Albumin formulations and uses thereof

Abstract

The present invention relates to a self-buffering albumin formulation and uses thereof. In particular, the present invention relates to a stable, self-buffering albumin formulation whereby the albumin is substantially free from aggregation and provides buffering without the need for small molecule buffers.

Description

[0001] ALBUMIN FORMULATIONS AND USES THEREOF

[0002] FIELD

[0003] The present invention relates to a self-buffering albumin formulation and uses thereof. In particular, the present invention relates to a stable, self-buffering albumin formulation whereby the albumin is substantially free from aggregation and provides buffering without the need for small molecule buffers.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0006] BACKGROUND

[0007] The invention relates to a new formulation of albumin and to uses of the albumin formulation.

[0008] Albumin is the most abundant protein in plasma, constituting approximately 60% of total plasma proteins (Exec. Comm, of the German Med. Assoc., et al,. 2016). Albumin has been described and characterised from many mammals and birds. Albumin is believed to have a role in maintaining correct osmotic pressure and it also has a role in transport of various compounds in the blood stream. Albumin is a protein which is used to treat patients with severe burns, shock or blood loss. It is also used as an excipient for pharmacologically active compounds, many of which need to be stabilised for example to reduce the formation of soluble aggregates and / or insoluble aggregates. Furthermore, albumin is used to supplement media used for growing higher eukaryotic cells, including stem cells. Albumin fusion proteins are a fusion, such as a genetic fusion, of a protein to albumin, or to a variant or fragment thereof, and may increase or decrease the half-life of the protein, for example increased in vivo half-life. Conjugation partners, e.g. proteins or chemicals, can be conjugated to albumin to increase or decrease the half-life of the conjugation partner, for example increased in vivo half-life. At present albumin is obtained from blood products, such as serum, or produced recombinantly in microorganisms such as yeast e.g. WO 96 / 037515, WO 00 / 044772) or from transgenic plants or animals. Typically, albumin is purified from the production source in order to provide a product which is sufficiently pure to meet the user's needs and / or to achieve a high yield of product. In some technical areas, such as cell culture or pharmaceuticals, there is a desire for products to be substantially free or completely free of animal derived components.

[0009] Purified albumin in a final liquid form is relatively unstable (compared to albumin in solid form) and so in order to maximise its shelf life it is either lyophilised and / or stabilisers are added to the final liquid formulation. However, lyophilization can add significantly to the overall cost of the preparation and can be inconvenient to the end user who would need to resuspend the lyophilised product if they need a liquid product. For the preferred liquid product, stabilisers that are commonly added to albumin are n-acetyl-tryptophan, octanoic acid (octanoate, caprylate) and / or polysorbate 80 (e.g. Tween® 80). The albumin of WO 00 / 044772 is stabilised by octanoic acid. Octanoic acid and n-acetyl-tryptophanate is shown to stabilise heat-induced aggregation of bovine serum albumin (Arakawa & Kita, 2000). Heat-treated human albumin is also stabilised by octanoic acid and n-acetyl-tryptophanate (Hosseini et al., 2002).

[0010] Most stable protein formulations for biological uses such as in cell media or in therapeutics, include a small molecule buffering species to control pH and hence maintain stability. Buffers are typically considered an integral part of protein formulations where they regulate pH and stabilise proteins by various mechanisms (Zbacnik et al., 2017). Lists of such formulations are available in the literature, with some examples of buffer containing formulations below:

[0011] 1. Avastin® (bevacizumab) is a monoclonal antibody formulated with a 51 mM sodium phosphate buffer, pH 6.2.

[0012] 2. Rituxan® (rituximab) is a monoclonal antibody formulated with a 7.35 mg / mL sodium citrate dihydrate buffer, pH 6.5.

[0013] 3. Human Serum Albumin (30%), (SigmaAldrich, Merck KGaA, Darmstadt, Germany, product code 12667-M) formulated with 20 mM Tris-HCI, pH 6-8.

[0014] 4. Neupogen® (filgrastim) is a recombinant human granulocyte-colony stimulating factor protein (rhG-CSF) formulated with 10 mM sodium acetate buffer, pH 4.0.

[0015] 5. Aranesp® (darbepoetin alfa) is an erythropoiesis-stimulating protein formulated with an 18 mM sodium phosphate buffer, pH 6.2 ±0.2.

[0016] In general, the technical literature strongly supports the inclusion of smallmolecule buffers in protein formulations. However, the presence of octanoic acid, polysorbate 80 and / or small-molecule buffering species at significant concentrations ranging from 1-100 mM can cause compatibility issues with some uses of human albumin, particularly in cell media. The very common, phosphate "Good's Buffers" (Good et al., 1966) are a prime example of this whereby the phosphate often chelates metal ions and may cause precipitation of the metal ions. Octanoic acid (and conjugate base octanoate) is a very effective and commonly used protein fold stabiliser or "classical stabiliser" of human albumin with binding driving conformational change and leads to higher thermal aggregation temperatures (Ballou et al., 1944). Other classical stabilisers include n-acetyl tryptophan. There is therefore a need for improved albumin formulations for use in cellbased techniques, biological media, pharmaceutical formulations and the coating of medical devices or implants.

[0017] The inventors have identified self-buffering properties of albumin, leading to the development of a platform albumin formulation that can be combined with an additional array of excipients to yield new commercially applicable formulations. The inventors have surprisingly found that albumin can provide superior buffering capacity compared to an equivalent molar concentration of small molecule buffer. Additionally, the inventors have found that such a formulation remains stable for at least 6 months with a high degree of free thiol retention and a low degree of aggregation. Therefore, the invention provides for an albumin formulation that can be used to partially or fully substitute small molecule buffers.

[0018] All documents referred to herein are incorporated by reference in their entirety. The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0019] SUMMARY OF INVENTION

[0020] The invention provides a liquid formulation of self- buffering, stable albumin, wherein the albumin provides for the buffering capacity of the formulation and remains substantially free from aggregation. The invention also provides uses of the formulation, such as in cell-based techniques, biological media, pharmaceutical formulations and the coating of medical devices or implants.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] Definitions

[0023] The term "cell-based techniques" includes any procedure undertaken in the handling of cells, preparation of cells for further processing, and / or storage. The term can encompass but is not limited to cell culture, cell storage, cell washing, cell purification and cell selection. In some instances, "cell-based techniques" can refer to the handling, preparation, further processing and storage of ex vivo tissue. Such cellbased techniques may be used to produce products such as small molecules, large molecules, assemblies and cell therapies.

[0024] The term "biological medium" is defined as a solution suitable for use in biological applications. For example, this may be in the form of a growth media for use in cell culture, storage medium for pharmaceutical products or solution used in the processing of biologies. The term "free from aggregation" refers to a protein formulation that does not contain aggregated protein, for example does not contain aggregated protein within recognised pharmacopoeia limits and / or is unlikely to form aggregates in typical product lifespans in typical storage conditions due to the absence of aggregation competent species. Protein aggregates can include "insoluble" particles ranging from 100 nm to 100 pm, which in some instances may be observable by eye or by analytical means including spectroscopic techniques measuring absorbance at 300-900 nm. Other imaging techniques can also be employed such as membrane imaging and microflow imaging microscopy (Zdlls et al., 2013) with instruments such as Halo Aura and ProteinSimple™ Micro-Flow Imaging. Aggregates can also include "soluble" or smaller aggregates, often characterised as colloids or micelle-like particles, associated through supramolecular or covalent interaction. This type of aggregation can be detected by dynamic light scattering (DLS), multi-angle light scattering (MALS), size-exclusion chromatography (SEC) / gel permeation chromatography (GPC), asymmetric flow field flow fractionation (AF4), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), Fourier Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), mass photometry, electron microscopy, atomic force microscopy and other similar methodologies.

[0025] Accordingly, the term "substantially free from aggregation" refers to a protein formulation that contains a minimal percentage of aggregated protein. For example, the percentage of aggregated protein can be less than or equal to 10% of the total protein mass. In assessing a protein formulation's overall aggregation state, the skilled person would expect the degree of aggregation to increase as a function of time and / or temperature and would appreciate that wider ranges may also be considered substantially free from aggregation.

[0026] By the term "buffer" and "provides buffering across a pH range", we refer to the property of a substance or mixture of substances which resist changes in pH upon challenge, such as titration, with acid or base. In particular, we refer to the pH range in which the substance or mixture of substances exhibits this property. For a small molecule buffer, this would be expected to be in agreement with the calculated pKa. For example, the buffering range would be the pKa ± 1 pH unit.

[0027] By the term "buffering capacity", we refer to the following formula: P = n / ApH, wherein p (buffering capacity) is a function of the number of moles (n) of strong acid or strong base required to change the pH of a solution (ApH). The related term "combined mean buffering capacity" is the combined mean obtained from separate titrations of acid and base. The skilled person will appreciate that the unit can be weighted according to molar mass, strength of the acid or base, or other characteristics. The term "Good's buffer" refers to a series of known buffers characterised in Good, N., et al., (1966), Good, N., et al., (1972) and Ferguson, W. J., et al., (1980). Good's buffers were originally selected based upon high water-solubility, low cell membrane permeability, consistent acid-base dissociation constants, low metal chelating capability, high chemical stability and low absorption spectra in UV and visible regions. Good's buffers can include ACES, Acetamidoglycine, ADA, BES, Bicine, Cholamine chloride, DIPSO, Glycinamide, Glycylglycine, HEPES, HEPPS, HEPPSO, MES, MOPS, MOPSO, phosphate, PIPES, POPSO, TAPS, TAPSO, TES, Tricine and Tris.

[0028] The term "small molecule buffer" (and synonymous terms: "non- macromolecular buffer", "traditional buffer" and "classical buffer") refers to small compounds, e.g. less than 1000 Daltons, capable of resisting changes in pH. The terms include organic and inorganic buffers whereby "organic" refers to any compound containing carbon and the contrasting term "inorganic" refers to any compound not containing carbon. Such buffers can include but are not limited to ACES, Acetate, ADA, AMP, AMPD, AMPSO, BES, BES, Bicarbonate, Bicine, Bis-Tris, Bis-Tris Propane, Borate, CABS, CAPS, CAPSO, CHES, Citrate, DIPSO, EPPS, Gly-Gly, HEPBS, HEPES, HEPPSO, Histidine-HCI, Imidazole, MES, MOBS, MOPS, MOPSO, MOPSO, phosphate, PIPES, POPSO, TABS, TAPS, TAPS, TAPSO, TEA, TES, Tricine, Tris, Tris-Citrate and Tris-HCl. The terms can extend to "zwitterionic compounds", characterised by molecules that contain an equal number of positively and negatively charged functional groups. Additionally, the terms can encompass commonly used biological substances such as amino acids. Examples can include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Specifically, the terms exclude macromolecular buffers.

[0029] The term "macromolecular buffer" refers to macromolecules in solution capable of resisting changes in pH. The term "macromolecule" refers to a molecule containing, for example, more than 1000 Daltons, such as a protein (typically more than 8000 Daltons), nucleic acid, or synthetic polymer. In the context of the present invention, the skilled person will appreciate that the term "macromolecular buffer" particularly relates to albumin formulations due to the self-buffering capacity of albumin.

[0030] The term "strong acid" refers to an acid which ionizes completely in a solution of water. Examples of strong acids can include but are not limited to Hydrobromic acid (HBr), Nitric acid (HNO3), Hydrochloric acid (HCI), Sulfuric acid (H2SO4), Perchloric acid (HCIO4), Hydroiodic acid (HI), and Chloric acid (HCIO3). In the context of the present invention, a strong acid would relate to a concentrated strong acid solution commonly used in the art to adjust the pH of a buffer. For example, a 1 M solution of HCI may be used.

[0031] The term "strong base" refers to a base which dissociates completely into its respective ions in water. Many strong bases are metal hydroxides composed of a metal ionically bonded to a hydroxyl ion. Examples can include but are not limited to sodium hydroxide (NaOH), potassium hydroxide (KOH) and calcium hydroxide (Ca(OH)z). In the context of the present invention, a strong base would relate to a concentrated strong base solution commonly used in the art to adjust the pH of a buffer. For example, a 1 or 0.5 M solution of NaOH may be used.

[0032] The term "classical stabiliser" refers to chemical agents commonly used to increase the stability (for example, preventing aggregation) and / or shelf life. These agents would be recognisable to the skilled person and can include sugars, polyols (e.g. PEG), fatty acids, surfactants, amino acids, small organics, and derivates or combinations thereof. Specific examples can include octanoic acid, polysorbate 80, and acetyl-tryptophan.

[0033] The term "GP-HPLC" also referred to as "GPC" or "gel permeation chromatography" is a type of size-exclusion chromatography (SEC), that separates high molecular species or colloidal aggregates based on hydrodynamic volume. The method is commonly used to assess aggregation state in protein preparations.

[0034] The term "albumin" means a protein having the same and / or very similar tertiary structure as human serum albumin (HSA) or one or more HSA domains and has similar properties to HSA or the relevant domain(s). Similar tertiary structures are, for example, the structures of the albumins from the species mentioned under "parent albumin". Some of the major properties of albumin are i) its ability to regulate plasma volume, ii) a long plasma half-life of around 19 days ± 5 days, iii) ligandbinding, e.g. binding of endogenous molecules such as acidic, lipophilic compounds including bilirubin fatty acids, hemin and thyroxine (see also Table 1 of Kragh-Hansen etal., 2002, hereby incorporated by reference), iv) binding of small organic compounds with acidic or electronegative features e.g. drugs such as warfarin, diazepam, ibuprofen and paclitaxel (see also Table 1 of Kragh-Hansen et al., 2002, hereby incorporated by reference). Not all of these properties need to be fulfilled to in order to characterise a protein or fragment as an albumin. If a fragment, for example, does not comprise a domain responsible for binding of certain ligands or organic compounds the variant of such a fragment will not be expected to have these properties either. The term "albumin" includes variants, and / or derivatives such as fusions and / or conjugations of an albumin or of an albumin variant. In some instances, the term is further abbreviated to "HA". The term "polymeric albumin" means a high molecular weight form of, or aggregate of albumin, for example which elutes in the void volume of a gel permeation HPLC column which separates molecules in the molecular weight range 10000 to 500000 Da, e.g. a TOSOH TSK G3000SWXL column. "Polymer" does not include dimer or trimer. "Polymer" includes tetramer and larger species. Naturally occurring albumin polymers are found in very low concentrations in fresh serum (less than 1% of total albumin) and can be formed through prolonged or improper storage (Wright et al., 1987).

[0035] The term "variant" means a polypeptide derived from a parent albumin comprising an alteration, i.e., a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position. The altered polypeptide (variant) can be obtained through human intervention by modification of the polynucleotide sequence encoding the parental albumin. 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2 and maintains at least one of the major properties of the parent albumin or a similar tertiary structure as HSA. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0036] The term "wild-type" (WT) albumin means an albumin having the same amino acid sequence as the albumins naturally found in an animal or in a human being. SEQ ID NO: 2 is an example of a mature wild-type albumin from Homo sapiens. SEQ ID NO: 3 is an example of an immature wild-type albumin from Homo sapiens.

[0037] The term "parent" or "parent albumin" means an albumin to which an alteration is made to produce the albumin variants which may be used in the present invention. The parent may be a naturally occurring (wild-type) polypeptide or an allele thereof or a variant thereof such as a variant described in WO 2011 / 051489, WO 2011 / 124718, WO 2012 / 059486, WO 2012 / 150319, WO 2014 / 072481, WO 2013 / 135896, WO 2015 / 036579, WO 2010 / 092135, WO 2013 / 075066, WO

[0038] 2014 / 179657, WO 2009 / 126920, WO 2010 / 059315, WO 2011 / 103076, WO

[0039] 2012 / 112188, WO 2015 / 063611, and WO 2017 / 029407 (the contents of which are incorporated herein by reference in their entirety, and in particular references to albumin variants).

[0040] The term "fusion" means a genetic fusion of albumin (or a variant or fragment thereof) and a non-albumin protein. The non-albumin protein may be a therapeutic, prophylactic, or diagnostic protein. Examples of albumin fusions are provided in EP624195, WO 01 / 079271, WO 03 / 059934, WO 03 / 060071, WO 2011 / 051489, WO 2011 / 124718 and EP11164955 (incorporated herein by reference in their entirety).

[0041] The term "recombinant" in the context of albumin protein in this application refers to artificially produced (and often purified) protein i.e. protein heterologously produced with exogeneous DNA. Therefore, references to "rHA" in the application should be taken to mean recombinant human albumin.

[0042] The term "conjugation" means an albumin (or a variant or fragment or fusion thereof) to which a non-albumin moiety is chemically conjugated. The non-albumin moiety may be a therapeutic, prophylactic, or diagnostic protein. Examples of albumin conjugations are provided in WO2011 / 124718 and EP11164955 (incorporated herein by reference in their entirety).

[0043] As used herein, the term "mammal" includes any human or non-human mammal, including but not limited to porcine (such as pigs), ovine (such as sheep), bovine (such as cattle, e.g. cows), rodents (such as rats, mice, guinea pigs, hamsters), ungulates, goats, deer, equine (such as mules, horses), primates (such as human primates and non-human primates), non-human primates (such as monkeys (e.g. macaques), chimpanzees, gorillas), dogs, cats, rabbits and birds (such as chickens).

[0044] The term "cell' includes any cell such as, but not limited to, any human or non- human mammalian cell as described herein. A cell may be a normal cell or an abnormal cell (e.g. transformed cells, established cells, or cells derived from diseased tissue samples). The cell may be a somatic cells such as a fibroblast or keratinocyte. Preferred cells are stem cells such as, but not limited to, embryonic stem cells, fetal stem cells, adult stem cells and pluripotent stem cells such as induced pluripotent stem cells. Particularly preferred cells are human embryonic stem cells, human fetal stem cells, human adult stem cells and human pluripotent stem cells such as induced human pluripotent stem cells.

[0045] Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains. The term "comprises" or "comprising" will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e. including, among other things). As such, the term "comprises" will include references to the component consisting essentially of (such as consisting of) the relevant features. The term "consists of" or "consisting of" will take its usual meaning in the art, namely indicating that the component includes and is limited to the relevant features.

[0046] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" may mean plus or minus 5% of the stated number.

[0047] Where a numerical range is provided herein for any parameter, it is understood that all numerical subsets of that numerical range, and all the individual integer values contained therein, are provided as part of the invention.

[0048] As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and therefore includes both event(s) which occur, and events that do not occur.

[0049] The present invention provides a formulation of stable, self-buffering albumin and associated uses in cell-based techniques, biological media and pharmaceutical compositions.

[0050] A first aspect of the invention relates to a stable, self-buffering albumin formulation comprising about 50 mg / mL to about 300 mg / mL albumin, about 125 mM to about 500 mM cations, < 2.0 mM, preferably < 1.0 mM, octanoic acid; and wherein the pH is from about 6.4 to about 7. An advantage of such a formulation may be an absence of additional buffering components, such as phosphate which can have deleterious effects on downstream applications. This may include component crystallisation during storage, precipitation of metal ions and cell toxicity. The inventors have found that the albumin itself can act as a macromolecular buffer and remains stable in the absence of small molecule buffers and / or the absence of classical stabilisers.

[0051] Principally, proteins are comprised of a series of amino acids (termed "residues"). Amino acids, in addition to a-amino and a-carboxyl groups, can have a net positive or negative charge imparted by a side chain functional group. The common groups include the carboxyl groups, contributed by the sidechains of glutamic and aspartic acid; the amino groups on lysine sidechains; the guanidinium groups contributed by arginine sidechains; the imidazolium groups of histidyl residues; the phenolic groups contributed by tyrosyl residues; and the sulfhydryl groups of cysteine residues. Charged residues can interact, e.g. attract, bind or repel, and form an equilibrium with hydrogen and hydroxyl ions according to the Brpnsted-Lowry theory, and thus function as buffers. The combined contribution of surface exposed amino acids allows proteins to exhibit "self-buffering" properties. The "self-buffering" property of proteins has been studied in the context of therapeutic proteins such as antibodies. In Gokarn et al., (2008), self-buffering monoclonal antibodies, at concentrations of 60- 80 mg / mL, were able to surpass the buffering capacity of 10 mM acetate within a pH range of 4-6. The same study also showed that self- buffering antibodies were more resistant to aggregation compared to conventional buffering formulations.

