Multiple-dose antibody drugs using phenol or benzyl alcohol

Abstract

This specification provides a method for stabilizing Fc-containing protein preparations in aqueous solution. The disclosed method can stabilize Fc-containing proteins in pharmaceuticals, particularly liquid formulations, by using phenol or benzyl alcohol as a preservative.

Description

[Technical Field]

[0001] Reference to related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 463,179, filed on 1 May 2023. The above referenced application is incorporated herein by reference in its entirety.

[0002] The present invention provides an improved method for stabilizing an Fc-containing protein preparation in an aqueous solution using a preservative. [Background technology]

[0003] Multidose antibody drugs are often supplied in lyophilized form to extend their shelf life. Some antibody products are supplied in solution and require preservatives. However, preservatives can destabilize antibodies. Furthermore, while various antimicrobial preservatives are used in commercially available biological products, they are rarely used in monoclonal antibodies (mAbs).

[0004] Therefore, considering the effect of typical antimicrobial preservatives on the stability of several mAb liquid formulations, an object of the present invention is to provide an improved method for stabilizing and storing Fc-containing protein preparations in aqueous solution. Another object of the present invention is to provide an improved method for developing formulations containing preservatives with stable Fc-containing proteins in the pharmaceutical product, for multi-dose Fc-containing protein pharmaceuticals. [Overview of the project]

[0005] A method for stabilizing an Fc-containing protein preparation in an aqueous solution is provided. For example, a multi-dose container may contain a parenteral Fc-containing protein preparation. The preparation may contain at least one Fc-containing protein in an aqueous solution. The preparation may also contain phenol or benzyl alcohol in an aqueous solution.

[0006] One or more of the following exemplary features may be included: The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%).

[0007] The container may be a single-patient container. The container may have a capacity of 1 mL to 100 mL. The container may also have a capacity of 0.5 mL to 100 mL. The container may further have a capacity of 5 mL to 50 mL. The container may have an even larger capacity of 20 mL to 40 mL. The container may also have a capacity of 30 mL. The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The container may contain one, two, three, or more types of Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. The Fc-containing protein may also be a trap protein.

[0008] In another exemplary implementation, the parenteral Fc-containing protein preparation may contain at least one Fc-containing protein in aqueous solution. The parenteral Fc-containing protein preparation may also contain phenol or benzyl alcohol in aqueous solution.

[0009] One or more of the following exemplary features may be included. The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to about 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%). The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The preparation may contain one, two, three, or more types of Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. The Fc-containing protein may also be a trap protein.

[0010] Another exemplary embodiment provides a method for stabilizing an Fc-containing protein preparation, which may include the step of providing the Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol. The method for stabilizing an Fc-containing protein preparation may also include the step of filling a container with the Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol.

[0011] One or more of the following exemplary features may be included. Phenol or benzyl alcohol may be added to an aqueous solution containing an Fc-containing protein. The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to about 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%). The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The preparation may contain one, two, three, or more types of Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. Fc-containing proteins may also be trap proteins.

[0012] Further explanation of the present invention is provided below. [Brief explanation of the drawing]

[0013] [Figure 1A] This study demonstrates the effect of preservatives on the turbidity of formulations under stress conditions. When stored at 45°C for 3 months, formulations containing benzyl alcohol showed a relatively greater increase in turbidity than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects. [Figure 1B] This study demonstrates the effect of preservatives on the turbidity of formulations under stress conditions. When stored at 45°C for 3 months, formulations containing benzyl alcohol showed a relatively greater increase in turbidity than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects. [Figure 1C]This study demonstrates the effect of preservatives on the turbidity of formulations under stress conditions. When stored at 45°C for 3 months, formulations containing benzyl alcohol showed a relatively greater increase in turbidity than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects. [Figure 1D] This study demonstrates the effect of preservatives on the turbidity of formulations under stress conditions. When stored at 45°C for 3 months, formulations containing benzyl alcohol showed a relatively greater increase in turbidity than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects.

[0014] [Figure 2A] This shows the turbidity (ΔOD) of antibodies containing preservatives at 25°C. When pharmaceuticals were stored with the test preservatives at 25°C for 6 months, no significant instability was observed. An invisible line indicates zero. [Figure 2B] This shows the turbidity (ΔOD) of antibodies containing preservatives at 5°C. When pharmaceuticals were stored with the test preservatives at 5°C for 24 months, no significant instability was observed. An invisible line indicates zero.

