Methods for selecting excipients and for preparing high-concentration monoclonal antibody preparations and other preparations containing proteins.

Abstract

The present invention relates to a method for preparing a highly concentrated monoclonal antibody (mAb) formulation, comprising screening and selection of excipients to be included in the mAb formulation. The excipients include amino acids, salts, and polyols.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 541,455, filed on September 29, 2023, and U.S. Provisional Application No. 63 / 461,683, filed on April 25, 2023. The applications referenced above are hereby incorporated by reference in their entirety.

[0002] The present invention relates to a method for preparing a highly concentrated formulation of a target protein, including an Fc - containing protein (such as a monoclonal antibody (mAb)). The method includes screening and selecting excipients to be included in such a formulation.

Background Art

[0003] Fc - containing proteins, such as antibodies, are important therapeutic products in today's medicine. The widespread use of monoclonal antibody therapies has made highly concentrated formulations desirable for subsequent dilution into the final dosage form.

[0004] High - concentration monoclonal antibody formulations present quality issues for manufacturing, storage, and administration. High concentrations cause high viscosities and increase antibody aggregation via protein - protein interactions (PPIs).

[0005] Excipients and manufacturing methods play an important role in the characteristics of high - concentration monoclonal antibody preparations. Excipients include amino acids, salts, and polyols.

Summary of the Invention

[0006] The present invention provides a method for selecting excipients for inclusion in a high - concentration Fc - containing protein formulation, where monoclonal antibodies are an example of an Fc - containing protein and serve as an example.

[0007] A monoclonal antibody preparation may be manufactured by a method comprising: (a) combining at least one excipient with a monoclonal antibody preparation; (b) performing a controlled nucleation cycle on the monoclonal antibody preparation from step (a); (c) drying the monoclonal antibody preparation from step (b) under vacuum; (d) stopping with or without vacuum; and (e) evaluating the excipient performance. The method may avoid using secondary drying. Annealing may be omitted if necessary.

[0008] The method allows for screening and selection of excipients selected from the group consisting of amino acids, salts, polyols, and combinations thereof. Excipients may be polyols selected from the group consisting of sucrose, trehalose dihydrate, sorbitol, glycerol, and combinations thereof. Excipients may be salts selected from the group consisting of magnesium chloride, sodium sulfate, ammonium sulfate, sodium bromide, sodium chloride, calcium chloride, sodium perchlorate, and combinations thereof. Excipients may be amino acids selected from the group consisting of monosodium glutamate, glutamate, alanine, proline, glycine, lysine, phenylalanine, methionine, isoleucine, threonine, valine, serine, asparagine, histidine arginine hydrochloride, glutamate arginine, and combinations thereof. A monoclonal antibody preparation may, according to the method of the present invention, contain at least one excipient comprising (i) at least one polyol selected from the group consisting of sucrose, trehalose dihydrate, sorbitol, glycerol, and combinations thereof; (ii) at least one salt selected from the group consisting of magnesium chloride, sodium sulfate, ammonium sulfate, sodium bromide, sodium chloride, calcium chloride, sodium perchlorate, and combinations thereof; and (iii) at least one amino acid selected from the group consisting of sodium glutamate, glutamate, alanine, proline, glycine, lysine, phenylalanine, methionine, isoleucine, threonine, valine, serine, asparagine, histidine arginine hydrochloride, glutamate arginine, and combinations thereof. A monoclonal antibody preparation may contain, according to the method of the present invention, one or more salts and amino acids as excipients, one or more salts and polyols as excipients, one or more amino acids and polyols as excipients, or one or more amino acids, salts, and polyols as excipients. A monoclonal antibody preparation containing one or more excipients selected according to the method of the present invention is also provided. [Brief explanation of the drawing]

[0009] [Figure 1] Bar graph showing reconstitution time and viscosity (cP). Test excipients (amino acids, salts, and polyols) are at a concentration of 150 mM and evaluated using a 190 mg / ml monoclonal antibody.

[0010] [Figure 2] The data shows the effect of the reconstituted excipient on aggregation (Δ%HMW).

[0011] [Figure 3] Data from excipient screening (amino acids and salts) of 220 mg / ml mAb preparations are shown. Reconstitution time and viscosity (cP) are indicated.

[0012] [Figure 4] Data from viscosity screening of single excipient versus excipient combinations in a 1:1 ratio are shown. Monoclonal antibodies were present at 220 mg / ml.