[0052] In the present disclosure, the inventors have surprisingly identified a similar property in albumin and determined that suitably formulated albumin can provide high buffering capacity and resist aggregation and oxidation. As particularly demonstrated by example Formulations 1 and 2 in Examples 1-8, albumin formulated with 175 to 250 mM NaCI and adjusted to pH 6.75 with strong acid or base addition yielded a sufficiently stable solution for storage at 4-40°C for greater than 6 months.

[0053] In the present disclosure, a "stable, self-buffering albumin", refers to an albumin able to resist challenge with acid or base whilst maintaining desirable characteristics including monodispersity and redox homogeneity.

[0054] Hence, rHA Formulations 1 and 2 in the Examples herein can be considered as buffers at a concentration of 125 g / L (1.88 mM). This concentration is comparatively low in molar terms to small molecule buffers that are typically used at 5-100mM, but with an array of charged amino acid species, the protein can be considered a multivalent zwitterion. Major additions of strong acid or base are required to adjust the protein solution pH by small increments and the pH is highly controllable in the manner of a small molecule buffer, within ± 1.0 pH unit from the pKa of the buffer.

[0055] The albumin described in the first aspect can be any suitable albumin that yields a stable, self- buffering solution when formulated at about 50 to about 300 mg / mL with about 125 mM to about 500 mM cations, < 2.0 mM, preferably < 1.0 mM, octanoic acid; and wherein the pH is from about 6.4 to about 7. The skilled person will be aware of suitable albumins for use in implementing the disclosures herein.

[0056] For example, human albumin (e.g. GenBank AAA98797.1 or UniProt P02768-1 Version 2 (updated 1 April 1990), which is SEQ ID NO: 3 herein (immature protein), in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 609 are the mature sequence that is further recited as SEQ ID NO: 2 herein)), non-human primate albumin (such as chimpanzee albumin (e.g. GenBank predicted sequence XP_517233.3 (updated 13 May 2011) which is SEQ ID NO: 4 herein, in which residues 1 to 18 are predicted to be the signal peptide, residues 19 to 24 are predicted to be the propeptide and residues 25 to 609 are predicted to be the mature sequence), gorilla albumin or macaque albumin (e.g. GenBank NP_001182578.1, which is SEQ ID NO: 5 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence)), rodent albumin (such as hamster albumin (e.g. UniProt A6YF56 Version 1 (updated 21 August 2007), which is SEQ ID NO: 6 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence), guinea pig albumin (e.g. UniProt Q6WDN9 Version 1 (updated 05 July 2004), which is SEQ ID NO: 7 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence), mouse albumin (e.g. GenBank AAH49971.1 or UniProt P07724 Version 3 (updated 15 July 1999), which is SEQ ID NO: 8 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence, the mature sequence being provided herein as SEQ ID NO: 19) and rat albumin (e.g. GenBank AAH85359.1 which is SEQ ID NO: 20 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence, or UniProt P02770 Version 2 (23 January 2007), which is SEQ ID NO: 9 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence; for rat albumin, SEQ ID NO: 20 is preferred), bovine albumin (e.g. cow albumin UniProt P02769 Version 4 (updated 1 February 1996), which is SEQ ID NO: 10 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 607 are the mature sequence), equine albumin such as horse albumin (e.g. UniProt P35747 Version 1 (updated 1 June 1994), which is SEQ ID NO: 11 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 607 are the mature sequence) or donkey albumin (e.g. UniProt Q5XLE4 Version 1 (updated 23 November 2004), which is SEQ ID NO: 12 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 607 are the mature sequence), rabbit albumin (e.g. UniProt P49065 Version 2 (updated 1 March 2004), which is SEQ ID NO: 13 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence), goat albumin (e.g. GenBank ACF10391.1 (submitted 2 September 2008), which is SEQ ID NO: 14 herein, in which residues 1 to 583 are the mature sequence), sheep albumin (e.g. UniProt P14639 Version 1 (updated 1 April 1990), which is SEQ ID NO: 15 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 607 are the mature sequence), dog albumin (e.g. UniProt P49822 Version 3 (updated 21 December 2004), which is SEQ ID NO: 16 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 608 are the mature sequence), chicken albumin (e.g. UniProt P19121 Version 2 (updated 1 August 1992), which is SEQ ID NO: 17 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 23 are the propeptide and residues 24 to 615 are the mature sequence) and pig albumin (e.g. UniProt P08835 Version 2 (updated 1 February 2005), which is SEQ ID NO: 18 herein, in which residues 1 to 18 are the signal peptide, residues 19 to 24 are the propeptide and residues 25 to 607 are the mature sequence). Mature forms of albumin are particularly preferred and the skilled person is able to identify mature forms using publicly available information such as protein databanks and / or by using signal peptide recognition software such as SignalP (e.g., SignalP (Nielsen et al., 1997)). SignalP Version 4.0 (Petersen et al., 2011) or Version 6.0 is preferred (Teufel et al., 2022); SignalP Version 6.0 is more preferred.

[0057] Albumin has been described and characterised from a large number of mammals and birds (e.g. albumins listed in W02010 / 092135 (particularly Table 1) and WO2011 / 124718 (particularly page 9 and SEQ ID NO: 2, 4-19 and 31), both incorporated herein by reference in their entirety).

[0058] Human albumin as disclosed in SEQ ID NO: 2 or any naturally occurring allele thereof, is the preferred albumin of the albumin composition according to the invention. SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 1.

[0059] The albumin of the formulation, particularly the human albumin, may be a variant, or a derivative such as fusion of conjugation of an albumin or of an albumin variant. It is preferred that the albumin has at least 70% identity to HSA (SEQ ID NO: 2), more preferably at least 72, 73, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5 % identity to HSA. The albumin variant may have one or more point (several) mutations, e.g. K573P, K573Y, K573W, K500A compared to a parent albumin such as those provided in the sequence listing, particularly SEQ ID NO: 2 (mutations are described in relation to SEQ ID NO: 2 and the skilled person can identify equivalent mutations in other albumins by aligning an albumin sequence against SEQ ID NO: 2 using the EMBOSS software described herein). Therefore, the albumin in the formulation can include mammalian albumins, for example human albumin.

[0060] Advantageously, the composition may comprise a recombinant albumin. That is, the albumin may be sourced from a recombinant organism such as a recombinant microorganism, recombinant plant or recombinant animal. Since some users prefer animal-free ingredients, it is more preferred that the albumin is sourced from a nonanimal recombinant source, such as a recombinant microorganism or recombinant plant. Preferred microorganisms include prokaryotes and, more preferably the host is a eukaryote such as an animal, plant, fungus or yeast, for example, but not limited to, the following species in which albumins have been successfully expressed as recombinant proteins:

[0061] • fungi (including but not limited to Aspergillus (WO 2006 / 066595), Kluyveromyces (Fleer 1991), Pichia (Kobayashi et a!., 1998) and Saccharomyces (Sleep et al 1990)), bacteria (Pandjaitab et al., 2000));

[0062] • animals (Barash et al., 1993);

[0063] • plants (including but not limited to potato and tobacco (Sijmons et al., 1990 and Farran 2002) and rice e.g. Oryza sativa);

[0064] • mammalian cells such as CHO and HEK.

[0065] The concentration of albumin in the formulation can be from about 50 mg / mL to about 300 mg / mL. For example, the concentration of albumin in the formulation can be from about 50, 55, 60, 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, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, or 295 mg / mL, to about 55, 60, 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, 200, 205,

[0066] 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,

[0067] 295, or 300 mg / mL. The concentration of albumin in the formulation can be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,

[0068] 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,

[0069] 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295 or 300 mg / mL. Preferably, the albumin concentration in the formulation is about 75 to 150 mg / mL, about 75 to about 125 mg / mL, or about 100 to about 150 mg / mL. Most preferably, the albumin concentration in the formulation is about 110 to about 140 mg / mL. For example, the albumin concentration in the formulation can be about 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 mg / mL. In the art, protein concentration is often specified in mg / mL or the equivalent unit g / L. For conversion to molar units the concentration in g / L should be divided by the corresponding molecular weight. For example, HSA has a molecular weight of 66,438 Da. A 125 g / L solution of HSA would therefore constitute a 1.88 mM solution. In the case of albumin variants, fragments or derivatives thereof, the molecular weight will vary. A skilled person would appreciate this and substitute the molecular weight with one determined with the variant, fragment or derivative's sequence.

[0070] Recombinant production is advantageous compared to plasma derived albumin due to the minimisation of pathogen contamination and absence of impurities from human plasma. Accordingly, it is preferred that the albumin can be produced recombinantly. In some embodiments, the recombinant albumin is yeast derived. Yeast hosts represent an attractive means of manufacturing due to ease of genetic manipulation and amenability to scalable fermentation. Recombinant yeast-derived albumin also has several advantages over albumin derived from other sources, particularly in the context of cell-based techniques, for example as discussed in WO2018 / 065491. In some embodiments, the albumin recombinantly produced from yeast is a human albumin. In some embodiments, the albumin can be a variant or derivative of a native albumin.

[0071] Anions and cations interact with charged amino acids and therefore may be used to stabilise proteins such as albumin. The cations of the albumin formulation may be provided by any cation and may be provided by one or more (several) classes or species as described below. For example, the cations may be either mono or bivalent, monoatomic or polyatomic and may be provided by one or more (several) of an alkali metal (such as sodium, potassium), an alkaline earth metal (such as calcium, magnesium) or ammonium. It is preferred that the cations are provided by one or more (several) of ammonium, potassium, sodium, calcium, and magnesium, most preferably sodium. Optionally, the cations are provided by the dissociation of a salt yielding a solution with equimolar concentration of cations and anions. In these embodiments, the anion is provided by one or more (several) of phosphate, sulphate, acetate and chloride, most preferably chloride. Optionally, the salt used in the formulation is NaCI.

[0072] The cations are present from at least about 125 mM to about 500 mM. For example, the minimum cation concentrations can include 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, and 475 mM. The maximum cation concentrations can include e.g. 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175 and 150 mM. For example, the formulation may comprise about 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,

[0073] 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,

[0074] 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375,

[0075] 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460,

[0076] 465, 470, 475, 480, 485, 490, 495 or 500 mM cations. Preferably, the cations are present from about 130 to about 400 mM. Most preferably, the cations are present from about 150 to about 300 mM. For example, the formulation may comprise about 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,

[0077] 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,

[0078] 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,

[0079] 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,

[0080] 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,

[0081] 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 or 300 mM cations. Optionally, the cations are present from about 175 to about 250 mM.

[0082] The skilled person would appreciate that cations or anions can provide ionic strength and / or buffering capacity to a solution. A skilled person would also be aware that acids or bases can be provided in salt forms and would therefore be expected to contribute a greater amount of ionic strength to a solution compared to their free forms.

[0083] By "octanoic acid", we mean either octanoic acid, octanoate, or suitable ester derivatives. Octanoic acid is a known excipient compound and can be used to maintain albumin stability. Octanoic acid has a pKa of 4.894 (Wellen et al., 2017), and therefore provides a negligible effect on pH when the solutions are ±1 pH unit from the pKa. In some embodiments, the concentration of octanoic acid is less than 2 mM, preferably less than 1 mM. For example, the concentration of octanoic acid can be less than or equal to 1.5, 1.0, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.01, or 0.001 mM. In some embodiments, the concentration of octanoic acid will depend on protein purification method used and protein source. The skilled person will appreciate that, while the term "octanoic acid" is predominantly used herein, this will typically be in the form of octanoate (i.e. the conjugate base) at a pH above its pKa, typically about 1.0 pH unit above its pKa.

[0084] The pH of the albumin formulation is between about 6.4 to about 7. For example, the pH of the formulation may be from about 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9 to about 6.5, 6.6, 6.7, 6.8, 6.9 or 7. For example, the pH of albumin formulation can be about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.

[0085] In some embodiments, the albumin in the formulation is substantially free from aggregation. Aggregation can be detrimental to protein function and substantial aggregation can lead to precipitation. A formulation substantially free from aggregation would be advantageous in downstream applications. The formulation described in the present disclosure maintains stability in the absence of non-biological buffers. Methods to assess protein aggregation are well known in the art and can include GP-HPLC, SEC, dynamic light scattering and analytical ultracentrifugation (see the definition for "free from aggregation" above for more methods). Runaway precipitation can be observed by visual inspection of the formulation. A suitable method for assessing albumin aggregation by GC-HPLC would be understood by a skilled person as for example, but not limited to, the assay disclosed Example 8 or the assay disclosed in W02013 / 006675 (incorporated herein by reference). The oxidation state of albumin can also provide a measure of stability. Human albumin contains 35 cysteine residues, with a single unpaired residue: cysteine-34. The availability of one free thiol for site-selective chemical conjugation offers an alternative approach to current genetic fusion and association-based products. The redox state of cysteines in a protein, such as cysteine- 34 in human albumin, can be assessed with a simple colorimetry thiol-based assay. Suitable protocols would be understandable by a skilled person for example, but not limited to, the assay described in Tada et al., (2022), which is herein incorporated by reference.

[0086] Stability can be measured through any of the aforementioned methods; however, it is preferred that stability is assessed through GP-HPLC and thiol-based assays. Using these methods, the albumin described in the formulation may remain >80% monomeric as determined by GP-HPLC after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. For example, the percentage monomeric albumin, determined by GP-HPLC, can be at least 80, 81, 82,

[0087] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. Additionally, the albumin in the formulation may retain a population of free thiol (for example the free thiol provided by Cysteine-34 of human albumin) of >85% after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. For example, the percentage of free thiol (for example the free thiol provided by Cysteine-34 of human albumin) can be at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. In addition, the albumin in the formulation may remain >70% monomeric as determined by GP-HPLC after incubation of the formulation at 40°C for at least 1, 1.5, 2 or 3 months, such as for 3 months. For example, the percentage monomeric albumin, determined by GP-HPLC, can be at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% after incubation of the formulation at 40°C for at least 1, 2 or 3 months, such as for 3 months. Lastly, the albumin in the formulation may retain a population of free thiol (for example the free thiol provided by Cysteine-34 of human albumin) of >55% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. For example, the percentage of free thiol (for example the free thiol provided by Cysteine-34 of human albumin) can be at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,

[0088] 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. A high retention of free-thiol (for example the free thiol provided by Cysteine- 34 of human albumin) makes the formulation suitable for the stabilisation of cell culture media and viral vector formulations. A high retention of free-thiol makes the formulation suitable for efficient conjugation of moieties to the albumin, e.g. via Cysteine-34 of human albumin. The skilled person will appreciate that other albumins than human albumin may have one or more free thiol(s) provided by one or more cysteine residues; such residue(s) may include a cysteine that corresponds to Cysteine-34 of human albumin.

[0089] Accordingly, a formulation free from aggregation would be expected to have a low percentage of polymeric albumin. In some embodiments, the formulation has a percentage of polymeric albumin of < 10% after incubation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months. For example, the formulation can have less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% polymeric albumin after incubation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months.

[0090] In accordance with the present disclosure, the albumin concentration in the formulation may fall within a range from about 50 to about 300 mg / mL. For example, the albumin concentration may be from about 50, 60, 70, 75, 80, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 175, 180, 190, 200, 210, 220, 225, 230, 240, 250 mg / mL, to about 300, 250, 240, 230, 225, 220, 210, 190, 180, 175, 170, 160, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 90, 80, 75, 70, 60 mg / mL respectively. It will be understood that the concentration of albumin must be sufficient to maintain a buffering capacity. The inventors have demonstrated that an albumin concentration of 1.88 mM has a greater buffering capacity than an equivalent molar concentration of small molecule buffer. Similarly, the inventors have also shown that an albumin concentration of 0.75 mM can provide a greater buffering capacity than 1.88 mM of small molecule buffer. Therefore, albumin concentrations from about 0.75 to about 5 mM are suitable, such as from about 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 1.8, 1.88, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 to about 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 1.8, 1.88, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5 mM. An albumin concentration of about 0.75, 1.88, 3.01 or 3.96 mM, such as 1.88 mM, would be particularly suitable. Optionally, the concentration of albumin which maintains buffering capacity is about 50, 125, 200 or 263 mg / mL, such as 125 mg / mL.

[0091] A key advantage of the present disclosure over known formulations is that the albumin provides for substantially all of the buffering capacity of the formulation. The term "substantially all" in this context refers to the albumin accounting for all or almost all of the buffering capacity, such that the contribution from other components is zero or negligible. In applications such as cell culture, small molecule buffers have played a significant role. For example, sodium bicarbonate is the most used buffer in tissue culture medium. However, sodium bicarbonate suffers from two drawbacks: 1) poor buffering over the physiological pH range and 2) an increase in alkalinity proportional to carbon dioxide release. Good's buffers can also suffer from similar drawbacks. For example, 0.1 M HEPES can significantly increase osmotic pressure of the medium. Additionally, HEPES can breakdown into cytotoxic free radicals and H2O2 upon exposure to light (UV light).

[0092] In some embodiments, the formulation is substantially or completely free of all other buffers except for albumin.

[0093] By the term "substantially free", we mean that the presence of other buffering compositions or components in the albumin formulation is negligible. Due to the propensity of proteins and especially albumin to non-specifically bind small molecules, the skilled person will understand that complete removal during protein isolation and purification is not always possible. In the case of albumin, which acts as a carrier protein, a small amount of the following ligands can remain bound during purification : fatty acids, amino acids, ions, metals, and for albumin derived from mammalian hosts residual ligands deriving from commercial preparations of mammalian albumin and small molecule buffers. The term can also refer to cases in which buffering compositions or buffering components have been added to the formulation but do not substantially impact pH. In other instances, the term can refer to when the albumin formulation has been prepared in the presence of a small molecule buffer and diluted prior to use whereby the final concentration of buffering composition or buffering components is negligible. In some embodiments, the other buffering compositions or buffering components can be present at concentrations below 1 mM, for example the buffering composition or buffering components are less than 0.5, 0.1, 0.01, 0.001, or 0.0001 mM.

[0094] As described above, some cations and anions can contribute both ionic strength and buffering capacity to a solution. A skilled person would appreciate the properties of each anion or cation and, with a view to formulating a solution substantially free of all other buffers except for albumin, would select appropriate anions and cations accordingly. For example, in the case of acetate, the skilled person would appreciate that it may provide buffering capacity at a range near or overlapping with albumin and / or may participate in the formulation's acid-base equilibria. In some instances, achieving a formulation that is substantially or completely free of all other buffers except for albumin may require the selection of free or salt forms of the cations or anions of interest. In some instances, this may require adjusting the formulation to a final pH outside of the buffering range of the cation or anion. Preferably, as described in the examples herein, the anions and cations are dissociated from a neutral salt. Neutral salts include salts of sodium (e.g. sodium chloride, sodium sulphate, sodium thiosulphate), salts of potassium e.g. potassium chloride, potassium sulphate, potassium thiosulphate), salts of calcium (e.g. calcium chloride, calcium sulphate, calcium thiosulphate), salts of magnesium (e.g. magnesium chloride, magnesium sulphate, magnesium thiosulphate), or salts of ammonium (e.g. ammonium chloride, ammonium sulphate, ammonium thiosulphate). Sodium, potassium, magnesium and ammonium salts are preferred. Calcium salts, such as calcium chloride, are less preferred due to their tendency to precipitate in the presence of phosphate.

[0095] The salts may, for example, be monovalent or divalent.