[0015] [Figure 3A] This study demonstrates the effect of preservatives on high molecular weight (HMW) complex formation under stress conditions. When stored at 40°C for 3 months, formulations containing benzyl alcohol showed a relatively greater destabilizing effect than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects. [Figure 3B] This study demonstrates the effect of preservatives on high molecular weight (HMW) complex formation under stress conditions. When stored at 40°C for 3 months, formulations containing benzyl alcohol showed a relatively greater destabilizing effect than those containing phenol. Furthermore, different antibody (mAb) formulations exhibited different effects. [Figure 3C]Shows the effect of preservatives on the formation of high molecular weight (HMW) complexes under stress conditions. When stored at 40 °C for 3 months, a relatively greater destabilizing effect was observed in the formulation containing benzyl alcohol than in the formulation containing phenol. Also, different effects were observed with different antibody (mAb) formulations. [Figure 3D] Shows the effect of preservatives on the formation of high molecular weight (HMW) complexes under stress conditions. When stored at 40 °C for 3 months, a relatively greater destabilizing effect was observed in the formulation containing benzyl alcohol than in the formulation containing phenol. Also, different effects were observed with different antibody (mAb) formulations.

[0016] [Figure 4A] Shows the effect of preservatives on the HMW complex formation rate (%) when stored at 25 °C for 6 months. No significant destabilization was observed between formulations measuring the same amount of benzyl alcohol versus phenol. [Figure 4B] Shows the effect of preservatives on the HMW complex formation rate (%) when stored at 25 °C for 6 months. No significant destabilization was observed between formulations measuring the same amount of benzyl alcohol versus phenol. [Figure 4C] Shows the effect of preservatives on the HMW complex formation rate (%) when stored at 25 °C for 6 months. No significant destabilization was observed between formulations measuring the same amount of benzyl alcohol versus phenol. [Figure 4D] Shows the effect of preservatives on the HMW complex formation rate (%) when stored at 25 °C for 6 months. No significant destabilization was observed between formulations measuring the same amount of benzyl alcohol versus phenol.

[0017] [Figure 5] Shows the effect of antibodies on the % HMW complex formation rate (%) by preservatives stored at 5 °C. When the pharmaceutical product was stored at 5 °C for 24 months with the test preservatives, no significant instability was observed.

[0018] [Figure 6A]This study demonstrates the effect of preservatives on antibody charge variant formation under stress conditions. When the drug was incubated at 40°C for two months, no substantial destabilization effect was observed in antibody charge variants containing preservatives. [Figure 6B] This study demonstrates the effect of preservatives on antibody charge variant formation under stress conditions. When the drug was incubated at 40°C for two months, no substantial destabilization effect was observed in antibody charge variants containing preservatives. [Figure 6C] This study demonstrates the effect of preservatives on antibody charge variant formation under stress conditions. When the drug was incubated at 40°C for two months, no substantial destabilization effect was observed in antibody charge variants containing preservatives.

[0019] [Figure 7A] This study demonstrates the effect of preservatives on antibody charge variant formation at 25°C. When pharmaceuticals were stored at 25°C for 6 months with the test preservative, no substantial instability was observed in antibody charge variants with the preservative. [Figure 7B] This study demonstrates the effect of preservatives on antibody charge variant formation at 25°C. When pharmaceuticals were stored at 25°C for 6 months with the test preservative, no substantial instability was observed in antibody charge variants with the preservative. [Figure 7C] This study demonstrates the effect of preservatives on antibody charge variant formation at 25°C. When pharmaceuticals were stored at 25°C for 6 months with the test preservative, no substantial instability was observed in antibody charge variants with the preservative.

[0020] [Figure 8A] This figure shows the effect of preservatives on antibody charge variant formation at 5°C for 12 months. As can be seen in the figure, when the drug was stored at 5°C with the test preservative, no significant instability was observed in antibody charge variants with the preservative. [Figure 8B]This figure shows the effect of preservatives on antibody charge variant formation at 5°C for 24 months. As can be seen in the figure, when the drug was stored at 5°C with the test preservative, no significant instability was observed in the antibody charge variants with the preservative.

