second aspect of the present invention, and the preference for kits includes those described above in relation to the third aspect of the present invention.

[0448] A sixth aspect of the present invention provides a formulation for use in gene therapy, the formulation being as defined in the first or second aspect of the present invention and further comprising a virus. This aspect also includes a kit of components according to the third aspect of the present invention for use in gene therapy.

[0449] "Gene therapy" refers to techniques that modify target genes in order to treat, cure, or prevent a disease or disorder. Gene therapy can work through several mechanisms: for example, by replacing disease-causing genes with healthy copies of those genes, inactivating disease-causing genes that are not functioning properly, or by introducing new or modified genes into the body to help treat a disease. The introduced genetic material can alter how a single protein or group of proteins is produced by cells.

[0450] A seventh aspect of the present invention provides a formulation for use in cell therapy, the formulation being as defined in the first or second aspect of the present invention and further comprising a virus. This aspect also includes a kit of components according to the third aspect of the present invention for use in cell therapy.

[0451] "Cell therapy" encompasses the technique of transferring intact, living cells to a patient to treat, cure, or prevent a disease or disorder. Such cells are typically modified using viral vectors before being administered to the patient. The cells can originate from the patient (autologous cells) or a donor (allogeneic cells). Cells used in cell therapy can be classified by their ability to transform into different cell types.

[0452] The preference for formulations and viruses includes those described above in relation to the first or second aspect of the present invention, and the preference for kits includes those described above in relation to the third aspect of the present invention.

[0453] In one embodiment, gene therapy and cell therapy can be combined to treat a genetic disorder. Stem cells are modified in culture by (ex vivo) gene therapy to express relevant functional proteins. The improved stem cells are then administered to or returned to the patient.

[0454] In another embodiment, cells are modified before transplantation or transport using viral vectors. Stabilization of these vectors improves, for example, the loss of activity after thawing. Furthermore, if these vectors are produced, there may be technical problems related to their stability during transport, which are similarly addressed by stabilization formulations.

[0455] An eighth aspect of the present invention provides a formulation for use in vaccination and / or immunization, the formulation being as defined in the first or second aspect of the present invention and further comprising a virus. This aspect also includes a kit of components according to the third aspect of the present invention for use in vaccination and / or immunization.

[0456] Vaccination involves the administration of a vaccine, which is used to stimulate an immune response to recognize a pathogen (an organism that causes disease) or a part of a pathogen. Once the immune system is trained to recognize it, if the body is subsequently exposed to the pathogen, the pathogen will be removed from the body, destroyed, or otherwise inactivated or rendered harmless. Specifically, the immune system recognizes foreign "antigens," which are parts of a pathogen that are not normally found in the body, either on the surface or inside the pathogen. Different types of vaccines may include whole-pathogen vaccines or viral vector vaccines.

[0457] The preference for formulations and viruses includes those described above in relation to the first or second aspect of the present invention, and the preference for kits includes those described above in relation to the third aspect of the present invention.

[0458] A ninth aspect of the present invention provides a formulation for use in immunotherapy, the formulation being as defined in the first or second aspect of the present invention and further comprising a virus, preferably the immunotherapy being oncolytic virus therapy. This aspect also includes a kit of components according to the third aspect of the present invention for use in immunotherapy.

[0459] Immunotherapy encompasses the treatment or prevention of disorders or diseases that involve activation, enhancement, reduction, suppression, or desensitization of the immune system. Preferably, the disorder or disease is an autoimmune disorder, allergy, or cancer.

[0460] Oncolytic virotherapy (OV) refers to a form of immunotherapy that uses viruses capable of replicating to infect and destroy cancer cells. Preferably, the replicating virus specifically attacks tumor cells but not healthy cells.

[0461] Current OV platforms require large doses of viral product far exceeding vaccine doses to achieve effective delivery to tumor sites and therapeutic efficacy. Higher viral concentrations often lead to the accumulation of impurities such as residual cell host DNA. More extensive purification procedures are required to eliminate these impurities. Therefore, stabilizing higher-dose gene therapy products enables their systemic use. This is important in gene therapy, but especially in oncolytic virus therapy, where local administration has the limitation of not affecting metastatic sites.

[0462] The preference for formulations and viruses includes those described above in relation to the first or second aspect of the present invention, and the preference for kits includes those described above in relation to the third aspect of the present invention.

[0463] A tenth aspect of the present invention provides a method for stabilizing a virus, comprising combining the virus with a formulation defined in the first and second aspects of the present invention, or a pharmaceutical composition defined in the fourth aspect of the present invention.

[0464] The preferences for formulations include those described above with respect to the first and second aspects of the present invention. In preferred embodiments, the formulation contains 0.4 to 50 mg / mL of albumin, such as 0.5 to 50 mg / mL. For example, the formulation may contain about 0.4, 0.5, 1, 2.5, 5, 10, 15, 20, 25, 30, 35, or 40 to about 25, 30, 35, 40, 45, or 50 mg / mL of albumin. Albumin concentrations of about 1 to 20 mg / mL are particularly preferred, and albumin concentrations of about 1 mg / mL, about 2.5 mg / mL, and about 5 mg / mL are even more particularly preferred.

[0465] It will be understood that a kit according to a third aspect of the present invention may be useful in such a way. For example, the method may include (a) providing albumin, which is substantially in an agglutinative form; (b) providing a plurality of negatively charged anions housed on a flexible skeleton; and (c) contacting a virus with the albumin of (a) and the anions of (b) to stabilize the virus. The provision of the albumin and anions in steps (a) and (b) may correspond to the respective components (i) and (ii) of the kit according to the third aspect of the present invention, which may or may not be pre-mixed before contacting the albumin and anions with the virus. For convenience, for example, “albumin” and “anions” are provided in stock form, which can be diluted to provide working concentrations of “albumin” and “anions” suitable for stabilizing a virus. The method may include the use of buffers and / or sulfates and / or chloride salts (e.g., sodium chloride) or one or more other components, as described above with respect to the first and second aspects of the present invention.

[0466] The preferences for techniques for evaluating formulations, buffers, viruses, and stability include any of those described herein.

[0467] An eleventh aspect of the present invention provides a method for selecting a suitable albumin for use in stabilizing a virus, the method comprising determining whether the albumin is substantially in an agglutinating form.

[0468] If albumin is considered suitable for use in stabilizing a virus, it will be understood that albumin is also suitable for use in the formulation of a virus or a virus-containing formulation (e.g., a formulation containing a viral vector). Accordingly, a method of the eleventh aspect of the present invention may be considered a method for selecting a suitable albumin for use in the formulation of a virus or a virus-containing formulation, comprising determining whether the albumin is substantially in an agglutinating form.

[0469] The preference for techniques for evaluating albumin, anions, viruses, and stability includes any of those described herein.

[0470] In one embodiment, determining whether albumin is substantially in a non-aggregated form includes (i) evaluating whether less than 0.025% (w / w) of albumin is present in polymer form, optionally performed by high-performance liquid chromatography, and determining that the value of less than 0.025% (w / w) of albumin in polymer form indicates albumin that is substantially in a non-aggregated form, and / or (ii) evaluating whether the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, optionally performed by dynamic light scattering, and determining that the hydrodynamic radius that does not change by more than 4.5% when incubated at 40°C for a duration of 3 days indicates albumin that is substantially in a non-aggregated form, and / or (iii) evaluating whether the polydispersity index of albumin is 0.05 or less.

[0471] If this method identifies an albumin that satisfies one or more of the criteria in (i) to (iii) above, preferably two or more or all three of the criteria in (i) to (iii) above, it will be understood that the albumin is substantially in an agglutinative form and is therefore identified as having value for stabilizing viruses and / or formulating viruses, or for use in viral formulations (e.g., viral vector formulations).

[0472] It will be understood that any of the desirable properties of albumin described herein as beneficial for stabilizing viruses may be evaluated as part of a method according to an eleventh aspect of the present invention.

[0473] In yet another embodiment, a method according to the 11th aspect of the present invention further comprises formulating a plurality of negatively charged anions and albumin housed on a flexible skeleton, and optionally evaluating whether the formulation containing albumin and anions stabilizes a virus.

[0474] The present invention will now be described with reference to the following embodiments, drawings, and examples.

[0475] Preferred Embodiments of the Invention (Part B) 1. The use of a preparation for stabilizing the virus, wherein the preparation is (i) Albumin, which is substantially in an unaggregated form, (ii) Multiple negatively charged anions housed on a flexible skeleton, Includes, use.