[0096] By the term "completely free", we refer to when the concentration of all other buffering compositions or buffering components is below the detection threshold of an appropriate analysis technique known in the art. For example, there may be no significant or discernible detection by HPLC or mass spectrometry. Optionally, the other buffering compositions or buffering components may be present at concentrations below 0.1 pM, for example less than 0.01, 0.001, 0.0001, 0.00001, or 0.000001 pM. A skilled person would understand a suitable concentration for a component to have a biological effect and therefore understands the terms substantially free and completely free.

[0097] Another key advantage of the claimed formulation is high buffering capacity. Buffering capacity, as defined above, describes the number of moles of acid or base required to shift the pH of a solution by a predetermined amount. In Example 4 herein, the inventors demonstrate that an albumin formulation prepared in accordance with the present disclosure has a greater molar equivalent buffering capacity compared to a selection of small molecule buffers. For example, the albumin can provide greater buffering capacity compared to the equivalent molar concentration of a Good's buffer or a phosphate buffer.

[0098] In some embodiments, the albumin provides buffering capacity upon challenge with strong base, wherein the buffering capacity can be determined by strong base titrations which may scale according to the albumin concentration. For example, the albumin, such as at 125 g / L, in the formulation can provide a buffering capacity with respect to strong base (e.g. 0.5 M NaOH) from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a buffering capacity with respect to strong base of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a buffering capacity with respect to strong base of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. In some embodiments, the albumin provides buffering capacity upon challenge with strong acid, wherein the buffering capacity can be determined by strong acid titrations. For example, the albumin in the formulation can provide a buffering capacity with respect to strong acid (e.g. 1.0 M HCI) from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, to about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a buffering capacity with respect to strong acid of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a buffering capacity with respect to strong acid of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0099] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.

[0100] In some embodiments, the combined mean buffer capacity provided by albumin is within the same range as the buffering capacities with respect to acid and / or base. For example, the albumin can provide a combined mean buffer capacity of 1-35, such as 3-30, 4-30 or 10-25. The albumin in the formulation can provide a combined mean buffer capacity of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a combined mean buffering capacity of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0101] 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. For example, the albumin can provide a combined mean buffering capacity of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.

[0102] Buffering capacity with respect to acid or base can be determined by low volume titrations with an acceptable strong acid (e.g. 1 M HCI) or strong base (e.g. 0.5 M NaOH). The skilled person would appreciate that buffering capacity may scale according to the concentration of albumin (e.g. rHA) and strength of the titrating acid or base used. Buffering capacity can be determined by titrating known quantities of acid and / or base and measuring the change in pH. The pH can be measured by any suitable means in the art, including the use of electrochemical methods (e.g. electrodes and millivoltmeter, referred to as a pH meter) and colorimetric methods (e.g. indicator solutions or paper). A person skilled in the art would understand the parameters involved in measuring pH, including temperature and pressure. Such a person would appreciate, that in some instances, the pH of a solution varies with temperature. Standard pH measurements can take place at 25°C, and 1 atmosphere (101 kPa). Measurements can also be performed at the intended temperature or temperature range of use or storage temperature, with expected shifts in performance. In some embodiments, the formulation has an intended temperature range for use or storage between 2-40°C (inclusive). In some embodiments, the formulation has an intended temperature range for use or storage between 2-25°C (inclusive), 20-25°C (inclusive), or 2-8°C (inclusive). In some embodiments, the formulation has an intended temperature range for use or storage around 20°C, 25°C or 4°C, preferably 4°C.

[0103] The inventors have surprisingly found that albumin can provide buffering capacity even at pHs > 1 pH unit from its isoelectric point. As exemplified in the Examples below, albumin provides buffering capacity at a pH of 5.25 to 7.75.

[0104] In some embodiments, the albumin provides buffering across a pH range from about 5.25 to about 7.75. For example, the albumin can provide buffering at a pH from about 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, or 7.5, to about 7.75, 7.5, 7.25, 7, 6.75, 6.5, 6.25, 6, 5.75 or 5.5. For example, the albumin can provide buffering at a pH of about 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, or 7.75. A skilled person would understand that in some instances, the efficacy of albumin as a buffer will depend on factors such as the final formulation composition, salts, or temperature. In further embodiments, albumin can be the sole buffering component. In some embodiments, the formulation is substantially free of all other buffers. In some instances, this refers to small molecule buffers. In other instances, this may include other macromolecular buffers.

[0105] The skilled person would appreciate that the various ranges described in relation to the components of the first aspect can be combined in any way. Illustrative but non-limiting examples include:

[0106] ® In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 500 mM cations, < 1 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.3 mM.

[0107] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 400 mM cations, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.3 mM.

[0108] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 250 mM cations, < 1.0 mM octanoic acid; and has a pH of about 6.75. • In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.3 mM.

[0109] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.3 mM.

[0110] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 250 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.3 mM.

[0111] ® In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are potassium and the originating salt is KCI, < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0112] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are potassium and the originating salt is KCI, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0113] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are potassium and the originating salt is KCI, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0114] ® In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are calcium and the originating salt is CaCIz,

[0115] < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0116] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are calcium and the originating salt is CaCIz, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0117] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are calcium and the originating salt is CaCh, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0118] ® In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 1000 mM cations, wherein the cations are sodium and the originating salt is NazSC , < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0119] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 800 mM cations, wherein the cations are sodium and the originating salt is NazSC , < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0120] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 650 mM cations, wherein the cations are sodium and the originating salt is NazSC , < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0121] ® In one embodiment, the albumin formulation can comprise about 25 mg / mL to about 75 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are sodium and the originating salt is NaCI,

[0122] < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.2 mM.

[0123] ® In another embodiment, the albumin formulation can comprise about 30 mg / mL to about 70 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are sodium and the originating salt is NaCI,

[0124] < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.2 mM.

[0125] ® In a further embodiment, the albumin formulation can comprise about 40 mg / mL to about 60 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.5 mM; optionally, wherein the octanoic acid concentration is < 0.2 mM.

[0126] ® In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 300 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are sodium and the originating salt is NaCI,

[0127] < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.8 mM; optionally, wherein the octanoic acid concentration is < 0.65 mM.

[0128] ® In another embodiment, the albumin formulation can comprise about 150 mg / mL to about 250 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.8 mM; optionally, wherein the octanoic acid concentration is < 0.65 mM.

[0129] ® In a further embodiment, the albumin formulation can comprise about 175 mg / mL to about 225 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.8 mM; optionally, wherein the octanoic acid concentration is < 0.65 mM.

[0130] ® In an alternative embodiment, the albumin formulation can comprise about 230 mg / mL to about 290 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are sodium and the originating salt is NaCI, < 2 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 1.5 mM; optionally, wherein the octanoic acid concentration is < 0.9 mM. • In one embodiment, the albumin formulation can comprise about 100 mg / mL to about 150 mg / mL albumin, about 125 mM to about 500 mM cations, wherein the cations are magnesium and the originating salt is MgCh, < 1.0 mM octanoic acid; and has a pH of about 6.6 to about 6.9; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0131] ® In another embodiment, the albumin formulation can comprise about 110 mg / mL to about 140 mg / mL albumin, about 150 mM to about 400 mM cations, wherein the cations are magnesium and the originating salt is MgCh, < 1.0 mM octanoic acid; and has a pH of about 6.7 to about 6.8; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0132] ® In a further embodiment, the albumin formulation can comprise about 120 mg / mL to about 130 mg / mL albumin, about 175 mM to about 325 mM cations, wherein the cations are magnesium and the originating salt is MgCh, < 1.0 mM octanoic acid; and has a pH of about 6.75; optionally, wherein the octanoic acid concentration is < 0.75 mM; optionally, wherein the octanoic acid concentration is < 0.4 mM.

[0133] Formulations 1 to 9 described in the Examples herein, represent suitable exemplified embodiments, as follows:

[0134] ® An albumin formulation comprising about 125 mg / mL albumin, cations are sodium ions, and the originating salt is NaCI, wherein the NaCI is at a concentration of about 175 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

[0135] ® An albumin formulation comprising about 125 mg / mL albumin, cations are sodium ions, and the originating salt is NaCI, wherein the NaCI is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

[0136] ® An albumin formulation comprising about 125 mg / mL albumin, cations are potassium ions, and the originating salt is KCI, wherein the KCI is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0137] ® An albumin formulation comprising about 125 mg / mL albumin, cations are calcium ions, and the originating salt is CaCIz, wherein the CaCIz is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0138] ® An albumin formulation comprising about 125 mg / mL albumin, cations are sodium ions, and the originating salt is NazSC , wherein the NazSC is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0139] ® An albumin formulation comprising about 50 mg / mL albumin, cations are sodium ions, and the originating salt is NaCI, wherein the NaCI is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.2 mM.

[0140] ® An albumin formulation comprising about 200 mg / mL albumin, cations are sodium ions, and the originating salt is NaCI, wherein the NaCI is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.8 mM; most preferably wherein the concentration of octanoic acid < 0.65 mM.

[0141] ® An albumin formulation comprising about 263 mg / mL albumin, cations are sodium ions, and the originating salt is NaCI, wherein the NaCI is at a concentration of about 250 mM, wherein the formulation has < 2 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 1.5 mM; most preferably wherein the concentration of octanoic acid < 0.9 mM.

[0142] An albumin formulation comprising about 125 mg / mL albumin, cations are magnesium ions, and the originating salt is MgClz, wherein the MgClz is at a concentration of about 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM. The albumin formulation described herein can be used in cell-based techniques including cell culture, cell storage, cell washing, cell purification and cell selection. In the case of cell culture, albumin provides nitrogen and essential amino acids required for cell growth. The albumin formulation can be used to supplement biological media. For example, albumin can be used to supplement cell culture media to support the growth and health of cells used in the production of monoclonal antibodies, recombinant proteins, and other biologies.

[0143] The albumin formulation can also be used in pharmaceutical compositions. For example, the albumin formulation can be used in pharmaceutical compositions to treat disorders such as hypovolemia, ascites, hypoalbuminemia including from burns, acute nephrosis, acute respiratory distress syndrome and cardiopulmonary bypass. Albumin can also be used as a pharmaceutical excipient, e.g. to improve viral stability for vaccines and gene therapies, or an API-excipient.

[0144] In some embodiments, the albumin formulation for use in a pharmaceutical composition can be further modified, for example, covalently conjugated to a small molecule ligand, peptide, antibody, antibody fragment, DNA, or RNA. In some embodiments, the albumin can be modified to non-covalently bind a small molecule ligand, peptide, antibody, antibody fragment, DNA, or RNA.

[0145] In some embodiments, the albumin formulation may be free of, or substantially free of, an active pharmaceutical ingredient (API) such as an antibody, a vaccine component, a viral vector (such as a virus) or lipid nanoparticle (LNP).

[0146] The albumin formulation can also be used for the coating of medical devices and / or implants, for example to increase biocompatibility, increase immunocompatibility, promote tissue formation, enhance material corrosion resistance, exhibit antimicrobial properties, or improve anti-fouling properties. Medical devices and / or implants can include but are not limited to oxygenators, bone grafts, sutures, heart valves, catheters, stents, guide wires, hip joints, intraocular lenses, dental implants and biosensors.

[0147] A second aspect of the invention relates to a stable, self-buffering formulation for cell-based techniques or for use as or in a biological medium, wherein the formulation comprises the stable, self- buffering albumin formulation in accordance with the first aspect of the invention.

[0148] It will be appreciated that the stable, self- buffering albumin formulation in accordance with the first aspect of the invention can be re-formulated with additional components to prepare a stable, self-buffering formulation for cell-based techniques or for use as a biological medium or for use in a biological medium. A skilled person understands the need to verify that additional reagents required in preparing a stable, self-buffering formulation for cell-based techniques or for use as or in a biological medium do not impair the stable, self-buffering properties of the albumin formulation.

[0149] The self-buffering formulation may be used to maintain a eukaryotic cell. Preferably, the eukaryotic cell is a mammalian cell, for example, a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is from a cell line. By "cell line" we include the meaning of a defined population of cells that can be maintained in culture for an extended period of time, retaining stability of certain phenotypes and functions. In some embodiments, the cell line is selected from one of the following: HeLa cell, a Huh7 cell, a HMEC cell, a human embryonic kidney cell (e.g., HEK293 cell), a HBEC cell (human bronchial epithelial cell), a HepG2 cell, a HT-29 cell, a T-cell (e.g., a Jurkat cell), a Saos-2 cell, a U2OS cell, a HUVEC cell, a mesenchymal stem cell (MSC) or a fibroblast cell.

[0150] In one embodiment, the stable, self-buffering albumin formulation in accordance with the first aspect of the invention can comprise from about 1% to about 99% of the stable, self- buffering formulation in accordance with the second aspect of the invention. For example, the stable, self-buffering albumin formulation in accordance with the first aspect of the invention can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0151] 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

[0152] 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

[0153] 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

[0154] 96, 97, 98, or 99% of the formulation in accordance with the second aspect of the invention.

[0155] A third aspect of the invention relates to a stable, self-buffering pharmaceutical composition, wherein the formulation comprises the stable, self-buffering albumin formulation in accordance with the first aspect of the invention.

[0156] A person skilled in the art will appreciate that the albumin formulation can fulfil several roles within a pharmaceutical composition. For example, in one embodiment, the albumin can be an API conjugate for drug delivery. In another embodiment, the albumin can be a non-covalent ligand. In another embodiment, the albumin can act as an API excipient such as a viral vector or antibody excipient. The excipient mechanism may be for example: a molecular crowding agent, chaotrope or kosmotrope.

[0157] A fourth aspect of the invention relates to a stable, self-buffering formulation for the coating of medical devices and / or implants, wherein the formulation comprises the stable, self-buffering albumin formulation in accordance with the first aspect of the invention.

[0158] It will be appreciated that the stable, self-buffering formulation in accordance with the fourth aspect of the invention may, for example, include additional components to facilitate coating, as will be known to those skilled in the art.

[0159] A fifth aspect of the invention relates to the use of an albumin formulation, substantially or completely free of inorganic phosphate, as a macromolecular buffer. A person skilled in the art will appreciate that suitable albumin formulations to be used in accordance with the second aspect of the invention will include albumin formulations in accordance with the first aspect of the invention. By "inorganic phosphate" we include both phosphate buffer or free phosphate. For example, the inorganic phosphate from which the albumin formulation should be substantially or completely free can include: (i) Mcllvaine buffer comprising disodium hydrogen phosphate (NazHPCh) and citric acid (Mcllvaine., 1921); (ii) sodium or potassium phosphate salt or combinations thereof, for example "PBS" buffer which typically contains disodium hydrogen phosphate (NazHPC ) and potassium dihydrogen phosphate (KH2PO4). Therefore, "substantially free" should be taken to mean that the final concentration of inorganic phosphate in the formulation is negligible. The concentration of inorganic phosphate could be around or lower than 1 mM. Preferably, the concentration of inorganic phosphate is less than 1 mM. In this context, the term "completely free" should be taken to mean that concentration of inorganic phosphate is below the detection threshold of an appropriate analysis technique known in the art. For example, no significant or discernible detection by HPLC or mass spectrometry techniques.

[0160] The use of a formulation substantially or completely free of inorganic phosphate can minimise one or more of enzyme inactivation, precipitation of cations, excipient crystallisation, viral inactivation or cell stress. By "enzyme inactivation" we refer to either enzyme denaturation, or when an enzyme is no longer catalytically competent due to a change in its active site or through ligand binding. The term can also refer to the complete or partial inhibition of an enzyme. Enzymes classes inhibited by phosphate can include carboxypeptidases, ureases, kinases and dehydrogenases. The term "precipitation of cations" refers to the formation of insoluble phosphate-cation complexes. The propensity of cations to precipitate in the presence of phosphate is well known in the art. Some cations have a greater propensity to precipitate in the presence of phosphate, for example calcium. The term "excipient" crystallisation refers to either the crystallisation of inorganic phosphate or associated salts. This can include temperature or pH induced crystallisation. "Viral inactivation" refers to the decrease in viral particle viability. The term "cell stress" refers to cytotoxicity induced by inorganic phosphate. For example, Ping et al., (2020) showed that phosphate concentrations greater than 40 mM induced cell damage in HEK293 and HeLa cells.

[0161] In some embodiments, the albumin formulation to be used in accordance with the second aspect of the invention is substantially or completely free of all other buffering composition or buffering components except albumin. The buffering composition or component can refer to any non-macromolecular buffer chemical / s as understood in the art. Accordingly, the albumin formulation can be substantially or completely free of non-macromolecular buffering compositions or non-macromolecular buffering components.

[0162] For the terms substantially or completely free, we refer to the discussion in respect of the first aspect of the invention.

[0163] In some embodiments, the albumin provides substantially all of the buffering capacity of the albumin formulation. Accordingly, the albumin would be able to resist acid and base changes without the need for additional buffering reagents. In some embodiments, the albumin confers a buffering capacity that is greater than an equivalent molar concentration of a Good's buffer or a phosphate buffer.

[0164] A sixth aspect of the invention relates to a method to allow tailoring of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants wherein the method comprises: (a) providing a stable, selfbuffering albumin formulation according to the first aspect of the invention; and (b) combining the stable, self-buffering albumin formulation with one or more components to produce the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation or the formulation for the coating of medical devices and / or implants. The method may include adjusting the formulation to a particular need or situation. The method may include preparing or mixing with one or more additional components.

[0165] Tailoring should be interpreted as including, for example, one or several of diluting, adding or combining (i) any albumin formulation described in accordance with the first aspect of the invention with, for example, (ii) a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants. The appropriate final concentration of albumin (volume / volume or weight / volume) would be determined by the skilled person.

[0166] A skilled person would understand which additional component(s) are appropriate to product a final formulation for any given specific use. For example, in embodiments relating to cell culture, the one or more components can include a basal media, antibiotics, and / or growth supplements. In embodiments relating to cell storage, the one or more components can include a basal freezing media, a basal recovery media and / or a cryopreservation solution. In embodiments relating to cell washing, the one or more components can include a cell washing solution. In embodiments relating cell purification, the one or more components can include cell dissociation solutions, enzymatic reagents and anti-aggregation solutions. In embodiments relating to cell selection, the one or more components can include transfection reagents, recovery media and reagents for density gradient separation.

[0167] In embodiments relating to a biological media, the one or more components can include a basal biological media, basal biological media components and / or basal biological media supplements.

[0168] In embodiments relating to a pharmaceutical formulation, the one or more components can include a pharmaceutical agent, a pharmaceutical active ingredient and / or appropriate excipients.

[0169] In embodiments relating to a formulation for the coating of medical devices and / or implants, the one or more components can include any medically suitable medical device or implant in need of an albumin coating to increase biocompatibility, increase immunocompatibility, promote tissue formation, enhance material corrosion resistance, exhibit antimicrobial properties, and / or improve anti-fouling properties of said medical device or implant.

[0170] A seventh aspect of the invention relates to a method of controlling, stabilising and / or buffering the pH of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises: (a) providing a stable, self-buffering albumin formulation according to the first aspect of the invention; and (b) including the stable, self-buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants.

[0171] Controlling, stabilising or buffering the pH of a solution refers to a process of providing a buffering substance to a liquid formulation to bring the measured pH within a desired range. Preferably, the desired range would be the range in which the buffering substance has optimal buffering capacity. To maximise the benefit of the selfbuffering capacity of albumin, preferably the formulations do not comprise additional buffers.

[0172] A skilled person would understand that preparing a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants would require an albumin formulation described in accordance with the first aspect of the invention and one or more components applicable to the specific formulation or its intended use, which would be identifiable to the skilled person.

[0173] For example, the one or more components can be as described above in relation to the sixth aspect of the invention.

[0174] An eighth aspect of the invention relates to a method to reduce and / or minimise precipitation of one or more medium components, such as metal ions, in a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises: (a) providing a stable, self-buffering albumin formulation according to the first aspect of the invention; and (b) including the stable, self-buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biological medium or the pharmaceutical formulation, completely or partially in replacement of one or more Good's buffer(s) and / or phosphate buffer(s).