[0013] [Figure 5] This study demonstrates the effect of pH on viscosity (cP) using a monoclonal antibody present at a concentration of 220 mg / ml.

[0014] [Figure 6] A bar graph showing the viscosity of a recomposed solution of monoclonal antibody at a concentration of 250 mg / ml with 75 mM and 150 mM potential viscosity-reducing excipients. WFI represents water for injection (without excipients).

[0015] [Figure 7] A bar graph showing the viscosity of a recomposed solution of monoclonal antibody at a concentration of 200 mg / ml with 75 mM and 150 mM potential viscosity-reducing excipients. WFI represents water for injection (without excipients). [Modes for carrying out the invention]

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains.

[0017] definition In the context of numbers and ranges, the term "approximately" refers to a value or range that approximates or is close to the enumerated value or range, so that the invention can be carried out as intended, for example, with a desired rate, quantity, density, degree, increase, decrease, percentage, value or presence of form, variant, temperature, or quantity of time. For example, "approximately" can indicate a value that is either above or below the stated value by approximately + / - 10% or less, depending on the ability to do so. Thus, the term encompasses values ​​other than those resulting simply from systematic errors.

[0018] The term "stoppering" refers to a function used in the freeze-drying process. Freeze-drying vials have stoppers on top of them for control. If the freeze-drying stopper is not fully inserted into the vial, there is a gap between the stopper and the vial opening, allowing freeze-drying to occur. Automatic freeze-dryers have a "stoppering" function in which a plate can push all the stoppers in. After this stoppering process, there is no gap between the stopper and the vial opening, and the vial is sealed from the atmosphere. Stoppering can be performed either while the freeze-dryer is still under vacuum (vacuum stoppering) or after the chamber has been filled with nitrogen gas.

[0019] The "protein of interest" or "polypeptide of interest" (POI) can have any amino acid sequence and includes any protein, polypeptide, or peptide that is desired to be expressed. This includes, but is not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins, and animal (including human) proteins. Protein types include, but are not limited to, antibodies, receptors, Fc-containing proteins, trap proteins (including mini-trap proteins), fusion proteins, antagonists, inhibitors, enzymes (such as those used in enzyme replacement therapy), factors, suppressors, activators, ligands, reporter proteins, selectable proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters, and contractile proteins. Also included are the above-mentioned derivatives, components, domains, chains, and fragments. The sequence can be a natural sequence, a semi-synthetic sequence, or a synthetic sequence. The protein of interest is encoded by the "gene of interest" (GOI).

[0020] An "antibody" (also referred to as an "immunoglobulin") is an example of a protein having multiple polypeptide chains and extensive post-translational modifications. A canonical immunoglobulin protein (e.g., IgG) contains four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine disulfide bond, and the two heavy chains are linked to each other via two cysteine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (e.g., asparagine residues) by various polysaccharides, which can vary by species and can affect the antigenicity of therapeutic antibodies. Butler and Spearman, “The choice of mammalian cell host and possibilities for glycosylation engineering”, Curr. Opin. Biotech. 30:107 - 112 (2014). Antibodies are often used as therapeutic biomolecules.

[0021] An antibody comprises an immunoglobulin molecule composed of four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDRl, LCDR2, and LCDR3. The term "high affinity" antibody refers to an antibody having a binding affinity for its target of at least 10 -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 M.

[0022] Antibodies can be based on all major antibody classes, namely IgG, IgA, IgM, IgD and IgE. IgG is the preferred class and includes subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Antibodies include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab’)2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

[0023] The antibody can be an IgG1 antibody. The antibody can be an IgG2 antibody. The antibody can be an IgG3 antibody. The antibody can be an IgG4 antibody. The antibody can be a chimeric IgG2 / IgG4 antibody. The antibody can be a chimeric IgG2 / IgG1 antibody. The antibody can be a chimeric IgG2 / IgG1 / IgG4 antibody. The above derivatives, components, domains, chains, and fragments are also included.

[0024] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. Bispecific antibodies generally contain two distinct heavy chains, each specifically binding to a different epitope on either two different molecules (e.g., antigens) or the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody can be located on the same target or on different targets (e.g., on the same protein or different proteins). Bispecific antibodies can be created, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs, followed by (from N-terminus to C-terminus) CH1 domain, hinge, CH2 domain, and CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and can bind to one or more epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain and can bind one or both heavy chains to one or both epitopes.