[0476] 2. (i) Less than 0.025% (w / w) of albumin is in polymer form, and / or (ii) The hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, and / or (iii) The polydispersity index of albumin is 0.05 or less, and / or (iv) Albumin is substantially non-aggregated when determined by a technique selected from the group consisting of high-performance liquid chromatography (HPLC) and / or fine spectral scattering (DLS). (v) Less than 75% of albumin molecules are post-translationally modified by means other than oxidation, and / or (vi) The total amount of lipids or fatty acids (excluding octanoates) in the preparation is 50 μg (micrograms) or less of lipids or fatty acids / mL per 100 mg / mL of albumin, and / or (vii) Albumin is recombinant albumin and / or (viii) The albumin is (a) yeast-derived albumin, and optionally the yeast is Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces lactis such as Kluyveromyces marxianus, Candida such as Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida utilis), or Zygosaccharomyces bailii, or (b) Plant-derived albumin such as rice-derived albumin, and / or (ix) The formulation is a liquid formulation and / or (x) The formulation contains at least 0.5 moles of free thiols per mole of albumin, and / or (xi) Albumin a. A composition comprising 130-160 mM, preferably 145 mM, sodium; 29-35 mM, preferably 32 mM, octanoate; 10-20 mg / L, preferably 10-15 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.7-7.3, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. b. A composition comprising 120-160 mM, preferably 145 mM, sodium; 4-12 mM, preferably 8 mM, octanoate; 0-50 mg / L, preferably 25-45 mg / L, polysorbate 80; and 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. c. A composition comprising 120-160 mM, preferably 145 mM, sodium; 8-24 mM, preferably 16 mM, octanoate; 0-100 mg / L, preferably 50-70 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. d. Provided by an albumin composition selected from the group consisting of any of the following compositions: containing 200-300 mM, preferably 230-260 mM, sodium; 0-3 mM, preferably 0-1 mM, octanoate; 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6-7, preferably 6.5; and / or having a percentage (w / w) of polymerized albumin of 0-1%, preferably less than 0.025%. (xii) The preparation contains 0.4 to 300 mg / mL of albumin, such as 0.4 to 50 mg / mL of albumin, and optionally the preparation contains 1 to 20 mg / mL of albumin, and / or (xiii) Multiple negatively charged anions housed on a flexible skeleton thermally stabilize albumin, and / or (xiv) Multiple negatively charged anions housed on a flexible skeleton prevent deamidation of albumin, and / or (xv) Multiple negatively charged anions housed on a flexible skeleton prevent N-terminal degradation of albumin, and / or (xvi) Multiple negatively charged anions housed on a flexible skeleton are polycarboxylic acids such as citrate, fumarate, tartaric acid, α-ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate, or sodium tripolyphosphate, or any intermediate anion of the citric acid cycle, and / or (xvii) Multiple negatively charged anions housed on a flexible skeleton are present at concentrations of 2–500 mM or 10–500 mM, optionally, anions are present at concentrations of 2–100 mM or 25–100 mM, optionally, anions are present at concentrations of approximately 2–60 mM, 10–60 mM, 40–60 mM, etc., such as approximately 2 mM, approximately 10 mM, or approximately 50 mM, and / or (xviii) The formulation further contains a buffer, optionally, (a) The buffer maintains or increases the stability of albumin in the presence of multiple negatively charged anions housed on a flexible skeleton, and / or (b) The buffer does not reduce the stability of albumin in the presence of multiple negatively charged anions housed on a flexible skeleton, and / or (c) The buffer is positively charged and / or (d) The buffer has a pKa higher than the pH of the formulation, and / or (e) The buffer is one or more of Tris, Bis-Tris, Bis-Trispropane, BTP, and / or HEPES, and / or (f) The formulation contains 0-250 mM or 1-250 mM buffer, optionally, the formulation contains 10-200 mM buffer, optionally, the formulation contains (fi) approximately 150 mM buffer, optionally, the buffer is Tris, or (fii) approximately 0-50 mM or approximately 30-50 mM buffer, optionally, the buffer is Tris, BTP, and / or HEPES, and / or (xix) formulation, (a) Magnesium chloride in a concentration of 0 to 50 mM, wherein the formulation optionally contains approximately 0 to approximately 10 mM magnesium chloride, such as approximately 25 mM magnesium chloride, or approximately 5 mM or approximately 10 mM magnesium chloride, and / or approximately 1 to approximately 10 mM magnesium chloride. (b) Sulfates of 0 to 500 mM, such as about 0 to 200 mM, and optionally, the formulation contains sulfates of about 25 to 250 or about 50 to 200 mM, such as about 50 mM or about 200 mM, and optionally, the sulfates are sodium sulfate, sodium bisulfate, sodium thiosulfate, ammonium sulfate, ammonium bisulfate, magnesium sulfate, magnesium bisulfate, potassium sulfate, potassium bisulfate, and / or (c) Disodium phosphate in concentrations of approximately 0 to 200 mM or 0 to 500 mM, wherein the formulation optionally contains approximately 10 mM disodium phosphate or approximately 200 mM disodium phosphate, and / or (d) EDTA of 0-50 mM, optionally, the formulation containing approximately 1 mM EDTA, and / or (e) Glutamic acid in a concentration of 0 to 200 mM, wherein the formulation optionally contains approximately 50 mM glutamic acid, and / or (f) Mannitol in a concentration of 0-500 mM, wherein the formulation optionally contains approximately 300 mM mannitol, and / or (g) Sucrose in a concentration of 0-500 mM, wherein the formulation optionally contains approximately 300 mM sucrose, and / or (h) Arginine in concentrations of approximately 0 to 200 mM, or 0 to 500 mM, wherein the formulation optionally contains approximately 25 mM to approximately 50 mM arginine, or the formulation contains approximately 200 mM arginine, and / or (i) Chloride salts of approximately 0 to 200 mM, etc., and optionally, the formulation contains chloride salts of approximately 25 to 250 mM, etc., such as approximately 200 mM or approximately 50 mM, (j) Histidine in a concentration of 0-200 mM, and optionally, the formulation contains histidine with a concentration of approximately 50 mM. (k) Glycine in a concentration of 0-200 mM, wherein the formulation optionally contains approximately 50 mM glycine or approximately 200 mM glycine, and / or (l) Glutamate salts of 0-200 mM, optionally, the formulation containing approximately 50 mM glutamate salt, and / or (m) Aspartates of 0 to 200 mM, optionally, the formulation containing approximately 50 mM aspartates, and / or (n) 0-0.01% (w / v) nonionic surfactant, wherein optionally the formulation contains about 0.001% or about 0.01% (w / v) of nonionic surfactant, and optionally the nonionic surfactant is a poloxamer or polysorbate such as Pluronic F-68, and / or (o) Use according to Embodiment 1, comprising sodium chloride, wherein the formulation optionally contains sodium chloride in a concentration of 0 to 500 mM, and the formulation contains sodium chloride in a concentration of approximately 25 to 250 mM or approximately 50 to 200 mM, such as sodium chloride in a concentration of approximately 200 mM or approximately 50 mM.

[0477] 3. The use according to Embodiment 1 or 2, wherein the pH of the formulation is approximately 4 to approximately 8, optionally, approximately 5 to approximately 7.5, and preferably approximately 6 or approximately 6.5.