[0175] A complete replacement can be considered the entire substitution of the or all Good's buffer(s) and phosphate buffer(s) with an albumin formulation according to the first aspect of the invention. In some instances, a complete replacement encompasses when the Good's buffer or phosphate buffer is present at a negligible concentration.

[0176] A partial replacement can be considered the incomplete substitution of a Good's buffer or phosphate buffer with an albumin formulation according to the first aspect of the invention, i.e. such that at least some of a Good's buffer or phosphate buffer is present in the resulting formulation. For example, an albumin formulation according to the first aspect of the invention can replace about 1% to about 99% of the concentration and / or buffering capacity of the Good's buffer or phosphate buffer in a desired formulation. For example, an albumin formulation according to the first aspect of the invention can replace about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0177] 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

[0178] 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

[0179] 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

[0180] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9% of the concentration and / or buffering capacity of the Good's buffer or phosphate buffer. Concentration can be weight / weight, weight / volume or volume / volume; weight / volume is preferred. For example, the skilled person may wish to have a partial replacement to reduce the concentration and / or buffering capacity of Good's buffer or phosphate buffer to avoid saturation and subsequent metal ion complexation.

[0181] Minimising or reducing the precipitation of one or more medium components can refer to a complete or partial reduction in precipitation of one or more medium components. This can include minimisation or reduction in precipitation during preparation, storage, and / or use of the formulation.

[0182] In one embodiment, the eighth aspect can relate to the adaptation and / or modification of an existing formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants to minimise precipitation of one or more medium components by including a stable, self- buffering albumin formulation according to the first aspect of the invention.

[0183] To adapt an existing formulation to reduce and / or minimise precipitation of one or more medium components, a skilled person would, for example:

[0184] (i) Identify a formulation with a known deficiency with respect to component precipitation;

[0185] (ii) Analyse the formula components to determine the role of the components, particularly buffer components;

[0186] (iii) Systematically replace one or more potentially undesirable buffer or buffer component (such as Good's and / or phosphate buffer components) by trialling concentration ratios of [potentially undesirable buffer or buffer component, such as Good's buffer and / or phosphate buffer]: [albumin formulation in accordance with the first aspect];

[0187] (iv) Analyse the resulting formulations to determine if the deficiency has been overcome; and

[0188] (v) Optionally, trial further ratios or suitable intermediate ratios to arrive at an acceptable formulation.

[0189] For example, the concentration ratios may involve a like-for-like replacement (e.g. 60% original buffer component and 40% albumin formulation) or the skilled person may take into account the different buffering capacity of the albumin and use a different proportion of albumin compared to the amount by which the original buffer component is reduced (e.g. 60% original buffer component and 20% albumin formulation).

[0190] The skilled person would appreciate that the concentration ratios would result in either a complete or partial replacement of one or more existing potentially undesirable buffer or buffer component such as Good's buffers and / or phosphate buffers. A complete replacement of all of the potentially undesirable buffer(s) or buffer component(s) such as Good's buffer(s) and phosphate buffer(s) would be appreciated as an entire substitution of a potentially undesirable buffer or buffer component such as Good's buffer or phosphate buffer with an albumin formulation according to the first aspect of the invention. Accordingly, the skilled person would appreciate that the formulation would be completely free or substantially free of all other buffers except albumin. The skilled person would also appreciate that a partial replacement of potentially undesirable buffer or buffer component such as Good's buffer or phosphate buffer would result in a formulation which may or may not be substantially free of all other buffers except albumin.

[0191] In view of the teachings of this aspect, the skilled person would see the advantage in completely or partially replacing one or more potentially undesirable buffer or buffer component such as Good's buffer(s) and / or phosphate buffer(s) with an albumin formulation according to the first aspect. A formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants in which component precipitation is minimised or reduced would be desirable. When generating a new formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, the skilled person in view of the present teachings herein might wish to avoid using one or more Good's and / or phosphate buffer components in favour of using an albumin formulation in accordance with the first aspect of the invention.

[0192] Accordingly, in one embodiment, the aspect can relate to the de novo designing of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, in which an albumin formulation in accordance with the first aspect of the invention is used, such that less or no potentially undesirable buffer or buffer component such as Good's buffer and / or phosphate buffer is required.

[0193] To prepare a de novo formulation, the skilled person would follow guidance in the art to select appropriate components required in a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants. This would include identifying components in the art that have a propensity to cause precipitation. In view of the new teachings therein, the skilled person would then be able to avoid using the one or more problematic components, substituting them with the albumin formulation according to the first aspect of the invention. A ninth aspect of the invention relates to a kit for tailoring of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or formulation for the coating of medical devices and / or implants, wherein the kit comprises (a) a stable, self-buffering albumin formulation according to the first aspect of the invention; and (b) one or more other components for a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants.

[0194] A skilled person would appreciate that the kit in accordance with the ninth aspect of the invention may be used to implement the method in accordance with the sixth aspect of the invention. Hence, the term tailoring can be understood as described above in relation to the sixth aspect of the invention.

[0195] A kit may further comprise of equipment such as containers (including but not exclusively comprised of plastic, glass, and / or metal) suitable or necessary for preparing or using a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants. For example, this may include a syringe or other device for administering a pharmaceutical formulation.

[0196] A kit may further comprise instructions or reference material, such as instructions or reference material for preparing a final formulation for use in cell-based techniques, as or in a biological medium, a pharmaceutical formulation or formulation for the coating of medical devices and / or implants.

[0197] Numbered Paragraphs

[0198] The invention is also further defined by reference to the following numbered paragraphs:

[0199] Paragraph 1. A stable, self- buffering albumin formulation comprising about 50 mg / mL to about 300 mg / mL albumin, about 125 mM to about 500 mM cations, < 2.0 mM, preferably < 1.0 mM, octanoic acid; and wherein the pH is from about 6.4 to about 7.

[0200] Paragraph 2. The formulation according to Paragraph 1, wherein the albumin is substantially free from aggregation.

[0201] Paragraph 3. The formulation according to Paragraph 1 or 2, wherein the albumin in the formulation has one or more of the following properties: (i) remains >80% monomeric as determined by GP-HPLC after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months;

[0202] (ii) retains a population of free thiol of >85% after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months;

[0203] (iii) remains >70% monomeric as determined by GP-HPLC after incubation of the formulation at 40°C for at least 1, 2 or 3 months, such as for 3 months; and / or

[0204] (iv) retains a population of free thiol of >55% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months; optionally, wherein the free thiol is provided by the side chain of Cysteine-34 of human albumin.

[0205] Paragraph 4. The formulation according to any one of Paragraphs 1-3, wherein the formulation has a percentage of polymeric albumin of <10% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months.

[0206] Paragraph 5. The formulation according to any one of Paragraphs 1-4, wherein the albumin is at a concentration of 100 to 150 mg / mL, or at a concentration of 110 to 140 mg / mL, or at a concentration of 120 to 130 mg / mL, optionally at a concentration of about 125 mg / mL.

[0207] Paragraph 6. The formulation according to any one of Paragraphs 1-5, wherein the albumin provides substantially all of the buffering capacity of the formulation.

[0208] Paragraph 7. The formulation according to any one of Paragraphs 1-6, wherein the albumin provides a combined mean buffer capacity of 1-35, 3-30, 4-30 or 10-25, wherein the combined mean buffer capacity is defined as the mean buffer capacity calculated for independent acid and base titrations.

[0209] Paragraph 8. The formulation according to any one of Paragraphs 1-7, wherein the albumin provides:

[0210] (i) a buffering capacity of 1-35, 3-30, 4-30 or 10-25 upon titration with strong acid; and / or

[0211] (ii) a buffering capacity of 1-35, 3-30, 4-30 or 10-25 upon titration with strong base.

[0212] Paragraph 9. The formulation according to any one of Paragraphs 1-8, wherein the formulation is substantially free of all other buffers except albumin. Paragraph 10. The formulation according to any one of Paragraphs 1-9, wherein the formulation is substantially free of non-macromolecular buffers.

[0213] Paragraph 11. The formulation according to any one of Paragraphs 1-10, wherein the albumin confers a buffering capacity that is greater than an equivalent molar concentration of a Good's buffer or a phosphate buffer.

[0214] Paragraph 12. The formulation according to any one of Paragraphs 1-11, wherein the formulation has a pH of about 6.6 to about 6.9, or a pH of about 6.7 to about 6.8, optionally wherein the formulation has a pH of about 6.75.

[0215] Paragraph 13. The formulation according to any one of Paragraphs 1-12, wherein the albumin provides buffering across a pH range of 5.25 to 7.75.

[0216] Paragraph 14. The formulation according to any one of Paragraphs 1-13, wherein the cations are at a concentration of about 125 mM to about 500 mM, or about 150 mM to about 400 mM, or about 175 mM to about 325 mM, optionally wherein the concentration of cations is from about 175 mM or about 250 mM.

[0217] Paragraph 15. The formulation according to any one of Paragraphs 1-14, wherein the cation is selected from ammonium, potassium, sodium, calcium, and magnesium; preferably wherein the cation is sodium; optionally wherein the originating salt is NaCI.

[0218] Paragraph 16. The formulation according to any one of Paragraphs 1-15, wherein the cations are in solution with a corresponding anion, wherein the anion is selected from a list including phosphate, sulphate, acetate and chloride; preferably wherein the anion is chloride; optionally wherein the originating salt is NaCI.

[0219] Paragraph 17. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 175 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

[0220] Paragraph 18. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

[0221] Paragraph 19. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are potassium ions and the originating salt is KCI, wherein the KCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0222] Paragraph 20. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are calcium ions and the originating salt is CaCIz, wherein the CaCIz is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0223] Paragraph 21. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium ions and the originating salt is NazSC , wherein the NazSC is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0224] Paragraph 22. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 50 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.2 mM.

[0225] Paragraph 23. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of 200 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.8 mM; most preferably wherein the concentration of octanoic acid < 0.65 mM. Paragraph 24. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of about 125 mg / mL, wherein the cations are sodium ions and the originating salt is MgCh, wherein the MgCh is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

[0226] Paragraph 25. The formulation according to any one of Paragraphs 1-16, wherein the albumin is at a concentration of about 263 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 2 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 1.5 mM; most preferably wherein the concentration of octanoic acid < 0.9 mM.

[0227] Paragraph 26. The formulation according to any one of Paragraphs 1-25, wherein the albumin is a mammalian albumin.

[0228] Paragraph 27. The formulation according to any one of Paragraphs 1-26, wherein the albumin is human albumin.

[0229] Paragraph 28. The formulation according to any one of Paragraphs 1-27, wherein the albumin is a recombinant albumin.

[0230] Paragraph 29. The formulation according to Paragraph 28, wherein the recombinant albumin is a recombinant yeast-derived albumin.

[0231] Paragraph 30. The formulation according to Paragraph 29, wherein the recombinant albumin is a recombinant yeast-derived human serum albumin.

[0232] Paragraph 31. The formulation according to any one of Paragraphs 17 to 25, wherein the albumin is recombinant yeast-derived human serum albumin.

[0233] Paragraph 32. The formulation according to any one of Paragraphs 1-31, wherein the albumin is a variant or derivative of a native albumin. Paragraph 33. A stable, self-buffering formulation for cell-based techniques or for use as or in a biological medium, wherein the formulation comprises the stable, selfbuffering albumin formulation of any one of Paragraphs 1-32.

[0234] Paragraph 34. A stable, self- buffering pharmaceutical composition, wherein the formulation comprises the stable, self- buffering albumin formulation of any one of Paragraphs 1-32.

[0235] Paragraph 35. The pharmaceutical composition according to Paragraph 34, wherein the albumin is an albumin-API conjugate, albumin-API non-covalent ligand, API excipient, viral vector excipient or antibody excipient.

[0236] Paragraph 36. A stable, self-buffering formulation for the coating of medical devices and / or implants, wherein the formulation comprises the stable, self-buffering albumin formulation of any one of Paragraphs 1-32.

[0237] Paragraph 37. A method to allow tailoring of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:

[0238] (a) providing a stable, self-buffering albumin formulation according to any one of Paragraphs 1-32; and

[0239] (b) combining the stable, self- buffering albumin formulation with one or more components to produce the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants.

[0240] Paragraph 38. A method of controlling, stabilising and / or buffering the pH of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:

[0241] (a) providing a stable, self-buffering albumin formulation according to any one of Paragraphs 1-32; and

[0242] (b) including the stable, self- buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants. Paragraph 39. A method to reduce and / or minimise precipitation of one or more medium components, such as metal ions, in a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:

[0243] (a) providing a stable, self-buffering albumin formulation according to any one of Paragraphs 1-32; and

[0244] (b) including the stable, self- buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants, completely or partially in replacement of one or more potentially undesirable buffer or buffer component such as Good's buffer(s) and / or phosphate buffer(s).

[0245] Paragraph 40. Use of an albumin formulation that is substantially or completely free of inorganic phosphate, as a macromolecular buffer.

[0246] Paragraph 41. The use according to Paragraph 40, wherein the albumin provides substantially all of the buffering capacity of the albumin formulation.

[0247] Paragraph 42. The use according to Paragraph 40 or 41, wherein the albumin formulation is substantially free of all other buffering composition or buffering components except albumin.

[0248] Paragraph 43. The use according to any one of Paragraphs 40-42, wherein the albumin formulation is substantially free of non-macromolecular buffering compositions or non- macromolecular buffering components.

[0249] Paragraph 44. The use according to any one of Paragraphs 40-43, wherein the albumin confers a buffering capacity that is greater than an equivalent molar concentration of a Good's buffer or a phosphate buffer.

[0250] Paragraph 45. The use according to any one of Paragraphs 40-44, wherein the albumin formulation is a stable, self- buffering albumin formulation according to any one of Paragraphs 1-32.

[0251] Paragraph 46. The use according to any one of Paragraphs 40-45, wherein being substantially or completely free of inorganic phosphate minimises one or more of enzyme inactivation, precipitation of cations, excipient crystallisation, viral inactivation or cell stress.

[0252] Paragraph 47. A kit for tailoring of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the kit comprises:

[0253] (a) a stable, self- buffering albumin formulation according to any one of Paragraphs 1- 32; and

[0254] (b) one or more other components for a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants.

[0255] All citations are incorporated herein by reference in their entirety.

[0256] BRIEF DESCRIPTION OF THE FIGURES

[0257] Figure 1 shows the effect of low volume incremental additions of 1.0 M NaOH. The dashed line and solid lines show the linear regression fits obtained for two repeats showing significant variation with additions of this high concentration base. See Figure

[0258] 2 for further data from 0.5 M NaOH additions.

[0259] Figure 2 shows the effect of addition of low volumes of strong acid and base on Formulation 1 and Formulation 2. Data presented from 3 to 4 titration replicates and globally fitted. (A) Formulation 1 : pH shifts with additions of 1.0M HCI yielding |3 of 13.7 with an R2of 0.77, (B) Formulation 1 : pH shifts with additions of 0.5 M NaOH yielded |3 of 16.9 with an R2of 0.87, (C) Formulation 2: pH shifts with additions of 1.0M HCI yielded |3 of 18.6 with an R2of 0.86, and (D) Formulation 2: pH shifts with additions of 0.5 M NaOH yielded |3 of 20.32 with an R2of 0.92.

[0260] Figure 3 shows the effect of addition of low volume, low molarity incremental additions of strong acid and base on PIPES and Mcllvaine buffer. Data presented from

[0261] 3 to 4 titration replicates and globally fitted. (A) PIPES buffer: pH shifts with additions of 0.04 M NaOH yielded |3 of 0.60 with an R2of 0.91, (B) PIPES buffer: pH shifts with additions of 0.04 M HCI yielding |3 of 0.33 with an R2of 0.84, (C) Mcllvaine buffer (sodium phosphate-citrate): pH shifts with additions of 0.04 M NaOH yielding |3 of 0.53 with an R2of 0.94, and (D) Mcllvaine buffer: pH shifts with additions of 0.04 M HCI yielded |3 of 0.32 with an R2of 0.87.

[0262] Figure 4A shows the combined mean buffering capacity of 1.88 mM rHA formulations and 1.88 mM small molecule buffers as calculated by "mean of means". Error bars are calculated as Standard Error of the Mean (SEM) for all samples presented.

[0263] Figure 4B shows the combined mean buffering capacity of 1.88 mM rHA formulations and 1.88 mM small molecule buffers as calculated by "mean of all values". Error bars are calculated as Standard Error of the Mean (SEM) for all samples presented.

[0264] Figure 5 shows the combined mass weighted mean buffering capacity of 1.88 mM rHA formulations and small molecule buffers. Error bars are calculated as Standard Error of the Mean (SEM) for all samples presented.

[0265] Figure 6 shows the typical concentration weighted combined mean buffering capacity of 1.88 mM rHA formulations and the buffering capacity of small molecule buffers calculated equivalent to 25 mM based on data acquired for 1.88 mM buffers. Error bars are calculated Standard Error of the Mean (SEM) for all samples presented.

[0266] Figure 7 shows the degree of free-thiol (Free-SH) stability of Formulation 1 and 2, and of Comparator Formulations 1 and 2 after 6 months of storage at 5°C and 40°C.

[0267] Figure 8 shows the percentage of polymeric albumin within Formulation 1, Formulation 2 and Comparator Formulation 1 and 2 by GP-HPLC with long-term storage at 4±3°C. The T="x" values represent months of incubation at the target temperature.

[0268] Figure 9 shows the percentage of polymeric albumin within Formulation 1, Formulation 2 and Comparator Formulation 1 and 2 Polymeric State by GP-HPLC with long-term storage at 40°C. The T="x" values represent months of incubation at the target temperature.

[0269] Figure 10 shows the combined mean buffer capacity of 1.88 mM rHA formulations with an array of fully dissociated monovalent and divalent ions from neutral salts, namely Formulations 1 and 2 (NaCI), Formulation 3 (KCI), Formulation 4 (CaCIz), and Formulation 5 (NazSC ). Comparison data are provided for small molecule buffers, and Comparator Formulations 1 to 4. Example 9 data is presented in dot- patterned bars, with data from Example 4 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented.

[0270] Figure 11 shows the calculated combined mass weighted mean buffer capacity of 1.88 mM rHA formulations with an array of fully dissociated monovalent and divalent ions from neutral salts, namely Formulations 1 and 2 (NaCI), Formulation 3 (KCI), Formulation 4 (CaCIz), and Formulation 5 (NazSC ). Comparison data are also provided for small molecule buffers, and Comparator Formulations 1 to 4. Example 9 data is presented in dot-patterned bars, with data from Example 5 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented. Figure 12 shows the calculated typical concentration weighted combined mean buffer capacity of 1.88 mM rHA formulations with an array of fully dissociated monovalent and divalent ions from neutral salts, namely Formulations 1 and 2 (NaCI), Formulation 3 (KCI), Formulation 4 (CaCIz), and Formulation 5 (NazSC ). Comparison data are also provided for small molecule buffers, and Comparator Formulations 1 to 4. The data for the small molecule buffers was calculated equivalent to 25 mM based on the data acquired for 1.88 mM buffers. Example 9 data is presented in dot-patterned bars, with data from Example 6 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented.

[0271] Figure 13 shows the degree of free-thiol (Free-SH) stability of Formulation 3 (KCI), and Formulation 5 (NazSC ) after storage at (A) 4°C and (B) 40°C.

[0272] Figure 14 shows the percentage of polymeric albumin within Formulation 3 (KCI), Formulation 4 (CaCIz), Formulation 5 (NazSC ), and Comparator Formulation 2 by GP-HPLC with storage at 4°C. The T="x" values represent months of incubation at the target temperature.

[0273] Figure 15 shows the percentage of polymeric albumin within Formulation 3 (KCI), Formulation 4 (CaCIz), and Formulation 5 (NazSC ) by GP-HPLC with storage at 40°C. The T="x" values represent months of incubation at the target temperature.