[0021] [Figure 9A] This shows the subvisible particle formation of all pharmaceutical formulations with and without preservatives after storage at 5°C for 24 months. The invisible line was zero. [Figure 9B] This shows the subvisible particle formation of all pharmaceutical formulations with and without preservatives after storage at 25°C for 6 months. The number of invisible lines was zero. [Figure 9C] This shows the subvisible particle formation of all pharmaceutical formulations with and without preservatives after storage at 40°C for 3 months. The invisible line was zero.

[0022] [Figure 10] This graph shows the bioburden count (CFU / mL) of formulation 1. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 0.3% phenol stored at 20-25°C over a period of 28 days.

[0023] [Figure 11] This graph shows the bioburden count (CFU / mL) of formulation 2. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 1% benzyl alcohol stored at 20-25°C over a period of 28 days.

[0024] [Figure 12] This graph shows the bioburden count (CFU / mL) of formulation 3. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 0.3% phenol stored at 20-25°C over a period of 28 days.

[0025] [Figure 13] This graph shows the bioburden count (CFU / mL) of formulation 4. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 1% benzyl alcohol at 20-25°C over a 28-day period.

[0026] [Figure 14] This graph shows the bioburden count (CFU / mL) of formulation 5. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 0.3% phenol at 20-25°C over a 28-day period.

[0027] [Figure 15] This graph shows the bioburden count (CFU / mL) of formulation 6. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 1% benzyl alcohol at 20-25°C over a 28-day period.

[0028] [Figure 16] This graph shows the bioburden count (CFU / mL) of formulation 7. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 0.3% phenol at 20-25°C over a 28-day period.

[0029] [Figure 17] This graph shows the bioburden count (CFU / mL) of formulation 8. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in formulations containing 1% benzyl alcohol at 20-25°C over a 28-day period. [Modes for carrying out the invention]

[0030] I. Definition This disclosure is not limited to the compositions and methods described herein, nor to the experimental conditions described herein, and should therefore be understood to be subject to change. Furthermore, since the scope of this disclosure is limited only by the appended claims, it should be understood that the terms used herein are for the purpose of describing only certain aspects and are not intended to be limiting.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure pertains. However, any composition, method, and substance that is similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference in their entirety.

[0032] The enumeration of value ranges herein is intended, unless otherwise indicated herein, simply as a way of referring individually to each individual value included in the range, and each individual value is incorporated herein as if it were individually enumerated herein. The aforementioned ranges are intended to be clarified by context and no further limitation is implied. All methods described herein may be carried out in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent by context. Any and all examples or exemplary words (e.g., "etc.") provided herein are intended merely to better illustrate the invention and do not limit the scope of the invention unless otherwise claimed herein. No word herein should be construed as indicating any unclaimed element as essential to the practice of the invention.

[0033] In the context of numbers and ranges, the term "approximately" refers to a value or range that approximates or is close to the enumerated values ​​or ranges, as is evident from the teachings contained herein, so that the invention can be carried out, for example, with the presence of a desired rate, quantity, density, degree, increase, decrease, percentage, value, or form, variant, temperature, or time quantity. Therefore, the term encompasses values ​​other than those resulting from mere systemic errors. For example, "approximately" can indicate a value either above or below a specified value within approximately + / - 10% or more of the stated value, depending on the ability to carry it out.

[0034] Antibodies (mAbs) (plural) are also called immunoglobulins and are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. Antibodies are often used as therapeutic biomolecules. A standard immunoglobulin protein (e.g., IgG) contains four polypeptide chains, including two heavy (H) chains and two light (L) chains linked together by cysteine ​​disulfide bonds. Each light chain is linked to one heavy chain by one cysteine ​​disulfide bond, and the two heavy chains are linked to each other via two cysteine ​​disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody is defined as having at least 10% affinity, as measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA. -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 This refers to the antibodies that have binding affinity to each of M's targets.

[0035] The term “antibody” includes references to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express antibodies. The term “antibody” also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to more than one different epitopes. Bispecific antibodies are generally described in U.S. Patent No. 8,586,713, incorporated by reference in this application. Immunoglobulins produced in mammalian systems can also be glycosylated at different residues (e.g., asparagine residues) by various polysaccharides, which can be species-specific and may 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).