[0478] 4. (I) When the virus comes into contact with the formulation, the stability of the virus increases compared to the stability of the virus in the absence of the formulation, and optionally, the increased stability (a) as revealed by a decrease in viral agglutination over a given period, and / or (b) When the formulation is incubated at 40°C for a duration of at least 20 hours, the coefficient of variation of the hydrodynamic radius of the formulation containing 5% or less of the virus is evident, and / or (i) After freeze-thaw stress (for example, after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storage at a temperature of approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours), the hydrodynamic radius of the formulation containing the virus is no more than 6 times (preferably 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.5 times or less) of the hydrodynamic radius of the virus in monomeric form, as revealed by the hydrodynamic radius of the formulation containing the virus, and / or (d) After freeze-thaw stress (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storage at a temperature of approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours), the mass recovery of at least 50% of the virus, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, and / or (e) After freeze-thaw stress (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles of storage at a temperature of approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours), the mass recovery rate of the virus is shown to be increased or improved by at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percent points compared to the mass recovery rate of the virus in the absence of the formulation (e.g., the virus in a formulation that does not contain albumin and / or does not contain multiple negatively charged anions housed on a flexible skeleton, but is otherwise identical or substantially identical), and / or (f) The number of particles in a virus-containing formulation is less than the number of particles in a control solution of the virus after freeze-thaw stress (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles of storage at a temperature of approximately -20°C for at least 6 hours, followed by thawing at room temperature for approximately 3 hours) (e.g., a solution of the virus in the absence of albumin and / or in the absence of multiple negatively charged anions housed on a flexible skeleton), and / or (g) After storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7°C, for a duration of at least one month, e.g., at least 2, 3, 4, 5, or 6 months, e.g., at approximately 4°C, e.g., less than 15% particle coverage as measured by background membrane imaging (BMI), and / or (h) After storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7°C, for a duration of at least one month, e.g., at least 2, 3, 4, 5, or 6 months, e.g., at approximately 4°C, the particle count is less than 60,000 particles as determined by background membrane imaging (BMI), and the estimated circular diameter (ECD) of the particles is 2 μm (microns) or less, and / or (i) After storage or incubation at a temperature of 2-8°C, such as 2, 3, 4, 5, 6, or 7°C, for a duration of at least one month, e.g., at least 2, 3, 4, 5, or 6 months, e.g., at approximately 4°C, the particle count is less than 15,000 particles as measured by background membrane imaging (BMI), the estimated circle diameter (ECD) of the particles is 2 μm (microns) or less, the particles are thioflavin T positive, and / or (j) The particle count is less than 200,000 particles as indicated by bright-field side-illumination membrane intensity (SIMI) measured by background membrane imaging after at least four freeze-thaw cycles, and the estimated circular diameter (ECD) of the particles is 2 μm (microns) or less, and / or (k) revealed by the preservation of viral integrity, and / or (l) This is revealed by the retention of the virus's ability to bind to a specific virus-binding partner, and / or (m) This is revealed by the retention of the virus's ability to infect cells, and / or (II) The virus (a) Any of the following: virus particles, virus fragments, virus-like particles, virus vectors, or vector viruses, and / or (b) Simian virus, which, at the discretion of any choice, is an adeno-associated virus (AAV), recombinant AAV, adenovirus, herpes simplex virus, varicella stomatitis virus, maraba virus, vaccinia virus, measles virus, Newcastle disease virus, rotavirus, reovirus, poliovirus type I, coxsackievirus A21, Seneca Valley virus, dengue virus, yellow fever virus, West Nile virus, Zika virus, alphavirus, or coronavirus, and at the discretion of any choice, is an AAV2, AAV9, AAV8, or AAV5, and / or (III) The virus is a live virus, a weakened virus, a live weakened virus, or an inactivated virus, and / or (IV) The virus is in the form of a vaccine and / or (V) The virus is a hybrid or chimeric virus, and / or (VI) The virus that is stabilized is 1 × 10 6 ~1 × 10 24 The use described in any one of Embodiments 1 to 3, with a concentration of vg / mL.

[0479] 5. A kit of a formulation or component, (i) Albumin, which is substantially in an unaggregated form, (ii) A kit of formulations or components comprising a plurality of negatively charged anions housed on a flexible skeleton.

[0480] 6. The formulation or kit is (iii) A buffer that maintains or increases the stability of albumin in the presence of a plurality of negatively charged anions housed on a flexible skeleton, or (iv) The formulation or kit according to Embodiment 5, further comprising a buffer that does not reduce the stability of albumin in the presence of a plurality of negatively charged anions housed on a flexible skeleton.

[0481] 7. (i) Less than 0.025% (w / w) of albumin is in polymer form, and / or (ii) The hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, and / or (iii) The polydispersity index of albumin is 0.05 or less, and / or (iv) Albumin is substantially non-aggregated when determined by a technique selected from the group consisting of high-performance liquid chromatography (HPLC) and / or fine spectral scattering (DLS). (v) Less than 75% of albumin molecules are post-translationally modified by means other than oxidation, and / or (vi) The total amount of lipids (excluding octanoates) is 50 μg (micrograms) or less of lipids / mL in a 100 mg / mL solution of albumin, and / or (vii) Albumin is recombinant albumin and / or (viii) Albumin (a) Albumin derived from yeast, optionally, the yeast being Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida such as Candida utilis, or Zygosaccharomyces bailii, or (b) Plant-derived albumin such as rice-derived albumin, and / or (ix) The formulation or kit contains at least 0.5 moles of free thiols per mole of albumin, and / or (x) Albumin, a. A composition comprising 130-160 mM, preferably 145 mM, sodium; 29-35 mM, preferably 32 mM, octanoate; 10-20 mg / L, preferably 10-15 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.7-7.3, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. b. A composition comprising 120-160 mM, preferably 145 mM, sodium; 4-12 mM, preferably 8 mM, octanoate; 0-50 mg / L, preferably 25-45 mg / L, polysorbate 80; and 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. c. A composition comprising 120-160 mM, preferably 145 mM, sodium; 8-24 mM, preferably 16 mM, octanoate; 0-100 mg / L, preferably 50-70 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. d. Provided by an albumin composition selected from the group consisting of any of the following compositions: containing 200-300 mM, preferably 230-260 mM, sodium; 0-3 mM, preferably 0-1 mM, octanoate; 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6-7, preferably 6.5; and / or having a percentage (w / w) of polymerized albumin of 0-1%, preferably less than 0.025%. (xi) The preparation or kit contains approximately 50-300 mg / mL of albumin, approximately 100-200 mg / mL of albumin, or approximately 0.4-50 mg / mL of albumin, and optionally the preparation contains 1-20 mg / mL of albumin, and / or (xii) Multiple negatively charged anions housed on a flexible skeleton thermally stabilize albumin, and / or (xiii) Multiple negatively charged anions housed on a flexible skeleton prevent deamidation of albumin, and / or (xiv) Multiple negatively charged anions housed on a flexible skeleton prevent N-terminal degradation of albumin, and / or (xv) Multiple negatively charged anions housed on a flexible skeleton are polycarboxylic acids such as citrate, fumarate, tartaric acid, α-ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate, or sodium tripolyphosphate, or any intermediate anion of the citric acid cycle, and / or (xvi) Multiple negatively charged anions housed on a flexible skeleton are present at concentrations of 2–500 mM or 10–500 mM, and optionally, anions are present at concentrations of 2–100 mM or 25–100 mM, and / or The formulation or kit according to Embodiment 5 or 6, wherein the (xvii) anion is present at concentrations of 2 to 100 mM, such as 2 to 60 mM, 10 to 60 mM, or 40 to 60 mM, such as about 2 mM, about 10 mM, or about 50 mM.

[0482] 8. (I) The buffer is positively charged, and / or (II) The buffer has a pKa higher than (a) the pH of the formulation, or (b) the pH of components (i) and / or (ii) of the kit, and / or (III) The buffer is one or more of Tris, Bis-Tris, Bis-Trispropane, BTP, and / or HEPES, and / or (IV) The formulation or kit (a) 0-250 mM buffers such as 1-250 mM, and optionally, the formulation or kit contains 10-200 mM buffer, and / or (b) A buffer of approximately 150 mM, optionally, the buffer is a buffer of approximately 0 to 50 mM, such as Tris or approximately 30 to 50 mM, optionally, the buffer is any of Tris, BTP, and / or HEPES, and / or (c) Magnesium chloride of 0 to 50 mM, wherein the formulation or kit optionally contains magnesium chloride of about 0 to about 10 mM, such as magnesium chloride of about 25 mM, or magnesium chloride of about 5 mM or about 10 mM, and / or magnesium chloride of about 1 to about 10 mM. (d) Sulfates of 0 to 500 mM, such as 0 to 200 mM, and optionally, the formulation or kit contains sulfates of approximately 50 to 500 mM, such as approximately 25 to 250 mM, such as approximately 50 mM or approximately 200 mM sulfates, and optionally, the sulfates are sodium sulfate, sodium bisulfate, sodium thiosulfate, ammonium sulfate, ammonium bisulfate, magnesium sulfate, magnesium bisulfate, potassium sulfate, potassium bisulfate, and / or (e) Disodium phosphate in concentrations of 0 to 500 mM, such as approximately 10 to 250 mM, and optionally, the formulation or kit contains approximately 10 mM disodium phosphate or approximately 200 mM disodium phosphate, and / or (f) EDTA of 0-50 mM, wherein optionally the formulation or kit contains approximately 1 mM EDTA, and / or (g) Glutamic acid in a concentration of 0-200 mM, wherein the formulation optionally contains approximately 50 mM glutamic acid, and / or (h) Mannitol in a concentration of 0-500 mM, wherein the formulation optionally contains approximately 300 mM mannitol, and / or (i) Sucrose in a concentration of 0 to 500 mM, optionally, the formulation containing approximately 300 mM sucrose, and / or (j) Arginine in concentrations of 0 to 500 mM, such as approximately 25 to 250 mM, and optionally, the formulation or kit contains approximately 25 mM to approximately 50 mM arginine, or the formulation or kit contains approximately 200 mM arginine, and / or (k) Chloride salts of 0 to 500 mM, such as approximately 25 to 250 mM, and optionally, the formulation or kit contains a chloride salt of approximately 50 mM or approximately 200 mM, and / or (l) Histidine in a concentration of 0-200 mM, wherein the formulation or kit optionally contains approximately 50 mM histidine, and / or (m) Glycine in a concentration of 0 to 200 mM, wherein the formulation or kit optionally contains approximately 50 mM glycine or approximately 200 mM glycine, and / or (n) Glutamate salts in concentrations of 0 to 200 mM, wherein the formulation or kit optionally contains approximately 50 mM glutamate salt, and / or (o) Aspartates in concentrations of 0-200 mM, wherein the formulation or kit optionally contains approximately 50 mM aspartates, and / or (p) 0-0.01% (w / v) nonionic surfactant, wherein optionally the formulation or kit contains about 0.001% or about 0.01 (w / v) of nonionic surfactant, and optionally the nonionic surfactant is a poloxamer or polysorbate such as Pluronic F-68, and / or (q) A formulation or kit according to Embodiment 6 or 7, comprising sodium chloride in a concentration of 0 to 500 mM, such as approximately 25 to 250 mM, and optionally, the formulation comprising approximately 50 to 200 mM sodium chloride, such as approximately 200 mM or approximately 50 mM sodium chloride.