[0274] Figure 16 shows the combined mean buffering capacity of rHA formulations with a range of rHA concentrations (50, 125, 200 and 263 g / L, 0.75, 1.88, 3.01, 3.96 mM respectively, Formulations 6, 1, 7 and 8 respectively), all without small molecule buffering species and with the same 250mM NaCI base formulation. Data are also provided for small molecule buffers, Formulation 2 and Comparator Formulations 1 to 4. Example 10 data is presented in dot-patterned bars, with data from Example 4 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented.

[0275] Figure 17 shows the calculated combined mass weighted mean buffering capacity of rHA formulations with a range of rHA concentrations (50, 125, 200 and 263 g / L, 0.75, 1.88, 3.01, 3.96 mM respectively, Formulations 6, 1, 7 and 8 respectively), all without small molecule buffering species and with the same 250mM NaCI base formulation. Data are also provided for small molecule buffers, Formulation 2 (175 mM NaCI) and Comparator Formulations 1 to 4. Example 10 data is presented in dot- patterned bars, with data from Example 5 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented.

[0276] Figure 18 shows the calculated typical concentration weighted combined mean buffering capacity of rHA formulations collected with a range of rHA concentrations (50, 125, 200 and 263 g / L, 0.75, 1.88, 3.01, 3.96 mM respectively, Formulations 6, 1, 7 and 8 respectively), all without small molecule buffering species and with the same 250mM NaCI base formulation. Data are also provided for small molecule buffers (calculated equivalent to 25 mM based on data acquired for 1.88 mM buffers), Formulation 2 (175 mM NaCI) and Comparator Formulations 1 to 4. Example 10 data is presented in dot-patterned bars, with data from Example 6 presented in unfilled bars for comparison. Error bars are calculated as SEM for all samples presented.

[0277] Figure 19 shows the degree of free-thiol (Free-SH) stability of Formulations 6, 7 and 8 after storage at (A) 4°C and (B) 40°C.

[0278] Figure 20 shows the percentage of polymeric albumin within Comparator Formulation 2 and Formulations 6, 7 and 8 by GP-HPLC with storage at 4°C. The T="x" values represent months of incubation at the target temperature.

[0279] Figure 21 shows the percentage of polymeric albumin within Formulations 6, 7 and 8 by GP-HPLC with storage at 40°C. The T="x" values represent months of incubation at the target temperature.

[0280] Figure 22 shows percentage confluence of MSCs with media containing Formulation 1, Formulation 2, Comparator Formulation 2 and 2% FBS base medium control. Error bars equal ±1 standard deviation.

[0281] Figure 23 shows percentage confluence of HEK293 cells with media containing Formulation 1, Formulation 2, Comparator Formulation 2 and 2% FBS base medium control. Error bars equal ±1 standard deviation.

[0282] Figure 24 shows cell counts of T cells with media containing Formulation 1, Formulation 2, Comparator Formulation 2 and 2% FBS base medium control. Error bars equal ±1 standard deviation.

[0283] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0284] EXAMPLES

[0285] Example 1: Preparation of albumin formulations

[0286] Formulation 1 was prepared by TFF diafiltration of Recombumin® Elite (i.e. yeast-derived recombinant wild-type sequence human albumin (SEQ ID NO: 2) at about 100 mg / mL in about 250 mM Na+, < 1 mM octanoic acid, pH 6.0-7.0, (Sartorius Albumedix Limited, UK)) using a PALL Omega T-Series Cassette (P / N :OS010T12) into a 50 mM NaCI solution. Diafiltrated material had its Na+concentration determined by Atomic Absorption Spectroscopy (Perkin Elmer PinAAcle 900T THGA / Flame AA System) and protein concentration determined by the Kjeldahl method (Kjeltec™ 8400, Foss Analytical). Using the determined values, [rHA] and [Na+] (where "[x]" means concentration of "x") was adjusted by addition of 5M NaCI and pyrogen-free water (PFW) to achieve the formulation described in Table 1, prior to 0.2 pm filtration through a polyvinylidene fluoride (PVDF) membrane.

[0287] Formulation 2 was prepared from Formulation 1 material by dilution with PFW to yield a 100 mM NaCI, 50 g / L rHA followed by concentrating 3-fold with 10 kDa molecular weight cut-off (MWCO) Sartorius Vivaspin® 20 centrifugal concentrator polyethersulfone units. [Na+] was measured as previously described to be 110 mM, with [rHA] determined by SEC-HPLC and UV 280 nm detection. rHA was reformulated by addition of 5M NaCI and PFW to achieve the formulation described in Table 1, prior to 0.2 pm filtration through a PVDF membrane.

[0288] To prepare Comparator Formulation 1, Recombumin® Prime (i.e. yeast-derived recombinant wild-type human albumin (SEQ ID NO: 2) at about 200 mg / mL in about 145 mM Na+, about 32 mM octanoic acid, about 15 mg / L Polysorbate 80, pH 6.7-7.3 (Sartorius Albumedix Limited, UK)) was diluted from 200 to 125 g / L with additions of 5.0 M NaCI, PFW and 1.0 g / L Polysorbate 80, to yield the final excipient formulation concentrations excluding octanoic acid which is retained by the protein through the Gibbs-Donnan effect. Comparator Formulation 1 was filtered through a 0.2 pm PVDF membrane into a clean lidded polythene storage tube. Comparator Formulation is described in Table 1.

[0289] To prepare Comparator Formulation 2, Recombumin® Elite was concentrated to >125 g / L with a 10 kDa MWCO Sartorius Vivaspin® 20 unit, [rHA] determined by SEC- HPLC and UV 280 nm detection, and then diluted with 250 mM NaCI and 20 mM sodium phosphate buffer to achieve the formulation described in Table 1. Comparator Formulation 2 was filtered through a 0.2 pm PVDF membrane into a clean lidded polythene storage tube.

[0290] To prepare Comparator Formulation 3, Recombumin® Elite was concentrated with a 10 kDa MWCO Sartorius Vivaspin® 20 unit from 100 to 135 g / L with the concentration determined by SEC-HPLC. The rHA was re-formulated to 125 g / L with additions of 5.0M NaCI, PFW, octanoic acid preparation (28.8% W / W octanoic acid, 7.61% W / W NaOH) and 0.2 M HCI titrated to attain pH 6.0, to yield the final formulation as described in Table 1. Comparator Formulation 3 was filtered through a 0.2 pm PVDF membrane into a clean lidded polythene storage tube.

[0291] To prepare Comparator Formulation 4, water and temperature destabilised albumin was prepared by initially diluting Recombumin® Elite 6-fold into PFW, with three equivalent rounds of diafiltration into PFW in a lOkDa MWCO Sartorius Vivaspin® 100 PES Centrifugal Concentrator. rHA concentration was determined by SEC-HPLC and PFW added to achieve a formulation of 125 g / L. Comparator Formulation 4 was filtered through a 0.2 pm PVDF membrane into a clean lidded polythene storage tube and then incubated at 50°C for 24 hours. Table 1: Compositions of each albumin formulation tested. Example 2: Methods for measuring buffering capacity and albumin stability 2.1 Comparative buffer preparation

[0292] Comparative small molecule buffers commonly used in biological applications were prepared to 1.88 mM concentration and included: DL-Histidine (pKa 6.0), MES ((2-(N-morpholino)ethanesulfonic acid, pKa 6.16), Citrate-Phosphate (Mcllvaine buffer (Mcllvaine, 1921), citrate pKa 6.40, Na2HPO4pKa 7.00), PIPES ((piperazine-N,N'- bis(2-ethanesulfonic acid)), pKa 6.75), sodium phosphate (Na2HPO4 / NaH2PO4, pKa 7.20) and HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pKa 7.50) buffers. The parameters and selection rationale are described in Table 2. The buffer stocks were prepared at or near the respective physiological pH value. This corresponds to the 20°C pKa values with the counter-ion pair, or if not stated then the counter ions were HCI and NaOH titrated as required.

[0293] Table 2: Characteristics of tested small molecule buffering agents

[0294] 2.2 Rationale for pH Measurement

[0295] To assess the self-buffering capability of albumin in Formulation 1, Formulation 2, and Comparator Formulations 1 to 4, the 125 g / L (1.88 mM) ImL WT rHA formulations were challenged with 2.5-10 pL volume additions of strong acid and base, namely HCI and NaOH. During testing, the formulation samples were constantly stirred by a micro magnetic stirrer bar at 300 rpm in a 2mL shallow conical bottomed polythene reaction tube. The pH was recorded with a temperature feedback adjusted Mettler Toledo InLab® Micro Pro-ISM (pH sensor P / N: 51344163 and Mettler Toledo Seven Compact pH meter at a nominal temperature of 20°C. Each acid or base addition was aimed to incrementally decrease or increase the pH towards a final shift of > ±1.0 pH unit from the starting pH as this is the range whereby sufficient concentrations of each acid and base species reside for notable equilibria to be maintained. The pH deviation of the formulations and buffers covered a nominal pH range of 5.75-7.75. The same measurement approach was applied to the described comparative small molecule buffers.

[0296] The measured pH deviation was plotted as acid or base concentration ([A] or [B]) in millimolar terms (y) versus ApH as (x). Linear fits established the gradient and a means of comparing buffer "strength" as Buffer Capacity (|3) (Slyke, 1922), generally as described in the equation |3 = [AB] / ApH where ApH describes the change in pH upon base addition [AB]. [AB] also describes [AA] in the negative form and therefore creates positive |3 values with both acid and base addition. A linear regression fit was applied up to the point of non-linearity (>>1.0 ApH) to exclude values where buffering equilibria were exceeded and enhance measurement variation handling.

[0297] 2.3 Free Thiol Measurement

[0298] High levels of free thiol indicate low oxidation and low levels of aggregation. High free thiol itself is also an important intrinsic property of rHA, with beneficial antioxidant activity. Free thiol monitoring is therefore a key performance indicator of the manufacture and formulation of rHA. The free thiol evaluation was performed using a colorimetric 4,4'-dithiodipyridine (DTDP) assay as described in Examples 8, 11 and 12.

[0299] 2.4 Measuring proportions of monomer, dimer, trimer, and polymer

[0300] Albumin over time oligomerises to dimers, trimers and can be subject to aggregation e.g. formation of polymers such as tetramers and larger species. Therefore, the potential for generation of oligomeric albumin species with time was measured using GP-HPLC which can be used to readily identify the multimeric species, as described in Examples 8, 11 and 12.

[0301] 2.5 Stability Study Sample Storage

[0302] Polythene screw-top, rubber sealed ImL cryo-vials were used to store the formulations at the specified temperatures. The long-term storage condition for rHA was 5±3°C. Data generated at 40±2°C was considered as an accelerated stability condition used to evaluate and compare the novel Formulations.

[0303] 2.6 Stability Testing Scope & Intervals

[0304] Samples were taken for each time point no later than 1 week following the due sampling date and were stored at 5±3°C pending analysis. All tests were initiated within 1 week of each time point specified. The stability study continued for 3 months at 40±2°C and 6 months at 5±3°C. For Examples 11 and 12, the stability study was continued for at least 1.5 months.

[0305] Example 3: rHA formulations required high molarity HCI and NaOH additions to generate significant pH shift but Molar equivalent small molecule buffers did not.

[0306] To assess the self-buffering capability of albumin in Formulation 1, Formulation 2 and Comparator Formulations 1 to 4, the formulations and comparative small molecule buffers were titrated by addition of small volumes of strong acid and base, namely HCI and NaOH. The aim was to produce experimentally valid incremental decreases or increases in pH towards a final shift of >1.0 pH unit from the starting pH. Small and approximately equivalent volumes of HCI and NaOH were required to avoid significant dilution (< 10%) of the buffering material. The concentrations of HCI and NaOH were empirically determined to yield experimentally sufficient pH-shift measurements in a reasonable experiment time-frame whilst also avoiding confounding variables such as rHA structural unfolding. Low volume (pl) incremental additions of 1.0 M NaOH shifted the starting formulation pH as shown in Figure 1. 1.0 M NaOH additions to >1.0 pH unit were nonideal with high numbers of additions required. A linear regression fit was then calculated and plotted with high and inconsistent gradients defining the Buffering Capacity (|3). Additions of this high concentration base yielded very slow pH equilibration and the pH change gradient was not consistent when compared with measurements conducted by addition of 0.5 M NaOH or lower concentrations. It is therefore hypothesised that with addition of 1.0 M NaOH, a sub-population of the rHA present may exhibit rapid rHA tertiary structure unfolding before complete sample mixing occurred. The non-conformity of pH shift potentially indicated that unfolded rHA may have a different charge state to fully folded rHA.

[0307] Using 0.5 M NaOH and 1.0 M HCI for titration yielded consistent experimental values and were empirically determined to be suitable for the generation of linear pH shifts in the rHA formulations. In comparison, low concentration 0.04 M additions of NaOH and HCI were used after optimisation to shift the pH of the comparative buffer systems in a similar manner.

[0308] Conclusion: rHA formulations required high molarity HCI and NaOH additions to generate significant pH shift but molar equivalent small molecule buffers did not. rHA Formulation 1 and Formulation 2 absent of small molecule buffers therefore act as highly effective buffering species of their own accord.

[0309] Example 4: rHA formulations had a significantly higher buffer capacity than Molar equivalent small molecule buffers.

[0310] Low volume (pl) incremental 0.5 M NaOH additions shifted the tested formulation pH by >1.0 pH unit. A linear regression fit was calculated and plotted with the gradient defining the Buffering Capacity (0) as shown in Figure 2. Formulation 1 pH shifts with additions of 1.0M HCI yielded a 3 of 13.7 with an R2of 0.77 and with additions of 0.5 M NaOH the |3 was 16.9 with an R2of 0.87. Formulation 2 pH shifts with additions of 1.0M HCI yielded a 3 of 18.6 with an R2of 0.86. Formulation 2 with additions of 0.5 M NaOH yielded a 3 of 20.3 with an R2of 0.92. Formulation 1 and Formulation 2 exhibit high buffering capacities and concurrently required high molarity HCI and NaOH additions.

[0311] In comparison, equivalent molar concentrations of small molecule buffers such as PIPES or Mcllvaine buffer (sodium phosphate / citrate) have a relatively weak buffering capacity and the plots are shown in Figure 3. For small molecule buffer buffering capacity determination, low volume (pl) and low molarity (0.04 mM) incremental additions shifted the formulation pH by >1.0 pH unit. A linear regression fit was calculated and plotted with the gradient defining the Buffering Capacity (3) ■ The mean buffering capacities are significantly lower than that of Molar equivalent rHA. PIPES buffer pH shifts with additions of 0.04 M NaOH yielded a 3 of 0.60 with an R2of 0.91 and additions of 0.04 M HCI yielded a 3 of 0.33 with an R2of 0.84. Mcllvaine buffer pH shifts with additions of 0.04 M NaOH yielding |3 of 0.53 with an R2of 0.94 and additions of 0.04 M HCI yielded a 3 of 0.32 with an R2of 0.87. Comparatively, Formulation 1 and Formulation 2 exhibit high buffering capacities and concurrently required high molarity HCI and NaOH additions.

[0312] Individual buffering capacity values determined from HCI and NaOH additions were calculated and combined into one mean value for simple comparison. This comparison presents the significantly higher buffering capacities of the tested rHA formulations versus small molecule buffers at the same molarity as shown in Figures 4A and 4B.

[0313] The results show that rHA formulations are significantly better buffers than small molecule buffers at equivalent molarities and so it can be considered that a single molecule of rHA is better at buffering than a single molecule of small molecule buffer. This effective single molecule buffering is very important when considered that at the molecular level, pH is a local effect and not a bulk solution effect. If one is trying to stabilise a low concentration active pharmaceutical ingredient (API) such as an antibody, a vaccine component, a viral vector or lipid nanoparticle (LNP), the local pH around the API is the more relevant parameter, not the bulk solution pH although this is what can be readily measured as a proxy for local pH. This localised pH property becomes even more important when localised concentrations of rHA molecular species increase above the bulk concentration in events such as rHA binding and / or coating of the API or a material surface and acting as a protective or surface modifying agent. This binding or coating concentrates and amplifies the localised pH effect by concentrating the point charges into a locus. In the case of single or multiple rHA weakly binding to an API, this could lead the API to exhibit significantly different electrostatically charged or non-polar interaction properties and so a highly effective buffer is preferably considered in the context of individual molecules rather than as a bulk solution property.

[0314] Conclusion: rHA formulations had a significantly higher buffer capacity than Molar equivalent small molecule buffers.

[0315] Example 5: rHA formulations have a lower buffering capacity than mass equivalent small molecule buffers.

[0316] Although buffers tend to be considered in molar terms, they may also be considered in mass terms e.g. where the large 66,438 Da molecular weight of rHA and small molecule imidazole at 68.0 Da are of a significantly different magnitude. Considering buffering capacity in mass terms may be especially suitable in fields such as chemistry where weight / weight (w / w) and weight / volume (w / v) are used more readily.

[0317] In consideration, the small molecule buffer typically has a high charge density and typically is completely solvent accessible. In comparison the individual amino acids composing rHA have an array of local and net charge states, non-polar regions and some amino acids alone are considered small molecule buffer components such as histidine. When combining these amino acids into the rHA globular protein, most of the amino acid non-polar properties and electrostatic charges are neutralised by burial and salt bridge formation in the protein structural core which has relatively low solvent accessibility. As such, the point surface charges presented readily to the solvent are relatively low compared to the mass of the protein molecule. Consideration of the buffering potential of protein is however not as straightforward as simply considering point charge solvent presentation as some buried charge can be spread or influence charge across the entire protein and so can experimentally yield surprising and unexpected results.

[0318] The complexity of protein buffering potential can also arise from the location of the point charges which can be non-uniform with charge and non-polar patches arrayed in an anisotropic manner. Protein charge can also be modified by dynamic interactions with small molecules such as salts, metal ions, small molecule buffers, fatty acids, ligands and other chemical entities in solution. To add further complexity, the charge state of a protein or complex can extend further than surface charge and is perhaps preferably considered as a long-range electrostatic field. This nature is exemplified by rHA charge variant ion-exchange chromatography where charge variation is significant enough to yield separation in ion-exchange chromatography and can also be used on megadalton proteinaceous viral vector presenting different net electrostatic charges depending upon the nucleic acid content. This complexity is highlighted by an array of protein charge modelling approaches in molecular dynamics simulations, and for macromolecular uses it is preferably determined empirically by bulk pH measurement as applied here.

[0319] For comparison, the mass equivalent buffering capacities have been calculated to 1 g / L from the experiments conducted at 1.88 mM (125 g / L) concentration. The values for this comparison were derived from individual buffering capacity values determined from HCI and NaOH additions were calculated individually and then combined into one mean value ("mean of means") for simple comparison. In this treatment, Formulation 1 and Formulation 2 provided combined buffering capacities of 0.13 and 0.15 respectively whereas PIPES or Mcllvaine buffer had buffering capacities of 0.82 and 1.20 respectively as shown in Figure 5. Whilst the protein formulations have a mean combined buffering capacity 12-fold lower than the average of the small molecule buffers, it may be considered that the mean molecular weight difference is 364-fold. When considering rHA and small molecule buffer at 1 g / L the mean concentration is 0.015 mM and 6.7 mM respectively, which is lower than would be typically used for either material, nor is it in a typical ratio of use in a combined formulation.

[0320] Conclusion: rHA formulations have a surprisingly high mass weighted buffering capacity.

[0321] Example 6: rHA formulations have a higher buffering capacity than small molecule buffers in typically used concentrations.