[0036] A "protein" refers to a molecule containing two or more amino acid residues linked to each other by peptide bonds. Proteins include polypeptides and peptides, and may also involve modifications such as glycosylation, lipid binding, sulfated molecules, gamma-carboxylation, alkylation, hydroxylation, and ADP-ribosylation of glutamate residues. Proteins can be of scientific or commercial interest, including protein-based drugs, and include, among other things, enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins. Proteins are produced by various types of recombinant cells using well-known cell culture methods and are generally introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-free sequences, etc.), where they may exist as episomes or be incorporated into the cell's genome.

[0037] "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.

[0038] An "Fc fusion protein" is a protein containing part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, and these proteins are not naturally found together. The preparation of fusion proteins containing a specific heterologous polypeptide fused to various parts of an antibody-derived polypeptide (including the Fc domain) has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 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" comprises one or more extracellular domains of a receptor bound to the Fc portion, which in some embodiments include a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein comprises two or more different receptor chains bound to one or more ligands or other molecules, depending on the type of Fc fusion protein. For example, the Fc fusion protein is a trap, such as an IL-1 trap or a VEGF trap.

[0039] The "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 participate 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. Fc fusion proteins include, for example, Fc fusion (N-terminal) proteins, Fc fusion (C-terminal) proteins, mono-Fc fusion proteins, and bispecific Fc fusion proteins.

[0040] The term "Fc-containing protein" includes antibodies and Fc fusion proteins (such as trap proteins, bispecific antibodies, antibody derivatives containing Fc, antibody fragments containing Fc, Fc fusion proteins, immunoadhesins, and other binding proteins containing at least functional portions of the immunoglobulin CH2 and CH3 regions). Fc-containing proteins such as antibodies may contain modifications to the immunoglobulin domain, which include modifications that affect one or more effector functions of the binding protein (e.g., modifications that affect FcyR binding, FcRn binding, and therefore half-life, and / or CDC activity). Such modifications refer to the EU numbering of the immunoglobulin constant region, as follows: 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, This includes the modifiers 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, and their combinations, but these are linear.

[0041] For example, but not limited to, the binding protein is an Fc-containing protein (e.g., an antibody) that exhibits an extended serum half-life (compared to the same Fc-containing protein without the listed modifications) and has modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or position 434 (e.g., H / F or Y), or positions 250 and / or 428, or positions 307 or 308 (e.g., 308F, V308F) and position 434. In another example, the modifications could include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 2591 (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F or 308P).

[0042] An "acidic-charged variant" is a variant of an Fc-containing protein (e.g., an antibody) that has a lower pH than the dominant peak morphology of the Fc-containing protein. Acidic-charged variants tend to have a greater negative charge.

[0043] A "basic charge variant" is a variant of an Fc-containing protein (e.g., an antibody) that has a higher pH than the dominant peak morphology of the Fc-containing protein. Basic charge variants tend to have more positive charge or less negative charge.

[0044] The "dominant peak morphology" of Fc-containing proteins (e.g., antibodies) is the dominant morphology of the Fc-containing protein and has a pH between the acidic charge variant and the basic charge variant.

[0045] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody generally contains 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). If 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. Epitopes recognized by a bispecific antibody can be located on the same target or on different targets (e.g., on the same protein or on different proteins). A bispecific antibody 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 with 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, then (from N-terminus to C-terminus) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either 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 allows one or both heavy chains to bind to one or both epitopes.

[0046] 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, as well as combinations thereof. A typical heavy chain has, following the variable domain (from N-terminus to C-terminus), 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.

[0047] 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, those that do not selectively bind to either a first antigen or a second antigen selectively bound by an antigen-binding protein. Suitable 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), and the light chain does not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include those that can bind to one or both epitopes bound by the antigen-binding domain of the antigen-binding protein.

[0048] The term "variable domain" includes an amino acid sequence (as desired, modified) of an immunoglobulin light or heavy chain containing the following amino acid region in order from N-terminus to C-terminus: 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 and second beta sheets.

[0049] 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 a wild-type organism. 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 as a result of, for example, sequence splicing or joining (e.g., VDJ recombination that forms a heavy chain CDR3).