[0483] 9. The pH of the formulation or kit is (I) pH of approximately 4 to approximately 8, and optionally, the pH of the formulation is approximately 5 to approximately 7.5, or (ii) about 6 or about 6.5, (iii) A formulation or kit according to any one of Embodiments 6 to 8, wherein the amount is approximately 4 or approximately 5.

[0484] 10. The formulation or kit further contains a virus, optionally the virus being one of a viral particle, viral fragment, virus-like particle, viral vector, or vector virus, optionally the virus being (i) Simian viruses, which, at the discretion of any choice, are adeno-associated viruses (AAV), recombinant AAV, adenovirus, herpes simplex virus, varicella stomatitis virus, maraba virus, vaccinia virus, measles virus, Newcastle disease virus, rotavirus, reovirus, poliovirus type I, coxsackievirus A21, Seneca Valley virus, dengue virus, yellow fever virus, West Nile virus, Zika virus, alphavirus, or coronavirus, and which, at the discretion of any choice, are AAV2, AAV9, AAV8, or AAV5, and / or (ii) Live viruses, attenuated viruses, live attenuated viruses, or inactivated viruses, and / or (iii) The form of the vaccine, and / or (iv) A hybrid or chimeric virus, and / or (v) 1 × 10 6 ~1 × 10 24 A formulation or kit according to any one of Embodiments 5 to 9, present at a concentration of vg / ml.

[0485] 11. A pharmaceutical composition comprising a formulation according to any one of Embodiments 5 to 10, and a pharmaceutically active ingredient, carrier, or diluent thereof.

[0486] 12. A formulation or kit according to any one of Embodiments 5 to 10 for use in pharmaceuticals.

[0487] 13. (i) gene therapy, and / or (ii) Cell therapy, and / or (iii) Vaccination and / or immunization, and / or (iv) An immunotherapy, optionally an oncolytic virus therapy, comprising the formulation or kit described in Embodiment 10 for use in immunotherapy.

[0488] 14. A method for stabilizing a virus, comprising combining the virus with a formulation described in any one of Embodiments 5 to 10 or a pharmaceutical composition described in Embodiment 11.

[0489] 15. A method for selecting a suitable albumin for use in stabilizing a virus, comprising determining whether the albumin is substantially in an agglutinative form, and optionally, (i) Determining whether albumin is substantially in an unaggregated form is (a) an assessment of whether less than 0.025% (w / w) of albumin in polymer form is present, optionally, the assessment being performed by high-performance liquid chromatography, and the value of less than 0.025% (w / w) of albumin in polymer form indicates albumin that is substantially in an unaggregated form, and / or (b) Evaluate whether the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, optionally, the evaluation is carried out by dynamic light scattering, and the evaluation and / or the evaluation of albumin in which the hydrodynamic radius does not change by more than 4.5% when incubated at 40°C for a duration of 3 days indicates that th...

Claims

1. The use of a preparation for stabilizing a virus, wherein the preparation is (i) Albumin, which is substantially in an unaggregated form, (ii) Multiple negatively charged anions housed on a flexible skeleton, Includes, use.

2. The use according to claim 1, wherein less than 0.025% (w / w) of albumin is in polymer form.

3. The use according to claim 1 or 2, wherein the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days.

4. The use according to any one of claims 1 to 3, wherein the polydispersity index of albumin is 0.05 or less.

5. The use according to any one of claims 1 to 4, wherein the albumin is substantially in an agglomerated form when determined by a technique selected from the group consisting of high-performance liquid chromatography (HPLC) and fine spectral scattering (DLS).

6. The use according to any one of claims 1 to 5, wherein less than 75% of the albumin molecule is modified post-translation by means other than oxidation.

7. The use according to any one of claims 1 to 6, wherein the total amount of lipids or fatty acids (excluding octanoates) in the preparation is 50 μg (micrograms) or less of lipids or fatty acids / mL per 100 mg / mL of albumin.

8. The use according to any one of claims 1 to 7, wherein the albumin is recombinant albumin.

9. The aforementioned albumin, (i) Albumin derived from yeast, wherein the yeast is optionally Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Candida such as Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida utilis, or Zygosaccharomyces albumin derived from yeast, or (ii) The use according to any one of claims 1 to 8, wherein the plant-derived albumin is such as albumin derived from rice.

10. The use according to any one of claims 1 to 9, wherein the preparation is a liquid preparation.

11. The use according to any one of claims 1 to 10, wherein the formulation contains at least 0.5 moles of free thiols per mole of albumin.

12. The aforementioned albumin, a. A composition comprising 130-160 mM, preferably 145 mM, sodium; 29-35 mM, preferably 32 mM, octanoate; 10-20 mg / L, preferably 10-15 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.7-7.3, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. b. A composition comprising 120-160 mM, preferably 145 mM, sodium; 4-12 mM, preferably 8 mM, octanoate; 0-50 mg / L, preferably 25-45 mg / L, polysorbate 80; and 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. c. A composition comprising 120-160 mM, preferably 145 mM, sodium; 8-24 mM, preferably 16 mM, octanoate; 0-100 mg / L, preferably 50-70 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. d. The use according to any one of claims 1 to 11, provided by an albumin composition selected from the group comprising any of the following compositions: 200 to 300 mM, preferably 230 to 260 mM sodium; 0 to 3 mM, preferably 0 to 1 mM octanoate; 95 to 105 mg / mL, preferably 100 mg / mL albumin (w / v); a pH of 6 to 7, preferably 6.5; and a percentage (w / w) of polymerized albumin (w / w) of 0 to 1%, preferably less than 0.025%.

13. The use according to any one of claims 1 to 12, wherein the preparation contains 0.4 to 300 mg / mL of albumin, such as 0.4 to 50 mg / mL of albumin, and optionally the preparation contains 1 to 20 mg / mL of albumin.

14. The use according to any one of claims 1 to 13, wherein the plurality of negatively charged anions housed on a flexible skeleton thermally stabilize the albumin.

15. The use according to any one of claims 1 to 14, wherein the plurality of negatively charged anions housed on a flexible skeleton prevent the deamidation of the albumin.

16. The use according to any one of claims 1 to 15, wherein the plurality of negatively charged anions housed on a flexible skeleton prevent N-terminal degradation of the albumin.

17. The use according to any one of claims 1 to 16, wherein the plurality of negatively charged anions housed on a flexible skeleton are polycarboxylic acids such as citrate, fumarate, tartaric acid, α-ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate, or sodium tripolyphosphate, or any intermediate anion of the citric acid cycle.

18. The use according to any one of claims 1 to 17, wherein the plurality of negatively charged anions housed on a flexible skeleton are present at a concentration of 2 to 500 mM or 10 to 500 mM, and optionally, the anions are present at a concentration of 2 to 100 mM or 25 to 100 mM.

19. The use according to claim 18, wherein the anion is present at a concentration of 10 to 100 mM, 2 to 60 mM, 10 to 60 mM, or 40 to 60 mM, such as about 2 mM, about 10 mM, or about 50 mM.

20. The use according to any one of claims 1 to 19, wherein the formulation further comprises a buffer solution.

21. The use according to claim 20, wherein the buffer maintains or increases the stability of the albumin in the presence of a plurality of negatively charged anions housed on a flexible skeleton.

22. The use according to claim 20 or 21, wherein the buffer does not reduce the stability of the albumin in the presence of the multiple negatively charged anions housed on a flexible skeleton.

23. The use according to any one of claims 20 to 22, wherein the buffer solution is positively charged.

24. The use according to any one of claims 20 to 23, wherein the buffer solution has a pKa higher than the pH of the formulation.

25. The use according to any one of claims 20 to 24, wherein the buffer solution is one or more of Tris, Bis-Tris, Bis-Trispropane, BTP, and / or HEPES.