[0322] For comparison of buffer capacity in typically used concentrations as would be observed in a typical rHA formulation, 125 g / L (1.88 mM) rHA values and calculated 25 mM small molecule buffer values (based on the data acquired for 1.88 mM buffers) have been used. Individual buffering capacity values determined from HCI and NaOH additions were calculated individually, the buffering capacity was combined into one mean value for simple comparison ("mean of means") and adjusted for typical use equivalence at 125g / L rHA and 25mM buffer. In this treatment, Formulation 1 and Formulation 2 provided combined buffering capacities of 16.1 and 19.3 respectively whereas PIPES and Mcllvaine buffer had buffering capacities of 6.20 and 5.67 respectively as shown in Figure 6. This comparison shows that albumin has a higher buffering capacity than small molecule buffers at these typically used concentrations, but the difference is smaller than when compared on a molecule-to-molecule (Molar) basis. This shows that rHA has a higher buffering capacity than small molecule buffers, but at these typical use concentrations they are of a similar magnitude. This comparison lends further weight to the argument that rHA is a buffer that is better or similar to commonly used buffers at typical use concentrations. This comparison covers typical storage and supply concentrations of rHA and formulation buffer. In this treatment, rHA and small molecule buffers have a similar buffering capacity. Using buffers at 25 mM or 50 mM is commonplace for protein storage solutions and rHA formulations are typically available at 100-200 g / L. Considering these ranges, rHA remains the primary buffering component, but is close to equivalence of small molecule buffers and so it can be strongly argued that the rHA is acting as an effective buffering species.

[0323] Conclusion: rHA formulations have a higher buffering capacity than typically used concentrations of small molecule buffers. Example 7: rHA was surprisingly the dominant buffering species in all small molecule buffered rHA formulations.

[0324] Individual buffering capacity values determined from HCI and NaOH additions were calculated individually, the buffering capacity was combined into one mean value for simple comparison. The tested rHA formulations have the same magnitude of buffering capacity (|3).

[0325] Mean combined |3 was determined by calculating the mean buffer capacity of NaOH or HCI additions separately and then calculating the mean of these two values ("mean of means", plotted in Figure 4A). This shows that Formulation 1 has a mean combined |3 of 15.3, which is similar to Comparator Formulations 1 and 4 (13.5 and 11.8 respectively) and are considered to be not buffered by small molecules. Formulation 2 has a mean combined |3 of 19.6, and the Comparator Formulation 2 and 3 have similar |3 of 22.1 and 23.0 respectively. Comparator Formulation 2 and 3 contain a small molecule buffer and the mean difference between the aforementioned non-buffered and buffered formulations |3 is 7.48 or 33%.

[0326] Mean combined |3 ("mean of all values") was also calculated directly from the individual buffer capacity values from the NaOH or HCI additions and plotted in Figure 4B. This shows that Formulation 1 has a mean combined |3 of 15.6, which is similar to Comparator Formulations 1 and 4 (15.8 and 17.7 respectively) and are considered to be not buffered by small molecules. Formulation 2 has a mean combined |3 of 21.1, and the Comparator Formulation 2 and 3 have similar |3 of 21.3 and 23.2 respectively. Comparator Formulation 2 and 3 contain a small molecule buffer and the mean difference between the aforementioned non-buffered and buffered formulations |3 is 4.58 or 21%.

[0327] Therefore, it can be considered that the buffer in the albumin formulations is providing around 20% of the formulations' buffering capacity at a buffer concentration of 25 mM compared to the 1.88 mM albumin concentration. One could also consider the corollary that at an equivalent molar concentration, albumin is approximately 32- fold and 35-fold more effective as a buffer than a small molecule buffer, as determined by the first and second calculation methods above, respectively.

[0328] Irrespective of how mean combined |3 is calculated, the data confirm that albumin is a substantially better buffer than a small molecule buffer.

[0329] Conclusion: rHA was surprisingly the dominant buffering species in small molecule buffered rHA formulations.

[0330] Example 8: rHA formulations without small molecule buffers retained storage stability in a manner surprisingly comparable to small molecule buffered rHA formulations. High free-thiol content is indicative of albumin native protein-fold maintenance and can be used as a rapid test for albumin stability. The free-thiol level may be measured by a free-thiol assay such as a colourimetric free-thiol assay, for example a DTNB assay or a DTDP assay. A DTDP assay is preferred and used here.

[0331] The DTDP assay comprised the following method with the described components: a) Mixed 50 pL (microliters) of 50 mg.mL-1albumin and buffer (950 pL (microliters), (100 mM sodium phosphate, 1 mM EDTA, pH 7.4) to make an albumin 'sample', using 0.65 mM glutathione and 950 pL (microliters) buffer as a control, b) measured the absorbance of the sample, the control and a buffer 'blank' at a wavelength of 324 nm, c) added 50 pL (microliters) of 4 mM 4,4' - Dithiodipyridine (DTDP) to the sample, the control and the buffer 'blank', d) incubated for 10 minutes at room temperature 20±3°C, e) measured the increase in absorbance at a wavelength of 324 nm of the albumin sample, the control and the buffer 'blank', and f) determined the free thiol level of the sample using the equations below:

[0332] 1. The increase in A324 (A3) for the blanks and samples was calculated as follows:

[0333] (A3 = A2 - Ai) where Ai is the initial absorbance i.e. pre-DTDP where A2 is the final absorbance i.e. post-DTDP

[0334] 2. The true absorbance change (A A324) was calculated by subtracting the mean of the three blank values, A3, from each sample.

[0335] A324 = A3 sample - Mean A3 blank

[0336] 3. The measured Free thiol (nmol) was calculated for each sample and test control replicate: nmol Free SH = A A324 x 1.05 x 1000

[0337] E324 where E324 = 21.4 mM Tcm1for 4-TP at pH 7.4

[0338] 4. The quantity of albumin assayed in nmol was calculated:

[0339] [albumin! in diluted sample (mg.mL ) x 50 nL x 103

[0340] 66,438 (Molecular Weight of human albumin)

[0341] For example, 50 mg.mL-1albumin should be 37.6 nmols per cuvette 5. The quantity of glutathione in the test control solution (nmol) was calculated: glutathione mass (q) in 1000 mL x 50 (uL vol, of test control) x 100 307.32 (Molecular weight of glutathione)

[0342] 2 g glutathione in 1000 mL (i.e. 6.5 mM stock) is 32.5 nmols per cuvette

[0343] 6. The molar ratio of free-thiol to albumin (SH / Albumin) in each sample replicate was calculated:

[0344] Molar Ratio = nmol Free-SH produced nmol albumin used in assay

[0345] 7. The molar ratio of free thiol to glutathione in each test control replicate was calculated:

[0346] Molar Ratio = nmol Free SH produced nmol glutathione used in assay

[0347] 8. The mean of each of the triplicate molar ratios was calculated for each test sample and test control.

[0348] 9. A mean molar ratio for the test control in the range 0.95 to 1.04 indicated that the assay was valid.

[0349] As shown in Figure 7, rHA free-thiol species are maintained to a high degree in Formulation 1 and Formulation 2 at the typical storage temperature of 5°C. At 40°C the oxidation rate was higher, yet Formulations 1 and 2 retain comparable free-thiol to the small molecule buffered Comparator Formulation 2. Comparator Formulation 1 free-thiol content decreased at a higher rate than in other formulations, likely due to the expected action of an excipient. pH control is essential in the maintenance of the free-thiol state as small changes in pH can lead to significant changes in oxidation rate and this further indicates that the pH is controlled significantly by the albumin preparation and stabilising Formulations 1 and 2.

[0350] GP-HPLC oligomeric state yields a clear measure of the formulation stability and is readily accessible to most laboratories determining protein stability. Polymer level was determined by measuring its peak area relative to the total peak areas present in the sample. GP-HPLC analysis was conducted using an Agilent 1260 HPLC system with concentration detection by UV absorbance at 280nm and the column temperature maintained at 30°C. Separation was performed on formulations diluted to 40 mg / mL, with 25 pl injections at a flow rate of 1 mL.min-1 on a TSKgel G3000 SWxl Analytical Column, 7.8mm id x 30 cm length and TSK SWXL Guard Column, 6.0mm id x 4 cm length. The GP-HPLC mobile phase comprised of 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% sodium azide, pH 7.0, prepared with laboratory grade water and 0.22pm filtered prior to use.

[0351] GP-HPLC data was acquired from recent Good Manufacturing Practice (GMP) prepared Formulation 1 material stored in Thermo Fisher Bioprocess Containers (BPC), data is shown in Table 3a.

[0352] BPCs can be described typically as polyethylene plastic "bags" and represent a common, but materially less preferred presentation of albumin. BPCs present a large plastic surface area for which albumin is known to coat. As an alternative storage vessel, 0.9 mL polyethylene cryo-vials represent a preferred storage vessel and GP- HPLC values relating to cryo-vials are shown in Table 3b and Figures 8 and 9.

[0353] The data presented in Tables 3a and 3b and Figures 8 and 9, show that Formulation 1 is highly stable and remains predominantly monomeric and free from polymeric species after 6 months at 5°C and 25°C, and more than 80% monomeric after 3 months at 40°C.

[0354] Table 3a. Formulation 1 Human Albumin Oligomeric States from BPC storage by GP- HPLC. Figures are percentages of total population determined by area under the curve.

[0355] Recombinant human albumin was incubated in Thermo-Fisher Bioprocessing Containers (BPC) at an array of testing temperatures. GP-HPLC peak elution percentages show that Formulation 1 is highly stable and remains predominantly monomeric after 6 months at 5°C, and 25°C, and more than 80% monomeric after 3 months at 40°C, see Table 3a.

[0356] Tested formulations were also incubated in "preferred" polyethylene cryo-vials at the recommended storage temperature of 4±3°C. GP-HPLC peak elution percentages shows that all formulations are highly stable and substantially free from polymer after 6 months at the preferred storage temperature of 4°C, see Table 3b and Figure 8.

[0357] Tested formulations were also incubated in "preferred" polyethylene cryo-vials at an elevated storage temperature of 40±3°C. GP-HPLC peak elution shows that more than 80% was monomeric after 3 months at 40°C, see Table 3b and Figure 9. Formulation 1 is therefore highly stable and remains predominantly monomeric and substantially free from polymeric species after 6 months at 5°C, 25°C and 40°C in two different presentations. GP-HPLC peak elution percentages in Table 3b and Figure 9 show that Formulation 1 is highly stable and remains predominantly monomeric after 3 months at 40°C and its stability is very similar to that of stable Comparator Formulation 2. Formulation 2 is relatively stable in this accelerated condition, but is less stable than the other formulations. Comparator Formulation 1 is the most stable with no discernible polymer formation due to the expected action of an excipient.

[0358] Table 3b. Formulation 1, 2 and Comparator Formulation 1 and 2 Human Albumin Oligomeric States from storage in polyethylene cryo-vials and determined by GP-HPLC. Figures are geometric mean percentages of the total population determined by area under the curve. Conclusion: rHA formulations without small molecule buffers and substantially free from classical stabiliser, retained storage stability in a manner surprisingly comparable to small molecule buffered rHA formulations. In a similar manner to the Comparator Formulation 2 and Comparator Formulation 3, Formulation 1 and Formulation 2 surprisingly had similar stability properties that were namely: - having at least 0.80 moles free thiol per mole albumin and at most 0.5% albumin polymer after incubation at at least 40 °C for at least 2 weeks, more specifically after incubation at 37 to 43 °C for 2 weeks. having at least 0.80 moles free thiol per mole albumin and at most 0.5% albumin polymer after incubation at at least 25 °C for at least 2 months, more specifically after incubation at 22 to 28 °C for 2 months. having at least 0.90 moles free thiol per mole albumin and at most 0.5% albumin polymer after incubation at at most 8 °C for at least 6 months, more specifically after incubation at 2 to 8 °C for 6 months. rHA Formulation 1 and Formulation 2 can both be considered to be a buffer at the formulated albumin concentration of 125 g / L or 1.88 mM. This concentration is relatively low in molarity terms for a small molecule buffer which are typically 5-100 mM. Albumin has an array of charged amino acid species, and so the protein can be considered a multi-valent zwitterion. Major additions of strong acid or base are required to adjust the protein solution pH by small increments and the pH is controllable in the manner of a small molecule buffer, within ± 1.0 pH unit from the pKa of the buffer.

[0359] Formulation 1 and Formulation 2 yield a significant benefit in formulation flexibility and could be considered a platform for formulating with an additional array of excipients. It would be expected that the buffering capacity of the protein can mitigate pH shifts induced from small additions of excipient to a similar degree as a small molecule buffer.

[0360] Example 9: rHA formulations with different monovalent and divalent anions and cations also required high molarity HCI and NaOH additions to generate significant pH shift, highlighting that rHA buffering capacity is universally maintained with fully dissociating neutral salts.

[0361] To determine if the self-buffering capability of albumin is universally maintained with fully dissociated neutral salts, an array of rHA formulations with monovalent and divalent ions from such neutral salts were tested for their buffering capacity. Formulation 3 comprised 125 g / L rHA in 250 mM Potassium Chloride (KCI) with the monovalent cationic potassium ion being compared to the equivalent concentration of sodium (NaCI) in Formulation 1 to test the universality of formulations with monovalent cations for maintaining rHA stability and self-buffering capacity. Formulation 4 had 125 g / L rHA and 250 mM calcium chloride (CaCH) to test divalent cations (Ca2+) with an equivalent valence of chloride counterions. Formulation 5 had 125g / L rHA and 250 mM sodium sulphate (NazSO) which tested divalent anion (SO42) with an equivalent valence of sodium counterions and also enables comparison with chloride anions used in other formulations.

[0362] To prepare Formulation 3, Recombumin® Elite was diluted 5-fold in 250 mM KCI, then concentrated 6-fold with 10 kDa MWCO Sartorius Vivaspin® 100 units, diluted 6- fold, concentrated 9-fold, diluted 9-fold, then concentrated 9-fold through rounds of diafiltration and finally concentrated to 216 g / L rHA and 250 mM KCI. [rHA] was determined by SEC-HPLC and UV 280 nm detection. The rHA was formulated by dilution with 250 mM KCI to achieve the 125 g / L rHA formulation described in Table 4, prior to filtration through a 0.2 pm PES membrane. To prepare Formulation 4, Recombumin® Elite was first diluted 10-fold in 250 mM NaCI, then concentrated 10-fold to the original volume with 10 kDa MWCO Sartorius Vivaspin® 100 units and the dilution and concentration was repeated. This material was then diluted 9-fold with 250 mM CaCh and concentrated 9-fold in 3 rounds of this diafiltration to a final 250 mM CaCh and 189 g / L rHA with the exchange process reducing the sodium phosphate concentration and minimising calcium phosphate precipitation. [rHA] was determined by SEC-HPLC and UV 280 nm detection and the formulation diluted with 250 mM CaCh to achieve the desired 125 g / L rHA formulation as described in Table 4, prior to filtration through a 0.2 pm PES membrane.

[0363] To prepare Formulation 5, Recombumin® Elite was diluted 9-fold in 250 mM NazSC , then concentrated 6-fold with 10 kDa MWCO Sartorius Vivaspin® 100 units, and diluted 6-fold in 3 similar rounds of diafiltration with a final rHA concentration of 191 g / L and 250 mM Na2SO4. [rHA] was determined by SEC-HPLC and UV 280 nm detection and the formulation diluted with 250 mM Na2SO4 to achieve the desired 125 g / L rHA formulation as described in Table 4, prior to filtration through a 0.2 pm PES membrane.

[0364] Formulations prepared for stability studies were stored in 0.9 mL polyethylene cryo-vials with minimal ullage and stored at the defined temperatures and time.

[0365] Table 4: Compositions of each albumin formulation tested.

[0366] Formulations 3, 4 and 5 containing fully dissociated salts were pH titrated by addition of small volumes of strong acid and base, namely HCI and NaOH. The aim was to produce experimentally valid incremental decreases or increases in pH towards a final shift of >1.0 pH unit from the starting pH. Small volumes of HCI and NaOH at the highest feasible concentration were used to avoid significant dilution (< 10%) of the buffering material. Addition of 0.5 M NaOH and 1.0 M HCI to the rHA formulations was empirically determined to yield experimentally sufficient pH-shift measurements in a reasonable experiment time-frame whilst also avoiding confounding variables such as rHA structural unfolding or precipitation induced by localised temporary pH shifts before complete mixing was achieved. As in previous examples, a linear regression fit was calculated and plotted with the gradient defining the Buffering Capacity (|3). The data are provided in Figure 10 (|3 calculated by "mean of all values").

[0367] The results show that rHA formulations with an array of different fully dissociated monovalent and divalent ions from neutral salts are significantly better buffers than small molecule buffers at equivalent molarities and so it can be considered that a single molecule of rHA in such formulations is better at buffering than a single molecule of small molecule buffer. The combined mean buffer capacity is broadly similar for the neutral salt-based formulations of Formulation 1 and 2 (NaCI), Formulation 3 (KCI), Formulation 4 (CaCIz) and Formulation 5 (NazSC ). These formulations have a similar buffering capacity to that of Comparator Formulations 1 to 4.

[0368] As in Example 5, the mass equivalent buffering capacities have been calculated to 1 g / L from the experiments conducted at 1.88 mM (125 g / L) concentration in the array of neutral salt-based formulations. As shown in Figure 11, in this treatment, Formulation 1 and Formulation 2 (NaCI) provided combined buffering capacities of 0.13 and 0.15 respectively (as calculated by "mean of means"). Similarly, Formulation 3 (KCI), Formulation 4 (CaCIz) and Formulation 5 (NazSC ) had buffering capacities of 0.17, 0.10 and 0.17 respectively. In comparison, PIPES or Mcllvaine (Citrate- Phosphate) buffer had buffering capacities of 0.82 and 1.20, respectively. Whilst the protein formulations without small molecule buffer have a mean combined buffering capacity 14-fold lower than the average of the small molecule buffers, it may be considered that the mean molecular weight difference is 364-fold. When considering rHA and small molecule buffer at 1 g / L the mean concentration is 0.015 mM and 6.7 mM respectively, which is lower than would be typically used for either material, nor is it in a typical ratio of use in a combined formulation. This further confirms that rHA formulations have a surprisingly high mass weighted buffering capacity.

[0369] To enhance comparison of buffer capacity across typically used concentrations of rHA formulation and small molecule buffer, as would be observed in a typical rHA formulation, a simple mathematical treatment was applied. Experimental buffer capacity values from 125 g / L (1.88 mM) rHA formulations were compared to calculated 25 mM small molecule buffer values derived from 1.88 mM experimental values as used in Example 6 but compared here with the array of neutral salt-based containing formulations. As shown in Figure 12, in this treatment, Formulation 1 and Formulation 2 (NaCI) provided combined buffering capacities of 16.1 and 19.4 respectively whereas PIPES and Mcllvaine buffer had buffering capacities of 6.20 and 5.67, respectively (all calculated by "mean of means"). This comparison shows that albumin has a 3 times higher mean buffering capacity than small molecule buffers at these typically used concentrations. This comparison lends further weight to the argument that rHA is a buffer that is better or similar to commonly used small molecule buffers at typical use concentrations and works with an array of fully dissociated monovalent and divalent ion containing formulations. This comparison covers typical storage and supply concentrations of rHA and formulation buffer. Using buffers at 25 mM or 50 mM is commonplace for protein storage solutions and rHA formulations are commonly available at 100-200 g / L. Considering these ranges, rHA remains the primary buffering component and so it can be strongly argued that the rHA is acting as an effective buffering species.

[0370] Conclusion: rHA formulations with an array of monovalent and divalent ions from fully dissociating neutral salts required high molarity HCI and NaOH additions to generate significant pH shift indicative of high buffering capacity and highlights that rHA acts as a highly effective buffering species. The array of rHA formulations tested have a higher buffering capacity than typically used concentrations of small molecule buffers.

[0371] Example 10: Low and high rHA concentration formulations also required high molarity HCI and NaOH additions to generate significant pH shifts, showing that rHA self-buffering capacity extends from at least 50 g / L to more than 250 g / L rHA.

[0372] To determine if the self-buffering capability of albumin is maintained at low and high concentrations of rHA, 50 g / L (0.75 mM, Formulation 6), 200 g / L (3.01 mM, Formulation 7) and 263 g / L (3.96 mM, Formulation 8), preparations were generated in the same 250mM NaCI formulation of Formulation 1. The octanoate molarity increases with albumin concentration, but the stoichiometry (mol. mol) remains constant. This is due to octanoate being tightly bound to the albumin despite several concentration and dilution steps.