[0050] "Antibody derivatives and fragments" include, but are not limited to, antibody fragments (e.g., ScFv-Fc, dAB-Fc, semi-antibodies) and multispecific molecules (e.g., IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, triplicate molecules).

[0051] A "protein product" refers to the target protein, such as an Fc-containing protein (e.g., an antibody). Protein products can be produced by cells in culture, usually engineered mammalian cells. Typically, cells in culture, for example, in a bioreactor, produce the target protein, and these proteins become protein products. Protein products can then be subjected to other finishing steps such as purification, characterization, sterilization, formulation, and concentration or lyophilization, as well as packaging to form the final protein product. Protein products include formulation drugs (FDS).

[0052] The term "single-dose drug" refers to a container designed for use as a single injection and / or single intravenous infusion by a single patient.

[0053] The term "single-patient container" refers to parenteral preparations intended for multiple uses in a single patient. For "multi-dose containers" (as defined below) and single-patient containers, the results of antimicrobial efficacy testing are used to support the labeled use-by date (BUD) or disposal statement.

[0054] The terms “multi-dose medicine” or “multi-dose container or vial” refer to a parenteral preparation medicine or container that meets antimicrobial efficacy testing requirements or is exempt from such testing requirements by FDA regulations. Multi-dose medicines are intended to contain more than one dose of medicine; for example, a multi-dose vial contains 5 to 6 doses of medicine. Multi-dose medicines for articles or preparations are intended for parenteral administration only and typically contain antimicrobial preservatives. Multi-dose containers are generally intended to contain 30 mL or less of product. The buy-by-use (BUD) of an opened or punctured (e.g., punctured with a needle) multi-dose container containing antimicrobial preservatives is 28 days unless otherwise specified by the manufacturer. See, for example, Chapter 51 of the United States Pharmacopeia (USP), titled “Antibacterial Efficacy Testing.”

[0055] The term "kinetic degradation profiling" refers to the analysis of protein degradation over time (for example, the storage stability of Fc-containing proteins over 24 months at 5°C, 6 months at 25°C, 3 months at 40°C, and 3 months at 45°C).

[0056] The terms "Compound sterile preparation (CSP)" or "Ready-to-use compound sterile preparation" refer to a sterile medicine that does not contain preservatives and is therefore intended for immediate use after opening.

[0057] Cation exchange chromatography (CEX) is a form of ion exchange chromatography (IEX) used to separate molecules based on their net surface charge. More specifically, Cation exchange chromatography uses a negatively charged ion exchange resin that has an affinity for molecules with a net positive surface charge.

[0058] The term "mean or median fluorescence intensity (MFI)" is commonly used to define and describe the average intensity and level of antibody expression. Fluorescence intensity indicates how much light (photons) is emitted, and it depends on the concentration of the excited phosphor.

[0059] The term "microflow imaging (MFI)" refers to a process that uses flow microscopy to measure the size distribution, concentration, and morphology of microspheres, protein particles, silicone droplets, and other subvisible / visible particles.

[0060] The term "optical density (OD)" refers to the turbidity of a sample measured using a Molecular Devices SPECTRAmax 190 microplate spectrophotometer (for example, the optical density at 405 nm).

[0061] The term "relative humidity (RH)" is expressed as a percentage and indicates the current state of absolute humidity relative to the maximum humidity at the same temperature.

[0062] The terms "official assay" or "official test" refer to tests performed to determine whether a material meets specifications and / or to address anticipated regulatory concerns. These series of tests may also be applied to determine general drug compatibility or for routine quality control.

[0063] The terms "size exclusion ultrahigh performance liquid chromatography (SE-UPLC)" or "size exclusion high performance liquid chromatography (SE-HPLC)" refer to a high-throughput analytical method for determining and quantifying the levels of aggregates and fragments in purified antibodies through isocratic conditions. In other words, this method separates molecules based on their size and high molecular weight (HMW) by filtration through a gel containing pores of a specific size distribution. Separation occurs when molecules of different sizes are either contained within or excluded from the pores in the gel matrix.

[0064] The term "reverse-phase high-performance liquid chromatography (RP-HPLC)" includes the separation of molecules based on hydrophobicity.