[0025] The terms “heavy chain” or “immunoglobulin heavy chain” include the constant region sequence of an immunoglobulin heavy chain from any organism and, unless otherwise specified, the heavy chain variable domain. The heavy chain variable domain includes three heavy chain CDRs and four FR regions unless otherwise specified. Fragments of a heavy chain include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain followed (from the N-terminus to the C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of a heavy chain can specifically recognize an antigen (e.g., recognize an antigen in the micromolar, nanomolar, or picomolar KD range), can be expressed and secreted from cells, and include fragments containing at least one CDR.

[0026] The term “light chain” includes the constant region sequence of an immunoglobulin light chain from any organism, and unless otherwise specified, includes human kappa and lambda light chains. The variable light chain (VL) domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes a VL domain containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus, and a constant light chain domain. Light chains that can be used in these inventions include, for example, light chains that do not selectively bind to either a first or second antigen selectively bound by an antigen-binding protein. Preferred light chains include those that can be identified by screening for the light chains most commonly used in existing antibody libraries (wet libraries or in silico), which do not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Preferred light chains include those that can bind to one or both epitopes bound by the antigen-binding region of the antigen-binding protein.

[0027] The term “variable domain” includes an amino acid sequence (as desired, modified) of an immunoglobulin light or heavy chain, which, unless otherwise specified, includes the following amino acid regions in its N-terminal to C-terminal sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A “variable domain” includes an amino acid sequence that can fold into a canonical domain (VH or VL) having a double beta-sheet structure, where the beta sheets are connected by disulfide bonds between the residues of the first beta-sheet and the residues of the second beta-sheet.

[0028] The term “complementarity-determining region” or “CDR” typically refers to an amino acid sequence encoded by the nucleic acid sequence of an organism’s immunoglobulin gene that appears between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor) in wild-type animals. A CDR can be encoded by, for example, a germline sequence, or by a sequence that has been rearranged or not rearranged, for example, by a naive or mature B cell or T cell. In some situations (e.g., CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not adjacent (e.g., in an unrearranged nucleic acid sequence) but are adjacent in a B cell nucleic acid sequence, for example, as a result of sequence splicing or joining (e.g., VDJ recombination forming a heavy chain CDR3).

[0029] "Antibody derivatives and fragments" include, but are not limited to, antibody fragments (e.g., ScFv-Fc, dAB-Fc, semi-antibodies), multispecificity (e.g., IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, triplicate), and Fc fusion proteins (e.g., Fc fusion (N-terminus), Fc fusion (C-terminus), mono-Fc fusion, bispecificity Fc fusion).

[0030] The term "Fc-containing protein" includes antibodies, bispecific antibodies, antibody derivatives containing Fc, antibody fragments containing Fc, Fc fusion proteins, immunoadhesins, and other binding proteins containing at least a functional portion of the immunoglobulin CH2 and CH3 regions. "Functional portion" refers to the CH2 and CH3 regions that can bind to an Fc receptor (e.g., FcyR, or FcRn (neonatal Fc receptor)) and / or be involved in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any Fc receptor and from activating complement, then the CH2 and CH3 regions are not functional.

[0031] "Fc" represents a crystallizable fragment and is often referred to as the fragment constant. Antibodies contain an Fc region formed by two identical protein sequences. IgG has a heavy chain known as the γ chain. IgA has a heavy chain known as the α chain, and IgM has a heavy chain known as the μ chain. IgD has a heavy chain known as the σ chain. IgE has a heavy chain known as the ε chain. In nature, the Fc region is the same in all antibodies of a given class and subclass within the same species. Human IgG has four subclasses, sharing approximately 95% homology among them. In each subclass, the Fc sequence is the same. For example, human IgG1 antibodies have the same Fc sequence. Similarly, IgG2 antibodies have the same Fc sequence, IgG3 antibodies have the same Fc sequence, and IgG4 antibodies have the same Fc sequence. Changes in the Fc region result in charge fluctuations.