[0373] To prepare Formulations 6 and 7, Recombumin® Elite was diluted 5-fold in 250 mM NaCI, then concentrated 5-fold with 10 kDa MWCO Sartorius Vivaspin® 100 units and diluted in 2 similar rounds of 5-fold diafiltration and concentrated to a final concentration of 225 g / L rHA and 250 mM NaCI. [rHA] was determined by SEC-HPLC and UV 280 nm detection. The rHA was diluted by addition of 250 mM NaCI to achieve the 50 and 200 g / L rHA formulations described in Table 5, prior to 0.2 pm filtration through a PES membrane.

[0374] To prepare Formulation 8, Recombumin® Elite was diluted 5-fold in 250 mM NaCI, then concentrated 4-fold with 10 kDa MWCO Sartorius Vivaspin® 100 units and diluted 4-fold in 3 similar rounds of diafiltration and concentrated to a final concentration to 263 g / L rHA and 250 mM NaCI. [rHA] was determined by SEC-HPLC and UV 280 nm detection. The rHA formulations described in Table 5 were sterile filtered through a 0.2 m PES membrane.

[0375] Table 5: Compositions of each albumin formulation tested.

[0376] Formulations 6, 7 and 8 were titrated by addition of small volumes of strong acid and base, namely HCI and NaOH as conducted in Examples 3 and 9.

[0377] As in previous examples, a linear regression fit was calculated and plotted with the pH gradient defining the Buffering Capacity (P). The data are provided in Figures 16, 17 and 18. Formulation 6 was titrated with 0.2 M NaOH and 0.4 M HCI, whilst Formulation 7 and Formulation 8 were titrated with 0.5 M NaOH and 1.0 M HCI to yield consistent experimental values and the variation relates to the albumin concentration and concurrent p. The concentrations of base and acid used were empirically determined to be suitable for the generation of linear pH shifts in the rHA formulations.

[0378] The results show that rHA formulations with a range of rHA concentrations from 50 to more than 250 g / L are substantially better buffers than small molecule buffers at equivalent molarities and so it can be considered that a single molecule of rHA in such formulations is better at buffering than a single molecule of small molecule buffer. As shown in Figure 16, Formulation 6 (50 g / L rHA) has a buffering capacity (P) of 4.61. Formulation 1 (125 g / L rHA) from Example 4 has a 3 of 15.6, Formulation 7 (200 g / L rHA) has a P of 25.5 and Formulation 8 (263 g / L rHA) has a P of 17.7 calculated by "mean of all values"). These formulations have a similar buffering capacity to that of Comparator Formulations 1 to 4. Generally, a lower concentration of rHA resulted in a lower buffering capacity.

[0379] As in Examples 5 and 9, the mass equivalent buffering capacities have been calculated to 1 g / L from the experiments conducted at 50-263 g / L (0.75-3.96 mM) rHA concentrations. As shown in Figure 17, in this treatment, Formulation 6 (50 g / L) has a buffering capacity (P) of 0.10, Formulation 1 (125 g / L) has a P of 0.13, Formulation 7 (200 g / L) has a P of 0.13 and Formulation 8 (263 g / L) has a P of 0.07. These formulations have a similar buffering capacity to that of Comparator Formulations 1 to 4. In comparison, PIPES or Mcllvaine (Citrate-Phosphate) buffer had buffering capacities of 0.82 and 1.20 respectively. All p were calculated as "mean of means". Whilst the protein formulations without small molecule buffer have a mean combined buffering capacity 14-fold lower than the average of the small molecule buffers, it may be considered that the mean molecular weight difference is 364-fold. When considering rHA and small molecule buffers at 1 g / L the mean concentration is 0.015 mM and 6.7 mM respectively, which is lower than would be typically used for either material, nor is it in a typical ratio of use in a combined formulation.

[0380] For enhanced comparison of buffer capacity across the varied 50-263 g / L rHA formulations and against small molecule buffers, a mathematical treatment was applied to assess which was the better buffer in typical use concentrations. Experimental buffer capacity values from the 50-263 g / L rHA formulations were calculated to an equivalence of 125 g / L (1.88 mM) and compared to calculated 25 mM small molecule buffer values derived from 1.88 mM experimental values in the same manner as used in Example 6 and 9. As shown in Figure 18, in this treatment, Formulation 6 (50 g / L) has a buffering capacity (P) of 12.0, Formulation 1 (125 g / L) has a P of 16.1, Formulation 7 (200 g / L) has a P of 16.7 and Formulation 8 (263 g / L) has a P of 8.55. whereas PIPES and Mcllvaine buffer had buffering capacities of 6.20 and 5.67 respectively. All p were calculated as "mean of means". This comparison shows that albumin has a 3-fold higher mean buffering capacity than small molecule buffers at these typically used concentrations. This comparison lends further weight to the argument that rHA is a buffer that is better or similar to commonly used small molecule buffers at typical use concentrations and works with an array of fully dissociated monovalent and divalent ion containing formulations. This comparison covers typical storage and supply concentrations of rHA and formulation buffer. Using small molecule buffers at 25 mM or 50 mM is commonplace for protein storage solutions and rHA formulations are commonly available at 100-200 g / L. Considering these ranges, rHA remains the primary buffering component, but is close to equivalence of small molecule buffers and so it appears that the rHA is acting as an effective buffering species.

[0381] The array of rHA concentrations tested shows that rHA formulation flexibility enables use as an excipient in a variety of applications such as antibody formulations, vaccine formulations, stabilisation of viral vectors or lipid nanoparticles (LNPs), and in cell media which may benefit from a varied concentration of albumin for stability and function of the active pharmaceutical ingredients or cells.

[0382] Conclusion: rHA formulations with low and high rHA concentrations provided significant buffering capacity from at least 50 to more than 250 g / L rHA. Example 11: rHA formulations with various neutral salts, but without small molecule buffers, retained storage stability in a manner comparable to small molecule buffered rHA formulations.

[0383] As shown in Figure 13A, rHA free-thiol species are maintained to a high degree in Formulation 3 and Formulation 5 at the typical storage temperature of 4°C. As shown in Figure 13B, at 40°C the oxidation rate was higher, yet Formulations 3 and 5 retain comparable free-thiol to the small molecule buffered Comparator Formulation 2. Data for Comparator Formulation 2 is shown in Figure 7. Formulation 4 was also tested but the CaCh in the formulation caused significant precipitation with the sodium phosphate buffer in the assay and so the optical absorbance measurements were not reliable, therefore data is not presented. pH control is essential in the maintenance of the free-thiol state as small changes in pH can lead to significant changes in oxidation rate and this further indicates that the pH is controlled significantly by the albumin preparation and stabilising Formulations 3 and 5.

[0384] GP-HPLC oligomeric state of human albumin yields a clear measure of the formulation stability. As in Example 8, albumin polymer level was determined by measuring its peak area relative to the total peak areas present in the sample. GP- HPLC analysis was conducted using an Agilent 1260 HPLC system with concentration detection by UV absorbance at 280nm and the column temperature maintained at 30°C. Separation was performed on formulations diluted to 40 mg / mL in formulant, except for Formulation 4 which was diluted in laboratory grade water to the same concentration to avoid CaCh and sodium phosphate precipitation in the HPLC system. 25 pl sample injections were conducted at a flow rate of 1 mL.min1on a TSKgel G3000 SWxl Analytical Column, 7.8mm id x 30 cm length and TSK SWXL Guard Column, 6.0mm id x 4 cm length. The GP-HPLC mobile phase comprised of 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% sodium azide, pH 7.0, prepared with laboratory grade water and 0.22 pm filtered prior to use.

[0385] The data presented in Table 6 and Figure 14, shows that Formulations 3, 4, 5 and Comparator 2 was highly stable and remained predominantly monomeric and free from polymeric species after 1.5 month at 4°C, which is a typical storage temperature for protein biological products. The data presented in Table 6 and Figure 15, showed that Formulations 3, 4, 5 and Example 8 Comparator 2 were highly stable and remained predominantly monomeric and free from polymeric species after 1.5 month at 40°C. Formulation 4 in this accelerated and high temperature condition had 21% polymer after 1 month and 27.2 polymer after 1.5 months at 40°C, whilst Formulations 3 and 5 and Comparator Formulation 2 had up to 1.10% after 1 month and up to 2.3% at 1.5 months. Relative to other salts, such as other divalent salts, calcium has a greater propensity to precipitate in the presence of phosphate. This could explain the polymer level observed in Formulation 4 after 1 and 1.5 months at 40°C. It could be hypothesised that non-visible calcium salt precipitation has increased the rate of polymerisation at this high temperature, which would not be observed with salts less likely to precipitate. The 40°C incubation is an accelerated stability study and not a typical storage temperature for biological or pharmaceutical products. In certain circumstances, accelerated stability study conditions induce protein states that do not occur at lower temperatures. In contrast, following incubation at 4°C, i.e. a typical storage temperature, no substantial amount of polymer was observed in Formulation 4.

[0386] Table 6. Comparator Formulation 2 ("buffered", 250 mM NaCI), Formulations 3 (250 mM KCI), 4 (250 mM CaCh) and 5 (250 mM NazSC ) Human Albumin Oligomeric States from storage in polyethylene cryo-vials and determined by GP-HPLC. Figures are geometric mean percentages of the total population determined by area under the curve.

[0387] To rapidly assess and compare the stability of rHA formulations with divalent cations, rHA Formulation 9 with 250 mM MgCh was prepared as described in Table 7. Recombumin® Elite ( / .e. 100 g / L rHA) was diluted 17-fold in 250 mM MgCh, then concentrated 36-fold with 10 kDa MWCO Sartorius Vivaspin® 100 units, diluted 36- fold, and concentrated 36-fold to 202 g / L rHA and 250 mM MgCh. [rHA] was determined by SEC-HPLC and UV 280 nm detection. The rHA was formulated by dilution with 250 mM MgCh to 125 g / L, prior to filtration through a 0.1 pm PVDF membrane.

[0388] Table 7: Composition of albumin Formulation 9.

[0389] Temperature of aggregation onset (Tagg) analysis was used to assess formulation stability of rHA Formulation 4 (CaCh), Formulation 9 (MgCh) and Comparator Formulation 2 (small molecule buffered and with 250 mM NaCI). These formulations were adjusted to 50 g / L prior to analysis. The Taggof each formulation was measured with a 0.1 °C / min temperature ramp from 25.0 to 84.9°C using a Wyatt Dynapro II dynamic light scattering (DLS) plate reader controlled with Dynamics V.8.4.0.64 software. Samples of approximately 20 pl were measured in triplicate with a 10 pl silicon oil overlay and plate seal in a black uncoated cycloolefin polymer 396- well Aurora plate (Wyatt, UK). Instrument parameters included a laser wavelength of 826.1 nm, 5 s acquisition time, 5 acquisitions, 0% attenuation, 25% laser power and a temperature delta of 0.465 °C per scan. Analysis was performed within the instrument software, with a data filter applied with minimum amplitude of 0, maximum amplitude of 1, Baseline Limit 0.01 and maximum Rh of 150 nm. Temperature Dependence analysis was performed with Onset Analysis with a below threshold of 11.3°C, above threshold of 11.3°C and threshold percentage of 30%. The Taggvalues presented in Table 8. showed that Comparator Formulation 2 and Formulation 9 (MgCh) had a very similar Taggof 58.4 ±0.34 and 58.8 ±0.09 °C, respectively. Formulation 4 (CaCh) had a lower Taggof 54.5 ±0.07°C, with a substantial ATaggof 4.38 °C between Formulation 4 and Formulation 9, indicating that Formulation 4 has a lower than expected stability, potentially from the higher propensity of CaCh to precipitate.

[0390] Table 8. Taggof Divalent Cation rHA Formulations and Comparator Formulation 2 (SD: standard deviation)

[0391] The array of formulations with monovalent and divalent fully dissociated ions tested here shows rHA formulation flexibility with buffering and stability is maintained. This enhances potential uses in various applications such as antibody formulations, vaccine formulations, stabilisation of viral vectors or lipid nanoparticles (LNPs), and in cell medias which may benefit from such monovalent and divalent ions for stability and function of the active pharmaceutical ingredients or cells, even if certain ions are preferred for albumin stability.

[0392] Conclusion: rHA formulations with neutral salts, without small molecule buffers and substantially free from classical stabiliser, retained storage stability in a manner surprisingly comparable to small molecule buffered rHA formulations. In a similar manner to the Comparator Formulation 2 and Comparator Formulation 3, Formulations 3, 4 and 5 surprisingly had similar stability properties that were namely: having at least 0.80 moles free thiol per mole albumin and at most 0.5% albumin polymer after incubation at at most 8 °C for at least 1 month, more specifically after incubation at 2 to 8 °C, for 1 month.

[0393] Example 12: rHA formulations at low and high rHA concentrations with 250 mM NaCI, but without small molecule buffers, retained storage stability in a manner comparable to a 125 g / L rHA equivalent formulation.

[0394] As shown in Figure 19, rHA free-thiol species are maintained to a high degree in Formulations 6, 7 and 8 at the typical storage temperature of 4°C. At 40°C the oxidation rate was higher, yet Formulations 6, 7 and 8 retain comparable free-thiol. pH control is essential in the maintenance of the free-thiol state as small changes in pH can lead to significant changes in oxidation rate and this further indicates that the pH is controlled significantly by the different concentration albumin formulations.

[0395] GP-HPLC oligomeric state of human albumin yields a clear measure of the formulation stability. As in Examples 8 and 11, albumin polymer level was determined by measuring its peak area relative to the total peak areas present in the sample. GP- HPLC analysis was conducted using an Agilent 1260 HPLC system with concentration detection by UV absorbance at 280nm and the column temperature maintained at 30°C. Separation was performed on formulations diluted to 40 mg / mL with 25 pl sample injections conducted at a flow rate of 1 mL.min-1 on a TSKgel G3000 SWxl Analytical Column, 7.8mm id x 30 cm length and TSK SWXL Guard Column, 6.0mm id x 4 cm length. The GP-HPLC mobile phase comprised of 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% sodium azide, pH 7.0, prepared with laboratory grade water and 0.22 pm filtered prior to use.

[0396] The data presented in Table 9 and Figure 20, show that Formulations 6, 7, 8 and Comparator 2 were highly stable and remained predominantly monomeric and free from polymeric species after 1.5 months at 4°C, which is a typical storage temperature for purified protein solution products, data was not collected at 1.5 months for Formulation 6. The data presented in Table 9 and Figure 21, show that Formulations 6, 7, and 8 were highly stable and remained predominantly monomeric and free from polymeric species after 1.5 months at 40°C. Formulation 8 in this accelerated and high temperature condition had 7.2% polymer after 1 month and 0.26% after 1.5 months at 40°C. Formulation 6 had 0.39% polymer after 1 month at 40°C, data was not collected at 1.5 months for Formulation 6. Formulation 7 had 0.84% polymer after 1 month, and 1.21% after 1.5 months at 40°C. These results are surprising in that the rHA formulations are very stable in the high temperature 40°C condition, even at protein concentrations greater than 200 g / L and without high concentrations of common stabilisers such as sodium octanoate.

[0397] Table 9. Comparator Formulation 2 (buffered, 250 mM NaCI), Formulations 6 (50 g / L), 7 (200 g / L), and 8 (263 g / L) Human Albumin Oligomeric States from storage in polyethylene cryo-vials by GP-HPLC. Figures are geometric mean percentages of the total population determined by area under the curve. Conclusion: rHA formulations with 50 g / L to more than 250 g / L rHA without small molecule buffers and substantially free from classical stabiliser, retained storage stability in a manner surprisingly comparable to small molecule buffered rHA formulations of 125 g / L. In a similar manner to the Comparator Formulation 2, Formulation 6, Formulation 7 and Formulation 8 surprisingly had similar stability properties that were namely: having at least 0.80 moles free thiol per mole albumin and at most 10% albumin polymer after incubation at at least 40 °C for at least 2 weeks , more specifically after incubation at 37 to 43 °C for 2 weeks. having at least 0.80 moles free thiol per mole albumin and at most 0.5% albumin polymer after incubation at at most 8 °C for at least 1 month, more specifically after incubation at 2 to 8 °C for 1 month.

[0398] Example 13: rHA Formulation 1 and Formulation 2 support positive cell expansion with a range of commonly used cell types.

[0399] Materials and Methods

[0400] After thawing, all cells underwent one passage in commonly used culture conditions (outlined below) prior to being used in the experiment. Serum use was minimised, whilst retaining cell viability. Specific culture conditions are outlined below for each cell type.

[0401] Mesenchymal Stem Cells (MSCs)

[0402] MSC media were prepared according to Table 10 below. MSC-qualified fetal bovine serum (MSC FBS) was purchased from Fisher Scientific (Loughborough, U.K.). Standard MSC culture medium for first passage after thawing was DMEM + 10% MSC FBS.

[0403] Table 10. Component volumes used to make MSC culture media. DMEM : Dulbecco's Modified Eagle Medium. MSC FBS and DMEM were supplied by Fisher Scientific: Loughborough, UK. rHA was supplied by Sartorius Albumedix: Nottingham, UK. The rHA formulation used in each media is as stated in the column headings.

[0404] Media components were combined with an rHA final concentration of 5 g / L, and 5xl03cells were seeded in 200 pl medium per well in the wells of a 48-well tissue culture treated plate. The seeded well plate was placed into a Sartorius S3 Incucyte® live-cell imaging system within an incubator set to 37 °C and 5% CO2, and images were captured every 8 hours and analysed using Sartorius Incucyte® Base Analysis software. The media were changed on day 5 and the analysis ended after day 7.

[0405] HEK293 Cells

[0406] HEK293 media was prepared according to Table 11. Standard culture conditions for first passage after thawing were as in Table 11, but contained 10% FBS and no rHA.

[0407] Table 11. Component volumes used to make HEK293 culture media. solution (NEAA), 200 mM L-glutamine, and Penicillin-Streptomycin (10 000 U / mL) (PenStrep). FBS, EMEM, NEAA, L glutamine, and PenStrep were supplied by Fisher Scientific: Loughborough, UK. rHA was supplied by Sartorius Albumedix: Nottingham, UK. The rHA formulation used in each media is as stated in the column headings.

[0408] The media components were combined for a final rHA concentration of 5 g / L, and 3.5xl04cells in 200pL were seeded per well in the wells of tissue culture treated 48-well plate. The plate was placed into a Sartorius S3 Incucyte® live-cell imaging system within an incubator set to 37°C and 5% CO2, where images were captured every 8 hours and analysed using Sartorius Incucyte® Base Analysis software. The analysis ended after 3 days. T Cells

[0409] T cell base medium components were combined according to Table 12, and sterile filtered through a 0.2 pm filter. The resultant base medium was used to make T cell complete medium according to Table 13.

[0410] Table 12. T cell Base Medium components.

[0411] ITS (lOOx) contains 1.0 mg / ml recombinant human insulin, 0.55 mg / ml human transferrin (substantially iron-free) and 0.5 pg / ml sodium selenite. Table 13. T cell complete medium volumes

[0412] Stemcell Technologies (Cambridge, U.K.), and human serum from human AB male plasma ("AB serum") from Merck (Gillingham, U.K.). T-cell base medium was according to Table 8. The rHA formulation used in each media is as stated in the column headings.

[0413] T cells were seeded at lxlO6 / mL in ImL of complete T cell medium into each well of an untreated 12-well plate. T cells were grown at 37°C and 5% CO2 in suspension and clump as they proliferate. To accommodate this growth pattern, well contents were pipetted up and down to break up clumps prior to cell counting. Cells were counted using the Cellometer Auto 2000 (Revvity, Llantrisant, Wales) with Acridine Orange / Propidium Iodide fluorescent dye (ViaStain AO / PI Staining Solution, Revvity, Llantrisant, Wales) and diluted with fresh media accordingly on days 3 and 5 so that the cell concentration was 2.5xl05 / mL after dilution on each day.