26. The use according to any one of claims 20 to 25, wherein the formulation comprises a buffer solution of 0 to 250 mM, such as a buffer solution of 1 to 250 mM, and optionally comprises a buffer solution of 10 to 200 mM.

27. The use according to claim 26, wherein the formulation comprises about 150 mM buffer, optionally the buffer being Tris, or the formulation comprises about 0 to 50 mM buffer, such as about 30 to 50 mM, optionally the buffer being Tris, BTP, or HEPES.

28. The use according to any one of claims 1 to 27, wherein the formulation comprises 0 to 50 mM magnesium chloride, and optionally, the formulation comprises about 25 mM magnesium chloride, or about 5 mM or about 10 mM magnesium chloride, or about 5 to about 10 mM magnesium chloride.

29. (i) The formulation contains a sulfate in a concentration of 0 to 500 mM, such as 0 to 200 mM, and optionally, the formulation contains a sulfate in a concentration of approximately 50 to 500 mM, such as approximately 50 mM or approximately 200 mM, such as approximately 50 to 200 mM, and optionally, the sulfate is sodium sulfate, sodium bisulfate, sodium thiosulfate, ammonium sulfate, ammonium bisulfate, magnesium sulfate, magnesium bisulfate, potassium sulfate, potassium bisulfate, and / or (ii) The formulation contains 0 to 500 mM disodium phosphate, such as 0 to 200 mM, and optionally the formulation contains about 10 mM disodium phosphate or about 200 mM disodium phosphate, and / or (iii) The formulation comprises 0 to 50 mM EDTA, optionally comprising about 1 mM EDTA and / or (iv) The preparation contains 0 to 200 mM glutamic acid, optionally, the preparation contains about 50 mM glutamic acid and / or (v) The formulation comprises 0 to 500 mM mannitol, optionally comprising approximately 300 mM mannitol and / or (vi) The formulation contains 0 to 500 mM sucrose, optionally, the formulation contains about 300 mM sucrose, and / or (vii) The use according to any one of claims 1 to 28, wherein the formulation contains 0 to 500 mM sodium chloride, such as about 0 to 200 mM, and optionally the formulation contains about 50 to 200 mM sodium chloride, such as about 200 mM or about 50 mM sodium chloride.

30. The use according to any one of claims 1 to 29, wherein the formulation contains 0 to 500 mM arginine, such as about 0 to 200 mM, and optionally the formulation contains about 25 mM to about 50 mM arginine.

31. The use according to any one of claims 1 to 30, wherein the formulation contains a chloride salt in a concentration of 0 to 500 mM, such as 0 to 200 mM, and optionally, the formulation contains a chloride salt in a concentration of approximately 50 mM.

32. The use according to any one of claims 1 to 31, wherein the formulation comprises 0 to 200 mM histidine, and optionally, the formulation comprises about 50 mM histidine.

33. The use according to any one of claims 1 to 32, wherein the formulation comprises 0 to 200 mM glycine, and optionally, the formulation comprises about 50 mM glycine or about 200 mM glycine.

34. The use according to any one of claims 1 to 33, wherein the formulation comprises 0 to 200 mM glutamate, and optionally, the formulation comprises about 50 mM glutamate.

35. The use according to any one of claims 1 to 34, wherein the formulation comprises 0 to 200 mM aspartate, and optionally, the formulation comprises about 50 mM aspartate.

36. The use according to any one of claims 1 to 35, wherein the formulation comprises 0 to 0.01% (w / v) of a nonionic surfactant, optionally comprising about 0.001% or about 0.01% (w / v) of a nonionic surfactant, and optionally the nonionic surfactant being a poloxamer or polysorbate such as Pluronic® F-68.

37. The use according to any one of claims 1 to 36, wherein the pH of the formulation is approximately 4 to approximately 8, optionally, the pH of the formulation is approximately 5 to approximately 7.5, and preferably the pH of the formulation is approximately 6 or approximately 6.

5.

38. The use according to any one of claims 1 to 37, wherein when the virus comes into contact with the formulation, the stability of the virus increases compared to the stability of the virus in the absence of the formulation.

39. The use according to claim 38, wherein the increased stability is manifested by a reduction in the aggregation of the virus over a given period of time.

40. The increased stability is evident from the coefficient of variation of the hydrodynamic radius of the formulation containing the virus, which is 5% or less when the formulation is incubated at 40°C for a duration of at least 20 hours, according to the use of claim 38 or 39.

41. The use according to any one of claims 38 to 40, wherein the increased stability is evident by particle coverage of less than 15% as measured by background membrane imaging (BMI) after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7°C to 3, 4, 5, 6, 7, or 8°C, for example at about 4°C, for a duration of at least one month, for example at at least two, three, four, five, or six months.

42. The use according to any one of claims 38 to 41, wherein the increased stability is evident by a particle count of fewer than 60,000 particles measured by background membrane imaging (BMI) after storage or incubation at a temperature of approximately 4°C, such as 2, 3, 4, 5, 6, or 7 to 3, 4, 5, 6, or 8°C, for a duration of at least one month, for example, at least two, three, four, five, or six months, and the estimated circular diameter (ECD) of the particles is 2 μm (microns) or less.

43. The use according to any one of claims 38 to 42, wherein the increased stability is evident by a particle count of fewer than 15,000 particles measured by background membrane imaging (BMI) after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7 to 3, 4, 5, 6, 7, or 8°C, for example at about 4°C, for a duration of at least one month, for example at at least two, three, four, five, or six months, the estimated circular diameter (ECD) of the particles is 2 μm (microns) or less, and the particles are thioflavin T positive.

44. The use according to any one of claims 38 to 43, wherein the increased stability is evident from the particle count of fewer than 200,000 particles in the bright-field side-illumination membrane intensity (SIMI) measured by background membrane imaging after at least four freeze-thaw cycles, and the estimated circular diameter (ECD) of the particles is 2 μm (microns) or less.

45. The use according to any one of claims 38 to 44, wherein the increased stability is evident from the hydrodynamic radius of the formulation containing the virus, which is 6 times or less (preferably 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, or 1.5 times or less) the hydrodynamic radius of the virus in its monomeric form, after freeze-thaw stress (for example, after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storage at a temperature of about -20°C for at least 6 hours, followed by thawing at room temperature for about 3 hours).

46. The use according to any one of claims 38 to 45, wherein the increased stability is evident by a mass recovery rate of at least 50% of the virus, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, after freeze-thaw stress (for example, after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of storing at a temperature of about -20°C for at least 6 hours, followed by thawing at room temperature for about 3 hours).

47. The use according to any one of claims 38 to 46, wherein the increased stability is evident from the mass recovery rate of the virus after freeze-thaw stress (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles of storing at a temperature of about -20°C for at least 6 hours, followed by thawing at room temperature for about 3 hours), which is increased or improved by at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percent points compared to the mass recovery rate of the virus in the absence of the formulation (e.g., the virus in a formulation that does not contain albumin and / or contains a plurality of negatively charged anions housed on a flexible skeleton, but otherwise is identical or substantially identical).

48. The use according to any one of claims 38 to 47, wherein the increased stability is evident after freeze-thaw stress (for example, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles of storage at a temperature of about -20°C for at least 6 hours, followed by thawing at room temperature for about 3 hours), by the number of particles in the formulation containing the virus being less than the number of particles in a control solution of the virus (for example, a solution of the virus in the absence of the albumin and / or the multiple negatively charged anions housed on a flexible skeleton).

49. The use according to any one of claims 38 to 48, wherein the increased stability is evident from the preservation of viral integrity.

50. The use according to any one of claims 38 to 49, wherein the increased stability is evident in the retention of the virus's ability to bind to a specific binding partner of the virus.

51. The use according to any one of claims 38 to 50, wherein the increased stability is evident in the retention of the virus's ability to infect cells.

52. The use according to any one of claims 1 to 51, wherein the virus is (i) any of a viral particle, viral fragment, virus-like particle, viral vector, or vector virus, and / or (ii) a Simian virus, and optionally the Simian virus is adeno-associated virus (AAV), recombinant AAV, adenovirus, herpes simplex virus, vesicular stomatitis virus, Maraba virus, vaccinia virus, measles virus, Newcastle disease virus, rotavirus, reovirus, poliovirus type I, coxsackievirus A21, Seneca Valley virus, dengue virus, yellow fever virus, West Nile virus, Zika virus, alphavirus, or coronavirus.

53. The use according to claim 52, wherein the AAV is AAV2, AAV9, AAV8, or AAV5.

54. The use according to any one of claims 1 to 53, wherein the virus is a live virus, a weakened virus, a live weakened virus, or an inactivated virus.