[0032] Fc-containing proteins may contain modifications to their immunoglobulin domains, including modifications that affect one or more effector functions of the binding protein (e.g., modifications affecting FcyR binding, FcRn binding, and therefore half-life, and / or CDC activity). Such modifications include, but are not limited to, the following modifications and their combinations, with reference to the EU numbering of the immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309 ,311,312,315,318,320,322,324,326,327,328,329,330,331,332,333,334,335,337,338,339,340,342,344,356,358,359,360,361,362,373,375,376,378,380,382,383,384,386,388,389,398,414,416,419,428,430,433,434,435,437,438, and 439.

[0033] For example, but not limited to, the binding protein is an Fc-containing protein exhibiting an enhanced serum half-life (compared to the same Fc-containing protein without the listed modifications(s)) and having modifications at position 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W, or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D, or T), or at position 428 and / or 433 (e.g., L / R / SI / P / Q, or K) and / or 434 (e.g., H / F, or Y), or at position 250 and / or 428, or at position 307 or 308 (e.g., 308F, V308F), and 434. In another example, the modifiers could include 428L (e.g., M428L) and 434S (e.g., N434S) modifiers, 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modifiers, 433K (e.g., H433K) and 434 (e.g., 434Y) modifiers, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifiers, 250Q and 428L modifiers (e.g., T250Q and M428L), and 307 and / or 308 modifiers (e.g., 308F or 308P).

[0034] Some recombinant Fc-containing proteins include "receptor Fc fusion proteins," which refer to recombinant molecules containing a soluble receptor fused to an immunoglobulin Fc domain, containing a receptor or receptor fragment, ligand or ligand fragment that has a congenerally related binding partner in the biological system.

[0035] An "Fc fusion protein" contains part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which is not fused in its native state. Fc fusion proteins include Fc fusion (N-terminus), Fc fusion (C-terminus), mono-Fc fusion, and bispecific Fc fusion. The preparation of fusion proteins containing specific heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) is described, for example, in Ashkenazi et al., Proc. Natl. Acad. Sci USA 88:10535-39 (1991), Byrn et al., Nature 344:677-70, 1990, and Hollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11 (1992). A "receptor Fc fusion protein" contains one or more extracellular domains of a receptor bound to its Fc portion, which may include a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. An Fc fusion protein may contain two or more distinct receptor chains that bind to one or more ligands. Some receptor Fc fusion proteins may contain ligand-binding domains of multiple different receptors. Receptor Fc fusion proteins are also referred to as "traps," "trap molecules," or "trap proteins." For example, such trap proteins include IL-1 traps (e.g., Rilonacept, which contains an IL-1RAcP ligand-binding domain fused to the extracellular domain of IL-1R1 fused to the Fc of hlgGl; see U.S. Patent No. 6,927,044) or VEGF traps (e.g., Aflibercept, which contains the Ig domain 2 of VEGF receptor Fltl fused to the Ig domain 3 of VEGF receptor Flkl fused to the Fc of hlgG1; see Sequence ID No. 1, U.S. Patent Nos. 7,087,411 and 7,279,159).

[0036] Rilonocept and aflibercept are examples of commercially available trap proteins that antagonize IL1R (see U.S. Patent No. 7,927,583) and VEGF (see U.S. Patent No. 7,087,411), respectively. Other recombinant Fc-containing proteins include recombinant proteins containing peptides fused to the Fc domain. Recombinant Fc-containing proteins are described in C. Huang, “Receptor-Fc fusion therapeutics, traps, and MFMETIBODY technology,” 20(6)Curr.Opin.Biotechnol.692-9(2009).

[0037] Proteins lacking the Fc moiety, such as recombinantly produced enzymes and minitraps, can also be used according to the present invention. Minitraps are trap proteins that use a polymerizing component (MC) instead of the Fc moiety, as disclosed in U.S. Patents 7,279,159 and 7,087,411. The above derivatives, components, domains, chains, and fragments are also included.

[0038] All numerical limits and ranges shown herein include all numbers or values ​​around or between the numerical range or limit. The ranges and limits described herein expressly represent and indicate all integers, decimals, and fractional values ​​defined and encompassed by the range or limit. Thus, unless otherwise indicated herein, the enumeration of ranges of values ​​is intended merely as a way of individually referring to each distinct value that falls within a range, and each distinct value is incorporated herein as if it were individually enumerated herein.

[0039] explanation The present invention provides a method for selecting excipients for inclusion in high-concentration Fc-containing protein formulations, wherein monoclonal antibodies are a type of Fc-containing protein and serve as an example.