[0414] Results

[0415] Figure 22 shows that Formulations 1 and 2 support growth of MSC cells to a similar extent to the Comparator Formulation 2 and to the FBS control. Figure 23 shows that Formulations 1 and 2 support growth of HEK293 cells to a similar extent to the Comparator Formulation 2 and to the FBS control. Figure 24 shows that Formulations 1 and 2 support growth of T cells to a similar extent to the Comparator Formulation 2 and to the serum control at day 3 and day 5.

[0416] Discussion

[0417] Self-buffering combined with stability has not previously been established in albumin, including human albumin, and is surprising.

[0418] Although some self-buffering capacity of proteins has been theorised, discussion of this with respect to human albumin (HA) is controversial (Rossing, N., 1978; Dibartola, S.P., 2006; Caironi, P. et al., 2009; Gatz, R et al., 2011).

[0419] From Examples 1-12, the inventors here have discovered that HA has a demonstrable, and surprisingly high, self-buffering capacity, e.g. in a physiological pH range, which exceeds the molar equivalents of commonly used small molecule buffers. The inventors have also demonstrated that a surprising and novel feature of Formulation 1 and Formulation 2 is that the self-buffering capacity of HA adjusted to pH 6.75 with strong alkali or base and addition of 175 to 250 mM NaCI yields a sufficiently stable solution for storage at 4°C for greater than 6 months and at 25°C for greater than 6 months and 40°C for greater than 6 months. Similarly surprising self-buffering capacity is observed at 50 g / L HA, 200 g / L HA and 263 g / L HA in the equivalent 250 mM NaCI Formulations 6, Formulation 7 and Formulation 8 respectively.

[0420] Human albumin formulations ~125 g / L with fully dissociated monovalent and divalent cations and anions at 250 mM and 500 mM concentrations, e.g. Formulations 3 (KCI), 4 (CaCh) and 5 (NazSC ), surprisingly also yield equivalent self-buffering capacity and stability to equivalent HA NaCI based formulations. The same is expected for Formulation 9 (MgCh).

[0421] The addition of the neutral salt dramatically enhances albumin stability, without which the albumin formulation would fail to provide sufficient and uniform buffering, nor a useful, stable commercial product. The addition of any neutral salt such as NaCI, should not substantially change the total buffering capacity of a solution as they dissociate uniformly in solution and order around the protein, assuming protein stability is retained. The formulation claimed range has been defined experimentally and theoretically to maximise albumin stability and the maintenance of the buffering capacity of the albumin.

[0422] Conclusions

[0423] The experimental data of Examples 1 to 12 provide albumin formulations with self-buffering capacity and good stability. The observed buffering capacity exceeded the buffering capacity of molar equivalents of commonly used small molecule buffers. The experimental data of Example 13 also demonstrate the applicability of such formulations in cell culture such as use within therapeutic manufacturing and cell and gene therapies, e.g. HEK293 cells, stem cells (e.g. MSC), and for primary cells (e.g. T cells).

[0424] Such self-buffering albumin formulations contained fewer components than previously available albumin formulations, while providing good stability. Such selfbuffering albumin formulations provide greater flexibility to the user. For example, the albumin formulations allow buffering in the absence of components, such as phosphate or octanoate, which may be undesirable to some cell types.

[0425] The albumin formulations presented herein bring together (1) "self-buffering", (2) high ionic strength (125-500mM), and (3) "High-concentration" protein (50 to more than 250 mg / mL), surprisingly producing a stable formulation, despite counterintuitive factors.

[0426] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. REFERENCES

[0427] Arakawa, T., & Kita, Y. (2000). Stabilizing effects of caprylate and acetyltryptophanate on heat-induced aggregation of bovine serum albumin. Biochimica et Biophysica Acta (BBA). 1479(1-2), 32-36.

[0428] Ballou, G. A., Boyer, P. D., Luck, J. M. & Lum, F. G. (1944) The heat coagulation of human serum albumin. J. Biol. Chem. 153, 589-605.

[0429] Barash, I., Faerman, A., Baruch, A., Nathan, M., Hurwitz, D.R., Shani, M. (1993) Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice. Transgenic Research 2, 266-276.

[0430] Caironi, P. & Gattinoni, L. The clinical use of albumin: the point of view of a specialist in intensive care. Blood Transfus. (2009) doi: 10.2450 / 2009.0002-09.

[0431] Dibartola, S. P. Fluid, Electrolyte and Acid-Base Disorders in Small Animal Practice. (Saunders, Philadelphia, 2006).

[0432] Executive Committee of the German Medical Association on the Recommendation of the Scientific Advisory Board, (2016). Cross-Sectional Guidelines for Therapy with Blood Components and Plasma Derivatives: Chapter 5 Human Albumin - Revised. Transfus Med Hemother 2016;43:223-232

[0433] Farran, I., Sanchez-Serrano, J. J., Medina, J.F., Prieto, J., Mingo-Castel, A.M. (2002) Targeted expression of human serum albumin to potato tubers. Transgenic Research 11, 337-346.

[0434] Ferguson, W. J., Braunschweiger, K. I., Braunschweiger, W. R., Smith, J. R., McCormick, J. J., Wasmann, C. C., Jarvis, N. P., Bell, D. H., Good, N. E. (1980). Hydrogen Ion Buffers for Biological Research. Anal. Biochem. 104 (2): 300-310.

[0435] Fleer, R., Yeh, P., Amellal, N., Maury, I., Fournier, A., Bacchetta, F., Baduel, P., Jung, G., L'Hote, H., Becquart, J., Fukuhara, H., Mayaux, J.F. (1991) Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts. Bio / technology 9, 968-975.

[0436] Gatz, R. & Elbers, P. Albumin is not a buffer in plasma. Blood Transfus. 107-107 (2011) doi: 10.2450 / 2010.0015-10.

[0437] Gokarn, Y. R., Kras, E., Nodgaard, C., Dharmavaram, V., Fesinmeyer, R. M., Hultgen, H., Brych, S., Remmele Jr, R. L., Brems, D. N., Hershenson S., (2008) Selfbuffering antibody formulations. J Pharm Sci. 97(8):3051-66.

[0438] Good, N. E., Winget G. D., Winter W., Connolly, T. N., Izawa S., Singh, R. M. M., (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry 5 (2), 467-477. Good, N. E, Izawa, S. (1972). Hydrogen ion buffers. Methods Enzymol. Vol. 24. pp. 53-68. Hosseini, K.M., (October 2002). Study of the Heat-Treated Human Albumin Stabilization by Caprylate and Acetyltryptophanate. Iranian Biomedical Journal. 6 (4): 135-140.

[0439] Kobayashi, K., Nakamura, N., Sumi, A., Ohmura, T., Yokoyama, K. (1998) The development of recombinant human serum albumin. Therapeutic Apheresis 2(4), 257- 262.

[0440] Kragh-Hansen, U., Chuang, V.T.G., Otagiri, M. (2002) Practical Aspects of the Ligand- Binding and Enzymatic Properties of Human Serum Albumin. Biol. Pharm. Bull. 25(6), 695-704.

[0441] Mcllvaine, T. C. (1921). A buffer solution for colorimetric comparison. J. Biol. Chem. 49, 183-186.

[0442] Needleman, S.B. and Wunsch, C.D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48(3): 443- 453.

[0443] Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10(1): 1-6.

[0444] Pandjaitab, B., Swoboda, I., Brandejsky-Pichler, F., Rumpold, H., Valenta, R., Spitzauer, S. (2000) Escherichia coli expression and purification of recombinant dog albumin, a cross-reactive animal allergen. J. Allergy Clin. Immunol. 105(2)(l), 279- 285.

[0445] Petersen, T.N., Brunak, S., von Heijne, G., Nielsen, H. (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods (8): 785- 786.

[0446] Ping, H., Mann-Collura, O., Fling J., Edara N., Hetz R., Razzaque, M. S., (2020) High phosphate actively induces cytotoxicity by rewiring pro-survival and pro-apoptotic signalling networks in HEP293 and HeLa cells. FASEB. 35(1).

[0447] Rice, P., Longden, I., Bleasby, A. (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16(6): 276-277.

[0448] Rossing, N. (1978). Intra- and Extravascular Distribution of Albumin and Immunoglobulin in_Man._Lymphology 11, 138-142.

[0449] Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M, Hoekema, A. (1990) Production of correctly processed human serum albumin in transgenic plants. Biotechnology 8, 217-221.

[0450] Sleep, D., Belfield, G.P., Goodey, A.R. (1990) The secretion of human serum albumin from the yeast saccharomyces cerevisiae using five different leader sequences. Biotechnology 8, 42-46. Slyke, D. D. V., (1922). On the measurement of buffer values and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution. J. Biol. Chem. 52, 525-570.

[0451] Tada, S, Yasukawa K., Yatomi Y., Uchiki T., (2022) A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin. Practical Laboratory Medicine. 31, e00281.

[0452] Teufel, F., Almagro Armenteros, J. J., Rosenberg Johansen, A., Halldor Gislason, M., Irby Pihl, S., Tsirigos, K.D., Winther, O., Brunak, S., von Heijne, S., Nielsen, H. (2022) SignalP 6.0 predicts all five types of signal peptides using protein language models. Nature Biotechnology (40): 1023-1025.

[0453] Wellen, B. A., Lach, E. A. & Allen, H. C. (2017) Surface pKa of octanoic, nonanoic, and decanoic fatty acids at the air-water interface: applications to atmospheric aerosol chemistry. Phys Chem Chem Phys 19, 26551-26558.

[0454] Wright, T.L., Lysenko, N., Ockner, R. K., Weisiger, R. A.; (1987) Interaction of Natural and Synthetic Albumin Polymers with Hepatocytes. Hepatology. Vol. 7, No. 2, pp. 294- 301.

[0455] Zdlls, S., Weinbuch, D., Wiggenhorn, M., Winter, G., Friess W., Jiskoot, W., Hawe A., (2013). Flow Imaging Microscopy for Protein Particle Analysis— A Comparative Evaluation of Four Different Analytical Instruments. AAPS J 15, 1200-1211.

[0456] Zbacnik, T. J., Holcomb, R. E., Katayama, D. S., Murphy, B. M., Payne, R. W., Coccaro, R. C., Evans, G. J., Matsuura, J. E., Henry, C. S., & Manning, M. C. (2017). Role of Buffers in Protein Formulations. Journal of Pharmaceutical Sciences, 106(3), 713-733.

[0457] List of human albumin sequences described herein

[0458] >SEQ ID NO: 1 gacgctcacaagtccgaagtcgctcacagattcaaggacttgggtgaagaaaacttcaaggctttggtcttgatcgctttc g ctca a ta cttg ca a ca a tg tcca ttcg a a g a tea eg tea a g ttg g tea a eg a a g tta ccg a a ttcg eta a g a ettg tg tt g etg a eg a a teeg eg g a a a a etg tg a ca a g teettg ca ca ccttg ttcg g tg a ta a g ttg tg ta etg ttg eta ccttg a g a gaaacctacggtgaaatggctgactgttgtgctaagcaagaaccagaaagaaacgaatgtttcttgcaacacaaggac gacaacccaaacttgccaagattggttagaccagaagttgacgtcatgtgtactgctttccacgacaacgaagaaacctt cttg aagaagta cttg taegaaa ttg ctagaagacacccata etteta eg ctcca g a a ttg ttg ttetteg eta a g a g a ta c aaggctgctttcaccgaatgttgtcaagctgctgataaggctgcttgtttgttgccaaagttggatgaattgagagacgaa ggtaaggctagctccgcaaagcaaagattgaagtgtgcttccttgcaaaagttcggtgaaagagctttcaaggcttggg etg teg eta g a ttg tetea aagattcccaaagg etg aa ttcg etgaag ttteta a g ttg g tta etg a cttg a eta a g g ttca c actgaatgttgtcacggtgacttgttggaatgtgctgatgacagagctgacttggctaagtacatctgtgaaaaccaaga ctctatctcttccaagttgaaggaatgttgtgaaaagccattgttggaaaagtctcactgtattgctgaagttgaaaacgat gaaatgccagctga cttg cca tctttg g etg etg a etteg ttg a a teta a g g a eg tttg taagaactacgctgaagctaag g a eg tettettg g g ta tg ttcttg ta eg a a ta eg eta g a a g a ca ceca g a eta ctccg ttg tettg ttg ttg a g a ttg g eta a g a ccta eg a a a eta ccctcg a g a a g tg ttg tg etg etg etg a ccca ca eg a a tg tta eg eta a g g ttttcg a tg a a ttca a gccattggtcgaagaaccacaaaacttgatcaagcaaaactgtgaattgttcgaacaattgggtgaatacaagttccaa a a eg ctttg ttg g tta g a ta ca eta a g a a g g tccca ca a g tctcca cccca a ctttg g ttg a a g teteta g a a a cttg g g ta aggtcggttctaagtgttgtaagcacccagaagctaagagaatgccatgtgctgaagattacttgtccgtcgttttgaacc aattgtgtgttttgcacgaaaagaccccagtctctgatagagtcaccaagtgttgtactgaatctttggttaacagaagac ca tg tttctctg ctttg g a a g teg a eg a a a etta eg ttcca a a g g a a ttca a eg etg a a a etttea ccttcca eg etg a ta tc tgtaccttgtccgaaaaggaaagacaaattaagaagcaaactgctttggttgaattggtcaagcacaagccaaaggcta ctaaggaacaattgaaggctgtcatggatgatttcgctgctttcgttgaaaagtgttgtaaggctgatgataaggaaactt gtttcgctgaagaaggtaagaagttggtcgctgcttcccaagctgccttaggtttgtaataa

[0459] >SEQ ID NO: 2

[0460] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA KQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRA DLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIK QNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYL SVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEK ERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL GL >SEQ ID NO: 3

[0461] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVK LVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQH KDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLV

[0462] TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAAD PHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNL GKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVD ETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCC

[0463] KADDKETCFAEEGKKLVAASQAALGL

[0464] All of the remaining sequences listed in the application can be found on public databases (e.g. Genbank and / or Uniprot) as discussed above. It will be understood that newer versions and / or different isoforms or splice variants may also be suitable for carrying out the invention.

Claims

CLAIMS1. A stable, self-buffering albumin formulation comprising about 50 mg / mL to about 300 mg / mL albumin, about 125 mM to about 500 mM cations, < 2.0 mM octanoic acid; and wherein the pH is from about 6.4 to about 7.

2. The formulation according to Claim 1, wherein the albumin is substantially free from aggregation.

3. The formulation according to Claim 1 or 2, wherein the albumin in the formulation has one or more of the following properties:(i) remains >80% monomeric as determined by GP-HPLC after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months;(ii) retains a population of free thiol of >85% after incubation of the formulation at 5°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months;(iii) remains >70% monomeric as determined by GP-HPLC after incubation of the formulation at 40°C for 3 months; and / or(iv) retains a population of free thiol of >55% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months; optionally, wherein the free thiol is provided by the side chain of Cysteine-34 of human albumin.

4. The formulation according to any one of Claims 1-3, wherein the formulation has a percentage of polymeric albumin of < 10% after incubation of the formulation at 40°C for at least 1, 1.5, 2, 3, 4, 5 or 6 months, such as for 6 months.

5. The formulation according to any one of Claims 1-4, wherein the albumin provides substantially all of the buffering capacity of the formulation.

6. The formulation according to any one of Claims 1-5, wherein the formulation is substantially free of all other buffers except albumin.

7. The formulation according to any one of Claims 1-6, wherein the formulation is substantially free of non-macromolecular buffers.

8. The formulation according to any one of Claims 1-7, wherein the cations are at a concentration of about 125 mM to about 500 mM, or about 150 mM to about 40082mM, or about 175 mM to about 325 mM, optionally wherein the concentration of cations is from about 175 mM or about 250 mM.

9. The formulation according to any one of Claims 1-8, wherein the octanoic acid is present at < 1.0 mM.

10. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 175 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

11. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1.0 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.3 mM.

12. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are potassium ions and the originating salt is KCI, wherein the KCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

13. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are calcium ions and the originating salt is CaCIz, wherein the CaCIz is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

14. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 125 mg / mL, wherein the cations are sodium, ions and the originating salt is NazSC , wherein the NazSC is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferablywherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

15. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 50 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.5 mM; most preferably wherein the concentration of octanoic acid < 0.2 mM.

16. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of 200 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.8 mM; most preferably wherein the concentration of octanoic acid < 0.65 mM.

17. The formulation according to any one of Claims 1-9, wherein the albumin is at a concentration of about 125 mg / mL, wherein the cations are sodium ions and the originating salt is MgCh, wherein the MgCh is at a concentration of 250 mM, wherein the formulation has < 1 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 0.75 mM; most preferably wherein the concentration of octanoic acid < 0.4 mM.

18. The formulation according to any one of Claims 1-8, wherein the albumin is at a concentration of about 263 mg / mL, wherein the cations are sodium ions and the originating salt is NaCI, wherein the NaCI is at a concentration of 250 mM, wherein the formulation has < 2 mM octanoic acid, and wherein the pH is about 6.75; preferably wherein the concentration of octanoic acid is < 1.5 mM; most preferably wherein the concentration of octanoic acid < 0.9 mM.

19. The formulation according to any one of Claims 1-18, wherein the recombinant albumin is a recombinant yeast-derived human serum albumin.

20. A stable, self-buffering formulation for cell-based techniques or for use as or in a biological medium, wherein the formulation comprises the stable, self-buffering albumin formulation of any one of Claims 1-19.

21. A stable, self- buffer! ng pharmaceutical composition, wherein the formulation comprises the stable, self-buffering albumin formulation of any one of Claims 1-19.

22. A stable, self-buffering formulation for the coating of medical devices and / or implants, wherein the formulation comprises the stable, self-buffering albumin formulation of any one of Claims 1-19.

23. A method to allow tailoring of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:(a) providing a stable, self-buffering albumin formulation according to any one of Claims 1-19; and(b) combining the stable, self- buffering albumin formulation with one or more components to produce the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants.

24. A method of controlling, stabilising and / or buffering the pH of a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:(a) providing a stable, self-buffering albumin formulation according to any one of Claims 1-19; and(b) including the stable, self- buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biological medium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants.

25. A method to reduce and / or minimise precipitation of one or more medium components, such as metal ions, in a formulation for cell-based techniques, a formulation for use as or in a biological medium, a pharmaceutical formulation, or a formulation for the coating of medical devices and / or implants, wherein the method comprises:(a) providing a stable, self-buffering albumin formulation according to any one of Claims 1-19; and(b) including the stable, self- buffering albumin formulation as a component of the formulation for cell-based techniques, the formulation for use as or in a biologicalmedium, the pharmaceutical formulation, or the formulation for the coating of medical devices and / or implants, completely or partially in replacement of one or more potentially undesirable buffer or buffer component such as Good's buffer(s) and / or phosphate buffer(s).

26. Use of an albumin formulation that is substantially or completely free of inorganic phosphate, as a macromolecular buffer.

27. The use according to Claim 26, wherein the albumin provides substantially all of the buffering capacity of the albumin formulation.

28. The use according to Claim 26 or 27, wherein the albumin formulation is substantially free of all other buffering composition or buffering components except albumin.

29. The use according to any one of Claims 26-28, wherein the albumin formulation is substantially free of non-macromolecular buffering compositions or non- macromolecular buffering components.

30. The use according to any one of Claims 26-29, wherein the albumin confers a buffering capacity that is greater than an equivalent molar concentration of a Good's buffer or a phosphate buffer.

31. The use according to any one of Claims 26-30, wherein the albumin formulation is a stable, self-buffering albumin formulation according to any one of Claims 1-19.

32. The use according to any one of Claims 26-31, wherein being substantially or completely free of inorganic phosphate minimises one or more of enzyme inactivation, precipitation of cations, excipient crystallisation, viral inactivation or cell stress.

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