55. The use according to any one of claims 1 to 54, wherein the virus is in the form of a vaccine.

56. The use according to any one of claims 1 to 55, wherein the virus is a hybrid or chimeric virus.

57. The stabilized virus is 1 x 10 ６ ~1 x 10 ２４ The use according to any one of claims 1 to 56, wherein the concentration is vg / mL.

58. A pharmaceutical product, (i) Albumin, which is substantially in an unaggregated form, (ii) A formulation comprising a plurality of negatively charged anions housed on a flexible skeleton.

59. It is a kit of components, (i) Albumin, which is substantially in an unaggregated form, (ii) A kit of components comprising multiple negatively charged anions housed on a flexible skeleton.

60. The aforementioned formulation or kit (iii) The formulation according to claim 58, or the kit according to claim 59, further comprising a buffer, which maintains or increases the stability of the albumin in the presence of a plurality of negatively charged anions housed on a flexible skeleton.

61. The aforementioned formulation, (iii) The formulation according to claim 58, or the kit according to claim 59, further comprising a buffer, the buffer which does not reduce the stability of the albumin in the presence of a plurality of negatively charged anions housed on a flexible skeleton.

62. A formulation or kit according to any one of claims 58 to 61, wherein less than 0.025% (w / w) of albumin is in polymer form.

63. The formulation or kit according to any one of claims 58 to 62, wherein the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days.

64. The formulation or kit according to any one of claims 58 to 63, wherein the polydispersity index of albumin is 0.05 or less.

65. The formulation or kit according to any one of claims 58 to 64, wherein the albumin is substantially in an agglutinated form when determined by a technique selected from the group consisting of high-performance liquid chromatography (HPLC) and fine spectral scattering (DLS).

66. The formulation or kit according to any one of claims 58 to 65, wherein less than 75% of the albumin molecules are modified post-translation by means other than oxidation.

67. A formulation or kit according to any one of claims 58 to 66, wherein the total amount of lipids (excluding octanoates) is 50 μg (micrograms) of lipids / mL or less in a 100 mg / mL solution of albumin.

68. The formulation or kit according to any one of claims 58 to 67, wherein the albumin is recombinant albumin.

69. The albumin is yeast-derived albumin, and optionally the yeast is Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida such as Candida utilis, or Zygosaccharomyces A formulation or kit according to any one of claims 58 to 68, wherein bailii.

70. The formulation or kit according to any one of claims 58 to 68, wherein the albumin is a plant-derived albumin such as rice-derived albumin.

71. The formulation or kit according to any one of claims 58 to 70, wherein the formulation or kit contains at least 0.5 moles of free thiol per mole of albumin.

72. The aforementioned albumin, a. A composition comprising 130-160 mM, preferably 145 mM, sodium; 29-35 mM, preferably 32 mM, octanoate; 10-20 mg / L, preferably 10-15 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.7-7.3, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. b. A composition comprising 120-160 mM, preferably 145 mM, sodium; 4-12 mM, preferably 8 mM, octanoate; 0-50 mg / L, preferably 25-45 mg / L, polysorbate 80; and 95-105 mg / mL, preferably 100 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. c. A composition comprising 120-160 mM, preferably 145 mM, sodium; 8-24 mM, preferably 16 mM, octanoate; 0-100 mg / L, preferably 50-70 mg / L, polysorbate 80; and 190-210 mg / mL, preferably 200 mg / mL, albumin (w / v); having a pH of 6.4-7.4, preferably 7; and a percentage (w / w) of polymerized albumin of 0-1%, more preferably less than 0.025%. d. The formulation or kit according to any one of claims 58 to 71, provided by an albumin composition selected from the group comprising any one of the following compositions: 200 to 300 mM, preferably 230 to 260 mM sodium; 0 to 3 mM, preferably 0 to 1 mM octanoate; 95 to 105 mg / mL, preferably 100 mg / mL albumin (w / v); a pH of 6 to 7, preferably 6.5; and a percentage (w / w) of polymerized albumin (w / w) of 0 to 1%, preferably less than 0.025%.

73. The formulation or kit according to any one of claims 58 to 72, wherein the formulation or kit contains 0.4 to 300 mg / mL of albumin, such as about 50 to 300 mg / mL or about 100 to 200 mg / mL of albumin, or about 0.4 to 50 mg / mL of albumin, and optionally the formulation contains 1 to 20 mg / mL of albumin.

74. The formulation or kit according to any one of claims 58 to 73, wherein the plurality of negatively charged anions housed on a flexible skeleton thermally stabilize the albumin.

75. The formulation or kit according to any one of claims 58 to 74, wherein the plurality of negatively charged anions housed on a flexible skeleton prevent the deamidation of the albumin.

76. The formulation or kit according to any one of claims 58 to 75, wherein the plurality of negatively charged anions housed on a flexible skeleton prevent the N-terminal degradation of the albumin.

77. The formulation or kit according to any one of claims 58 to 76, wherein the multiple negatively charged anions housed on a flexible skeleton are polycarboxylic acids such as citrate, fumarate, tartaric acid, α-ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate, or sodium tripolyphosphate, or any intermediate anion of the citric acid cycle.

78. A formulation or kit according to any one of claims 58 to 77, wherein a plurality of negatively charged anions housed on a flexible skeleton are present at a concentration of 2 to 500 mM or 10 to 500 mM, and optionally, the anions are present at a concentration of 25 to 100 mM.

79. The formulation or kit according to any one of claims 58 to 78, wherein the anion is present at a concentration of 10 to 100 mM or 40 to 60 mM, such as about 50 mM.

80. The formulation or kit according to any one of claims 60 to 79, wherein the buffer solution is positively charged.

81. The formulation according to any one of claims 60 to 80, wherein the buffer solution has a pKa higher than the pH of the formulation, or the kit according to any one of claims 59 to 80, wherein the buffer solution has a pKa higher than the pH of component (i) and / or (ii) of the kit.

82. The formulation or kit according to any one of claims 60 to 81, wherein the buffer solution is one or more of Tris, Bis-Tris, Bis-Trispropane, BTP, and / or HEPES.

83. The formulation or kit according to any one of claims 60 to 82, wherein the formulation or kit comprises a buffer solution of 0 to 250 mM, such as a buffer solution of 1 to 250 mM, and optionally comprises a buffer solution of 10 to 200 mM.

84. The formulation or kit according to any one of claims 60 to 83, wherein the formulation or kit comprises about 150 mM buffer, optionally wherein the buffer is Tris, or the formulation or kit comprises about 0 to 50 mM buffer, such as about 30 to 50 mM buffer, optionally wherein the buffer is Tris, BTP, and / or HEPES.

85. The formulation or kit according to any one of claims 58 to 84, wherein the formulation or kit comprises 0 to 50 mM magnesium chloride, and optionally comprises about 25 mM magnesium chloride, or about 1 to about 10 mM magnesium chloride, such as about 5 mM or about 10 mM magnesium chloride.

86. (i) The formulation or kit contains a sulfate in a concentration of 0 to 500 mM, such as 0 to 200 mM, and optionally, the formulation or kit contains a sulfate in a concentration of approximately 50 to 500 mM, such as approximately 50 mM or approximately 200 mM, such as approximately 50 to 200 mM, and optionally, the sulfate is sodium sulfate, sodium bisulfate, sodium thiosulfate, ammonium sulfate, ammonium bisulfate, magnesium sulfate, magnesium bisulfate, potassium sulfate, potassium bisulfate, (ii) The formulation or kit contains 0 to 500 mM disodium phosphate, such as 0 to 200 mM, and optionally the formulation or kit contains about 10 mM disodium phosphate, or about 200 mM or about 500 mM disodium phosphate, and / or (iii) The formulation or kit comprises 0 to 50 mM EDTA, optionally comprising approximately 1 mM EDTA and / or (iv) The formulation contains 0 to 200 mM glutamic acid, optionally, the formulation contains about 50 mM glutamic acid, and / or (v) The formulation contains 0 to 500 mM mannitol, optionally, the formulation contains about 300 mM mannitol, and / or (vi) The formulation contains 0 to 500 mM sucrose, optionally, the formulation contains about 300 mM sucrose, and / or (vii) The formulation or kit according to any one of claims 58 to 85, wherein the formulation comprises 0 to 500 mM sodium chloride, such as about 0 to 200 mM, and optionally comprises about 50 to 200 mM sodium chloride, such as about 200 mM or about 50 mM sodium chloride.

87. The formulation or kit according to any one of claims 58 to 86, wherein the formulation or kit contains 0 to 500 mM arginine, such as about 0 to 200 mM, and optionally the formulation or kit contains about 25 mM to about 50 mM arginine, or the formulation or kit contains about 200 mM arginine.