[0040] High-concentration mAb formulations required for high-dose subcutaneous (SC) administration are subject to high viscosity and protein aggregation. Screening of excipients, pH, and buffers is often necessary to develop optimized high-concentration mAb formulations. However, this screening can be challenging because protein solutions without excipients at concentrations exceeding 200 mg / ml can reach their gelation point.

[0041] This invention utilizes lyophilization for screening high-concentration antibody formulations to select excipients and combinations of excipients for use with monoclonal antibodies. The lyophilized mAbs can be reconstituted to a desired protein concentration using any combination of excipients and buffers screened and selected according to this invention. Such screened and selected excipients are useful for high-concentration monoclonal antibody preparations. [Examples]

[0042] The present invention is further illustrated by the following embodiments which support and illustrate the invention, but does not limit the invention in any way.

[0043] Example 1 - Effect of different freeze-drying methods on reconstitution time Monoclonal antibodies were dialyzed into water at a moderate concentration (approximately 100 mg / ml), and pre-lyophilized buffer was added. The resulting solution was lyophilized in 6R vials (1.5 ml per vial) and reconstituted to the protein concentration required for the desired screening buffer (e.g., approximately 200 mg / ml). The pre-lyophilized buffer and lyophilization method were optimized to minimize both reconstitution time and the presence of high molecular weight (HMW) species. Using this method, different reconstituted buffers were screened to minimize solution viscosity while maintaining the other parameters mentioned above within acceptable limits.

[0044] Table 1 below shows the effect of vacuum stoppering on annealing and controlled nucleation freeze-drying in terms of reconstruction time. Conventional freeze-drying was used as a control. [Table 1]

[0045] Lyophilization of monoclonal antibody samples in water resulted in high levels of HMW species. Addition of buffer and sucrose (5 mM histidine, 1% sucrose, pH 5.9) or 1% sucrose alone reduced HMW species to acceptable levels (less aggregation). The lyophilization cycle was initially optimized by eliminating secondary drying, which was found to be unnecessary. Aggregation tended to increase with exposure to drying; therefore, eliminating secondary drying reduced aggregation.

[0046] To reduce reconstruction time, controlled nucleation and annealing, as well as vacuum stoppering, were tested. Controlled nucleation at -5°C combined with vacuum stoppering resulted in the shortest reconstruction time and acceptable HMW level. Annealing was less effective in reducing reconstruction time. Controlled nucleation can optionally be used with temperature-controlled freezing, for example, to reduce supercooling.

[0047] Example 2 - Screening of different viscosity reducers The protocol of Example 1 was implemented to screen different viscosity reducers, and it was found that several salts and charged amino acids, at a concentration of 150 mM, maintained short reconstitution times and acceptable levels of HMW species while reducing the viscosity of the reconstituted solution (190 or 220 mg / ml) to an acceptable level (<30 cps). The reconstitution buffer contained 10 mM histidine, 2% sucrose, 150 mM excipients, and 190 or 220 mg / ml mAb. The pH was 5.9. The results are shown in Figures 1 and 3.

[0048] Example 3 - Reconstitution time, viscosity, and Δ%HMW Table 2 discloses data from controlled nucleation and vacuum stoppering using monoclonal antibodies at concentrations of 190 mg / ml and 220 mg / ml. Changes in reconstitution time, viscosity, and percentage of high molecular weight species were measured. The results indicate that the change in percentage of high molecular weight species (Figure 2, Table 2) was reasonable for all tested excipients, confirming the usefulness of this screening method. Changes in reconstitution time in the presence of excipients followed changes in viscosity (Figures 1-2, Table 2). [Table 2]

[0049] Example 4 - Characterization of Excipients This example illustrates the characteristics of many excipients. Figure 2 shows data illustrating the effect of the reconstituted excipient on aggregation (Δ%HMW). The results confirm the usefulness of this screening method, indicating that the percentage change in high molecular weight species was moderate for all tested excipients. The results demonstrate that a lyophilized platform with minimal stabilizer (mAb to sucrose weight ratio of 10:1) can be applied to screen high-concentration mAb formulations for optimal compositions and pH with acceptable viscosity and stability.

[0050] Figure 3 shows data from excipient screening for a smaller number of amino acid and salt excipients for mAb preparations at 220 mg / ml. Reconstitution time and viscosity (cP) are shown.

[0051] Figure 4 shows data from viscosity screening of single excipient versus excipient combinations in a 1:1 ratio. Monoclonal antibodies were present at 220 mg / ml.

[0052] Figure 4 includes six groups. Each group presents data separately for each excipient, and then each excipient is combined in a 1:1 ratio (1:1). In some cases, a cumulative effect of the excipients on solution viscosity was observed. The results demonstrate that the platform can be applied to screen excipient combinations for high-concentration mAb formulations.

[0053] Figure 5 shows the effect of pH on viscosity (cP) using a monoclonal antibody present at a concentration of 220 mg / ml. Each excipient was tested at pH 5 or pH 6 to determine its effect on viscosity. In most cases, viscosity was lower at pH 5 compared to pH 6. The results demonstrate that the platform can be applied to screen pH / buffer levels for high-concentration mAb formulations.

[0054] Figure 6 is a bar graph showing the viscosity of a recomposed solution of monoclonal antibody at a concentration of 250 mg / ml with 75 mM and 150 mM potential viscosity-reducing excipients. Figure 7 is a bar graph showing the viscosity of a recomposed solution of monoclonal antibody at a concentration of 200 mg / ml with 75 mM and 150 mM potential viscosity-reducing excipients. WFI represents water for injection (without excipients).

[0055] Figures 6 (250 mg / ml mAb) and 7 (200 mg / ml mAb) using 75 mM or 150 mM excipients collectively show that some of the tested excipients (e.g., camphor sulfonic acid and phenyltrimethylammonium iodide) result in lower solution viscosity than arginine, a commonly used viscosity-reducing excipient. The results demonstrate that the platform can be applied to screening excipients and excipient combinations for high-concentration mAb formulations to find excipients or combinations that are more effective than commonly used viscosity-reducing agents (e.g., arginine hydrochloride).

[0056] Example 5 - Exemplary Antibody According to an additional aspect of the present invention, the antibody is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand 1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., as described in U.S. Patent No. 9,018,356) Anti-AngPtl3 antibodies, anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Patent No. 9,132,192) Anti-EGFRvIII antibodies (such as those described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-precursor protein-converting enzyme subtilisin kexin-9 antibodies (e.g., anti-PCSK9 as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-proliferative and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies as described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptor (For example, anti-GCGR antibodies as described in U.S. Patent Application Publication No. 2015 / 0337045A1 or No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies as described in U.S. Patent Application Publication No. 2014 / 0271681A1 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibodies (such as those described in Patent No. 880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., anti-IL33 antibodies as described in U.S. Patent Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies as described in U.S. Patent Publication No. 2014 / 0271653A1), anti-differentiation antigen group 3 (e.g., U.S. Patent Publication No. 2 Anti-CD3 antibodies (as described in U.S. Patent Publication No. 014 / 0088295A1 and No. 2015 / 0266966A1, and U.S. Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Publication No. 2014 / 0088295A1 and No. 2015 / 0266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation antigen group 48 (e.g., anti-CD48 antibodies as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody as described in U.S. Patent Publication No. 2015 / 0337029A1), anti-Ebola virus antibody (e.g., as described in U.S. Patent Publication No. 2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activator gene 3 antibody (e.g., anti-LAG3 antibody, or anti-CD223 antibody), anti-nerve growth factor antibody (e.g., U.S. Patent Publication No. 2016 / 0017029, and U.S. Patents No. 8,309,088 and 9,353,The bispecific antibody can be selected from the group consisting of anti-NGF antibodies (such as those described in Patent Publication No. 176) and anti-activin A antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibodies (such as those described in U.S. Patent Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1), anti-CD3 × anti-mucin 16 bispecific antibodies (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3 × anti-PSMA bispecific antibody). See also U.S. Patent Publication No. 2019 / 0285580A1. Furthermore, this includes MetxMet antibodies, agonist antibodies against NPR1, LEPR agonist antibodies, BCMAxCD3 antibodies, MUC16xCD28 antibodies, GITR antibodies, IL-2Rg antibodies, EGFRxCD28 antibodies, factor XI antibodies, antibodies against SARS-CoC-2 variants, Fel d 1 multi-antibody therapy, and Bet v 1 multi-antibody therapy. The above derivatives, components, domains, chains, and fragments are also included.