88. The formulation or kit according to any one of claims 58 to 87, wherein the formulation or kit contains a chloride salt in a concentration of 0 to 500 mM, such as 0 to 200 mM, and optionally, the formulation or kit contains a chloride salt in a concentration of about 50 mM.

89. The formulation or kit according to any one of claims 58 to 88, wherein the formulation or kit comprises 0 to 200 mM histidine, and optionally, the formulation or kit comprises about 50 mM histidine.

90. The formulation according to any one of claims 58 to 89, wherein the formulation or kit comprises 0 to 200 mM glycine, and optionally, the formulation or kit comprises about 50 mM glycine or about 200 mM glycine.

91. The formulation or kit according to any one of claims 58 to 90, wherein the formulation or kit comprises 0 to 200 mM glutamate, and optionally comprises about 50 mM glutamate.

92. The formulation or kit according to any one of claims 58 to 91, wherein the formulation or kit comprises 0 to 200 mM aspartate, and optionally comprises about 50 mM aspartate.

93. The formulation or kit according to any one of claims 58 to 92, wherein the formulation or kit comprises 0 to 0.01% (w / v) of a nonionic surfactant, optionally comprising about 0.001% or about 0.01% (w / v) of a nonionic surfactant, and optionally the nonionic surfactant being a poloxamer such as Pluronic F-68 or a polysorbate.

94. The formulation according to any one of claims 58 and 60 to 93, wherein the pH of the formulation is approximately 4 to approximately 8, and optionally, the pH of the formulation is approximately 5 to approximately 7.5, or the kit according to any one of claims 59 to 93, wherein the pH of component (i) and / or (ii) of the kit is approximately 4 to approximately 8, and optionally, the pH of the formulation is approximately 5 to approximately 7.

5.

95. The formulation according to any one of claims 58 and 60 to 94, wherein the pH of the formulation is (a) about 6 or about 6.5, or (b) about 4 or about 5, or the kit according to any one of claims 55 to 90, wherein the pH of component (i) and / or (ii) of the kit is (a) about 6 or about 6.5, or (b) about 4 or about 5.

96. The formulation or kit according to any one of claims 58 to 95, further comprising a virus, wherein the virus is optionally a viral particle, a viral fragment, a virus-like particle, a viral vector, or a vector virus.

97. The formulation or kit according to claim 96, wherein the virus is a Simian virus, and optionally the Simian virus is an adeno-associated virus (AAV), recombinant AAV, adenovirus, herpes simplex virus, varicella stomatitis virus, maraba virus, vaccinia virus, measles virus, Newcastle disease virus, rotavirus, reovirus, poliovirus type I, coxsackievirus A21, Seneca Valley virus, dengue virus, yellow fever virus, West Nile virus, Zika virus, alphavirus, or coronavirus.

98. The formulation or kit according to claim 97, wherein the AAV is AAV2, AAV9, AAV8, or AAV5.

99. The formulation or kit according to any one of claims 96 to 98, wherein the virus is a live virus, a weakened virus, a live weakened virus, or an inactivated virus.

100. The formulation or kit according to any one of claims 96 to 99, wherein the virus is in the form of a vaccine.

101. The formulation or kit according to any one of claims 96 to 100, wherein the virus is a hybrid or chimeric virus.

102. The aforementioned virus, 1 x 10 ６ -1 x 10 ２４ A formulation or kit according to any one of claims 96 to 101, present at a concentration of vg / ml.

103. A pharmaceutical composition comprising a formulation according to any one of claims 58 and 60 to 102, and a pharmaceutically active ingredient, carrier, or diluent thereof.

104. A formulation or kit according to any one of claims 58 to 102 for use in pharmaceuticals.

105. A formulation or kit according to any one of claims 96 to 102 for use in gene therapy.

106. A formulation or kit according to any one of claims 96 to 102 for use in cell therapy.

107. A formulation or kit according to any one of claims 96 to 102 for use in vaccination and / or immunization.

108. A preparation or kit for use in immunotherapy, wherein the immunotherapy is optionally oncolytic virus therapy, according to any one of claims 96 to 102.

109. A method for stabilizing a virus, comprising combining the virus with a formulation according to any one of claims 58 to 102 or a pharmaceutical composition according to claim 103.

110. A method for selecting a suitable albumin for use in stabilizing a virus, comprising determining whether the albumin is substantially in an agglutinating form.

111. Determining whether the albumin is substantially in an unaggregated form is (i) an assessment of whether less than 0.025% (w / w) of albumin in polymer form is present, optionally, the assessment being performed by high-performance liquid chromatography, and the value of less than 0.025% (w / w) of albumin in polymer form indicates that the albumin is substantially in an unaggregated form, and / or (ii) Evaluating whether the hydrodynamic radius of albumin does not change by more than 4.5% when incubated at 40°C for a duration of 3 days, optionally, the evaluation being carried out by dynamic light scattering, and the evaluation and / or the evaluation indicating that the albumin is substantially in a non-aggregated form, wherein the hydrodynamic radius does not change by more than 4.5% when incubated at 40°C for a duration of 3 days. (iii) The method according to claim 110, comprising evaluating whether the polydispersity index of albumin is 0.05 or less.

112. The method according to claim 110 or 111, further comprising: formulating a plurality of negatively charged anions housed on a flexible skeleton with the albumin; and evaluating whether the formulation containing the albumin and the anions stabilizes the virus.

113. The method according to claim 112, wherein the plurality of negatively charged anions housed on a flexible skeleton are as defined in any one of claims 14-19 and 74-79.

114. A liquid formulation of albumin or a fragment thereof, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or a fragment thereof, after storage or incubation at 40°C for at least 1, 2, 3, 4, 5, or 6 months.

115. A liquid formulation of albumin or a fragment thereof, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or a fragment thereof, after storage or incubation at 25°C for at least 1, 2, 3, 4, 5, 6, 9, or 12 months.

116. A liquid formulation of albumin or its fragments, comprising at least 0.75 moles of free thiols and up to 1% (w / w) of albumin polymer per mole of albumin or its fragments, after storage or incubation at a temperature of 2 to 8°C, such as 2, 3, 4, 5, 6, or 7°C, for a duration of at least 1, 2, 3, 4, 5, or 6 months or at least 1, 2, 3, 4, 5, or 6 years, such as 3, 4, 5, 6, or 8°C, for example, after storage or incubation at about 4°C.

117. A liquid formulation according to any one of claims 114 to 117, comprising at least 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, or 0.99 moles of free thiols per mole of albumin or a fragment thereof.

118. The liquid formulation according to claim 117, comprising at least 1 mole of free thiol per mole of albumin or a fragment thereof.

119. A liquid formulation according to any one of claims 114 to 118, comprising a theoretical free thiol level of at least 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

120. The liquid formulation according to claim 119, comprising a theoretical free thiol level of 100%.

121. A liquid formulation according to any one of claims 114 to 120, comprising approximately 2.5 to approximately 7.5 mM of fatty acids.

122. A liquid formulation according to claim 121, comprising approximately 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 to approximately 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5 mM fatty acids.

123. A liquid formulation according to claim 122, comprising approximately 4 to approximately 6 mM fatty acids.

124. A liquid formulation according to claim 123, comprising approximately 5 mM fatty acids.

125. A liquid formulation according to any one of claims 114 to 124, having a cation concentration of at least about 175 mM.

126. The liquid formulation according to claim 125, having a cation concentration of approximately 200, 225, 250, 275, or 300 mM to approximately 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mM.

127. The liquid formulation according to claim 126, having a cation concentration of approximately 200 to approximately 300 mM.

128. The liquid formulation according to claim 127, having a cation concentration of approximately 225 to approximately 275 mM.

129. A liquid formulation according to any one of claims 114 to 128, having a pH of approximately 5.5 to approximately 6.

5.

130. A liquid formulation according to claim 129, having a pH of approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 to approximately 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.

5.

131. The liquid formulation according to claim 130, having a pH of approximately 5.7 to approximately 6.

2.