[0057] Cells producing exemplary antibodies can be cultured according to the present invention. Exemplary antibodies include alirocumab, atorutivimab, maftivimab, odesibimab, odesibibumab-ebgn, cacilibimab, imdevimab, semiprimab and semiprimab-rwlc (human IgG4 monoclonal antibodies that bind to PD-1), dupilumab (human monoclonal antibody of the IgG4 subclass that binds to the IL-4R alpha (α) subunit and thereby inhibits interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling), evinacumab, evinacumab-dgnb, facinumab, fianlimab, galetosumab, itepecimab, nesbakumab, odronextumab, pozelimuab, sarilumab, trevoglumab, and reinukumab.

[0058] Additional exemplary antibodies include ravulizumab-cwvz, absiximab, adalimumab, adalimumab-atto, ad-trastuzumab, alemtuzumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotokisumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cetuximab, denosumab, dinutuximab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, entansine alirocumab, evolocumab, golimumab, and gsel. Examples include coumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nivolumab, obilutoxaximab, obinutuzumab, ocrelizumab, ofatumumab, oraratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, laxibakumab, reslizumab, linukumab, rituximab, secukinumab, siltuximab, tocilizumab, trastuzumab, ustekinumab, and vedolizumab.

[0059] It should be understood that the descriptions, specific examples, and data are given as illustrations and are not intended to limit the invention. Various changes and modifications within the invention, including combining the teachings in whole and in part, will be apparent to those skilled in the art from the discussions, disclosures, and data contained herein and are therefore considered part of the invention.

Claims

1. A method for selecting an excipient to be incorporated into a high-concentration Fc-containing protein preparation, wherein the method is (a) The step of combining at least one excipient with a preparation containing an Fc-containing protein, (b) A step of performing a controlled nucleation cycle on the preparation from step (a), (c) A step of drying the preparation from step (b) under vacuum, (d) A step of stopping regardless of whether there is a vacuum, A method comprising the step of (e) evaluating the excipient performance for use in an Fc-containing protein preparation.

2. The method according to claim 1, wherein secondary drying is not used.

3. The method according to claim 1 or 2, wherein the excipient is selected from the group consisting of amino acids, salts, polyols, and combinations thereof.

4. The method according to any one of claims 1 to 3, wherein the excipient is a polyol selected from the group consisting of sucrose, trehalose dihydrate, sorbitol, glycerol, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the excipient is a salt selected from the group consisting of magnesium chloride, sodium sulfate, ammonium sulfate, sodium bromide, sodium chloride, calcium chloride, sodium perchlorate, and combinations thereof.

6. The method according to any one of claims 1 to 5, wherein the excipient is an amino acid selected from the group consisting of sodium glutamate, glutamate, alanine, proline, glycine, lysine, phenylalanine, methionine, isoleucine, threonine, valine, serine, asparagine, histidinearginine hydrochloride, glutamate arginine, and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the Fc-containing protein is a monoclonal antibody.

8. The Fc-containing protein preparation is (i) at least one polyol selected from the group consisting of sucrose, trehalose dihydrate, sorbitol, glycerol, and combinations thereof, (ii) At least one salt selected from the group consisting of magnesium chloride, sodium sulfate, ammonium sulfate, sodium bromide, sodium chloride, calcium chloride, sodium perchlorate, and combinations thereof, and (iii) The method according to any one of claims 1 to 7, comprising at least one excipient selected from the group consisting of sodium glutamate, glutamate, alanine, proline, glycine, lysine, phenylalanine, methionine, isoleucine, threonine, valine, serine, asparagine, histidinearginine hydrochloride, glutamate arginine, and combinations thereof.

9. The method according to any one of claims 1 to 8, wherein the Fc-containing protein preparation comprises a salt and an amino acid as excipients.

10. The method according to any one of claims 1 to 8, wherein the Fc-containing protein preparation comprises a salt and a polyol as excipients.

11. The method according to any one of claims 1 to 8, wherein the Fc-containing protein preparation comprises an amino acid and a polyol as excipients.

12. The method according to any one of claims 1 to 8, wherein the Fc-containing protein preparation comprises an amino acid, a salt, and a polyol as excipients.

13. An Fc-containing protein preparation comprising one or more excipients selected according to the method described in any one of claims 1 to 12.

14. The Fc-containing preparation according to claim 13, wherein the Fc-containing protein is a monoclonal antibody.

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