132. A liquid formulation according to claim 131, having a pH of approximately 6.

133. A liquid formulation comprising albumin, fatty acids, and cations, wherein the formulation has the following formula: (a) Free thiol = 1.1786 - 0.05167 ＊ pH - 0.0001544 ＊ Cation - 0.01133 ＊ FA + (pH - 6) ＊ ((Cation - 198.3) ＊ 0.0002659) + (pH - 6) ＊ ((FA - 7.125) ＊ 0.003160) + (Cation - 198.3) ＊ ((FA - 7.125) ＊ 0.000001935) + (pH - 6) ＊ ((pH - 6) ＊ -0.0500) + (Cation - 198.3) ＊ ((Cation - 198.3) ＊ 0.000002583) + (FA - 7.125) ＊ ((FA - 7.125) ＊ 0.0001413) + (pH - 6) ＊ ((Cation - 198.3) ＊ ((FA - 7.125) ＊ -0.000009879)), and (b) Polymer = 9.1677 - 0.8417 ＊ pH-0.01044 ＊ Cation -0.2690 ＊ FA+ (pH-6) ＊ ((Cation-198.3) ＊ -0.004796)+(pH-6) ＊ ((FA-7.125) ＊ (0.06523) + (cation -198.3) ＊ ((FA-7.125) ＊ 0.001494)+(pH-6) ＊ ((pH-6) ＊ 2.25) + (cation -198.3) ＊ ((Cation-198.3) ＊ 0.00006768) + (FA-7.125) ＊ ((FA-7.125) ＊ 0.02490)+(pH-6) ＊ ((Cation-198.3) ＊ ((FA-7.125) ＊ It is located within the design space defined by both of 0.001237)) During the ceremony, "Free thiol" refers to the level of free thiols. "Cation" refers to the cation concentration in mM units. "FA" refers to a liquid formulation where the fatty acid concentration is expressed in mM units.

134. For example, the formulation according to claim 133, comprising, after storage or incubation at 40°C for at least 1, 2, 3, 4, 5, or 6 months, at least 0.75 moles of free thiol per mole of albumin or a fragment thereof, and up to 1% (w / w) of albumin polymer.

135. The above formulation is given by the following formula: (a) Free thiol = 1.1786 - 0.05167 ＊ pH-0.0001544 ＊ Cation -0.01133 ＊ FA+ (pH-6) ＊ ((Cation-198.3) ＊ 0.0002659)+(pH-6) ＊ ((FA-7.125) ＊ (0.003160) + (cation -198.3) ＊ ((FA-7.125) ＊ 0.000001935)+(pH-6) ＊ ((pH-6) ＊ (-0.0500) + (cation -198.3) ＊ ((Cation-198.3) ＊ 0.000002583)+(FA-7.125) ＊ ((FA-7.125) ＊ 0.0001413)+(pH-6) ＊ ((Cation-198.3) ＊ ((FA-7.125) ＊ -0.000009879), and (b) Polymer = 9.1677 - 0.8417 ＊ pH-0.01044 ＊ Cation -0.2690 ＊ FA+ (pH-6) ＊ ((Cation-198.3) ＊ -0.004796)+(pH-6) ＊ ((FA-7.125) ＊ (0.06523) + (cation -198.3) ＊ ((FA-7.125) ＊ 0.001494)+(pH-6) ＊ ((pH-6) ＊ 2.25) + (cation -198.3) ＊ ((Cation-198.3) ＊ 0.00006768) + (FA-7.125) ＊ ((FA-7.125) ＊ 0.02490)+(pH-6) ＊ ((Cation-198.3) ＊ ((FA-7.125) ＊ It is located within the design space defined by both of 0.001237)) During the ceremony, "Free thiol" refers to the level of free thiols. "Cation" refers to the cation concentration in mM units. A liquid formulation according to any one of claims 114 to 134, wherein "FA" means fatty acid concentration in mM units.

136. The aforementioned fatty acid, C ６ , C ８ , or C １０ fatty acids and other C ６ Selected from fatty acids or larger substances, preferably C ７ Fatty acids or larger substances, most preferably C ８ A liquid formulation according to any one of claims 121 to 135, wherein the liquid formulation is a fatty acid (octanoate).

137. The liquid formulation according to any one of claims 125 to 136, wherein the cation is selected from sodium, potassium, calcium, magnesium, and ammonium, and is preferably sodium.

138. A liquid formulation according to any one of claims 114 to 137, comprising about 225 to about 275 mM of cation, preferably sodium, and 4 to 6 mM of fatty acid, preferably octanoate, and having a pH of about 5.7 to about 6.

2.

139. The liquid formulation according to claim 138, wherein the formulation comprises about 250 mM of cation, preferably sodium, about 5 mM of fatty acid, preferably octanoate, and about pH 6.

0.

140. The liquid formulation according to any one of claims 114 to 139, wherein the albumin or a fragment thereof is fused to a partner.

141. The liquid formulation according to any one of claims 114 to 140, wherein the albumin, fragments or fusion thereof comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 free thiols.

142. The liquid formulation according to any one of claims 114 to 141, wherein one or more of the free thiols are provided by one or more cysteine ​​residues at one or more (e.g., several) positions in albumin or a fragment or fusion thereof.

143. The free thiols are those of sequence number 2, 34, 1, 2, 4, 38, 40, 48, 52, 55, 58, 60, 75, 76, 79, 80, 82, 83, 83, 86, 91, 104, 113, 115, 116, 121, 122, 124, 125, 129, 168, 169, 177, 229, 236, 266, 269, 270, 273, 283, 298, 300, 301, 303, 304, 308, 313, 314, 316, 318, 320, 321, 324, 325, 355, 360, 361, 364, 365, 368, 369, 371, 375, The liquid formulation according to claim 142, provided by cysteine ​​at a position corresponding to one or more (e.g., several) of the positions selected from 379, 386, 390, 396, 397, 435, 439, 443, 471, 478, 479, 490, 496, 498, 501, 503, 504, 505, 506, 508, 512, 538, 541, 542, 546, 549, 550, 558, 560, 562, 564, 565, 566, 567, 573, 574, 577, 578, 580, 581, 582, 584, and 585.

144. The liquid formulation according to claim 143, wherein the free thiol is provided by cysteine ​​at the position corresponding to position 34 of SEQ ID NO:

2.

145. The liquid formulation according to any one of claims 114 to 144, wherein the albumin or fragment thereof is preferably selected from the group consisting of mammalian albumin or fragment thereof, HSA (SEQ ID NO: 2), mouse (SEQ ID NO: 3), rat (SEQ ID NO: 4), macaque (SEQ ID NO: 5), cattle (SEQ ID NO: 6), pig (SEQ ID NO: 7), horse (SEQ ID NO: 8), or rabbit (SEQ ID NO: 9).

146. The liquid formulation according to any one of claims 114 to 145, wherein the albumin or its fragment or fusion has at least 70% sequence identity with SEQ ID NO:

2.

147. The liquid formulation according to claim 146, wherein the albumin or its fragment or fusion has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:

2.

148. The liquid formulation according to claim 146 or 147, wherein the albumin or its fragment or fusion has 1, 2, 3, 4, 5, 6, 7, 8, or 9 to 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences compared to SEQ ID NO:

2.

149. The liquid formulation according to claim 148, wherein the albumin or a fragment or fusion thereof contains SEQ ID NO 2.

150. The liquid formulation according to any one of claims 114 to 149, wherein the albumin fragment comprises at least 175 amino acids.

151. The liquid formulation according to any one of claims 114 to 150, wherein the albumin fragment comprises at least 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 500, or 575 amino acids.

152. The liquid formulation according to any one of claims 114 to 151, wherein the albumin fragment comprises at least one (e.g., several) of domain I, domain II, or domain III of HSA.

153. The liquid formulation according to any one of claims 114 to 152, wherein the albumin or its fragment or fusion is recombinant.

154. The liquid formulation according to any one of claims 114 to 153, wherein the albumin or its fragments or fusions are produced in a eukaryotic host or a prokaryotic host.

155. The liquid formulation according to claim 154, wherein the eukaryotic host is selected from fungi, plants, and mammals.

156. The liquid formulation according to claim 155, wherein the fungus is selected from Aspergillus, Kluyveromyces, Pichia (e.g., Pichia pastoris), and Saccharomyces (e.g., Saccharomyces cerevisiae).

157. The liquid formulation according to claim 156, wherein the Saccharomyces is Saccharomyces cerevisiae.

158. The liquid formulation according to claim 155, wherein the plant is selected from potatoes, tobacco, and rice.

159. The liquid formulation according to claim 158, wherein the aforementioned rice is Oryza sativa.

160. The liquid formulation according to claim 155, wherein the mammal is a mammalian cell.

161. The liquid formulation according to claim 160, wherein the mammalian cell is CHO or HEK.

162. The liquid formulation according to any one of claims 114 to 161, wherein the albumin is derived from human or animal serum.

163. The liquid formulation according to any one of claims 114 to 162, wherein the albumin or its fragments or fusions are present in an amount of 5 to 30% (w / v), preferably 10 to 20% (w / v), most preferably about 20%.

164. The liquid formulation according to any one of claims 114 to 163, wherein the protein content of the formulation is at least 50% albumin or its fragments or fusions, more preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% albumin or its fragments or fusions.

165. A liquid formulation according to any one of claims 114 to 164, which is substantially or completely free of proteins other than albumin, its fragments, or fusions.

166. The liquid formulation according to any one of claims 114 to 165, wherein the formulation is aqueous.

167. A liquid preparation according to any one of claims 114 to 166, presented in a container.

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