[0065] All numerical limits and ranges described herein include all numbers or values ​​around or between the numbers of the range or limit. The ranges and limits described herein expressly represent and indicate all integers, decimals, and fractional values ​​defined and encompassed by such range or limit.

[0066] II. Methods for stabilizing Fc-containing proteins Multi-dose antibody drugs are often supplied in lyophilized form to extend their shelf life. Some antibody drugs are supplied in solution, which requires preservatives to prevent or inhibit microbial growth. However, preservatives can destabilize the antibodies in the drug. Therefore, methods are provided to stabilize antibody drugs that require preservatives.

[0067] The present invention provides an approach for developing formulations that stabilize Fc-containing proteins prepared in aqueous solution and contain preservatives for stabilizing Fc-containing proteins in multi-dose antibody drugs. The storage stability of the Fc-containing proteins was evaluated at various temperatures, including 5°C, 25°C, 40°C, and 45°C, to enable kinetic degradation profiling, where applicable, as will be described in more detail later. One embodiment provides a multi-dose container comprising at least one Fc-containing protein and phenol or benzyl alcohol.

[0068] Once opened, a container becomes contaminated with bacteria and other microorganisms present in the air. Without preservatives, these bacteria and other microorganisms will grow rapidly within the container, necessitating its disposal. However, an ISO Class 5 or higher cleanroom contains far fewer bacteria than a typical room. Strictly speaking, for example, opened or needle-punctured single-dose containers such as bags, bottles, syringes, and vials of sterile products and compound sterile preparations (CSPs) must be used within one hour if the air quality in the room where the container was opened is lower than ISO Class 5. However, if the air quality in the room where the container was opened is equivalent to or better than ISO Class 5, the container must be discarded within six hours of opening. See Table 1. In comparison, single-dose containers (or vials) exposed to ISO Class 5 or cleaner air may be used for up to six hours after the initial needle puncture. [Table 1]

[0069] The ISO classification of particulate matter in indoor air is revised from the former Federal Standard No. 209E, General Services Administration, Washington, DC, 20407 (September 11, 1992), and ISO 14644-1:1999, Cleanrooms and related controlled environments - Part 1: Classification of air cleanliness. For example, 1 m 33,520 particles (ISO Class 5) of 0.5 μm or larger per 1 ft 3 This corresponds to 100 particles per unit (Class 100) (1m 3 = 35.2 ft 3 As shown in the diagram, the limit is 0.5 μm or larger particles per cubic meter [current ISO] and cubic foot [former Federal Standard No. 209E, FS 209E].

[0070] Multi-dose containers are designed to allow for multiple doses, as they typically contain antimicrobial preservatives. Unless otherwise specified by the manufacturer, the BUD (Body Under Use) of a multi-dose container is 28 days after the initial puncture or opening (e.g., puncture with a needle). See, for example, Chapter 51 of the United States Pharmacopeia (USP), titled "Antimicrobial Effectiveness Testing."

[0071] Another embodiment provides a parenteral Fc-containing protein preparation in an aqueous solution. This method comprises at least one Fc-containing protein and phenol or benzyl alcohol. In some embodiments, the Fc-containing protein can be provided in an aqueous solution containing phenol or benzyl alcohol, and the Fc-containing protein can be stabilized by filling a container with the aqueous solution containing phenol or benzyl alcohol.

[0072] Further details of the methods and systems to be disclosed are provided below.

[0073] A. Multiple-dose container for parenteral Fc-containing protein preparations In one embodiment, a multi-dose container for a parenteral Fc-containing protein preparation is provided. For example, the multi-dose container may contain a parenteral Fc-containing protein preparation. The preparation may contain at least one Fc-containing protein in aqueous solution. The preparation may also contain phenol or benzyl alcohol in aqueous solution.

[0074] One or more of the following exemplary features may be included. The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to about 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%). The container may have a capacity of 1 mL to 100 mL. The container may also have a capacity of 5 mL to 100 mL. The container may further have a capacity of 10 mL to 50 mL. The container may have a capacity of 20 mL to 40 mL. The container may also have a capacity of 30 mL. The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The container may contain two or more Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. The Fc-containing protein may also be a trap protein.

[0075] B. Method for preparing parenteral Fc-containing protein The disclosed systems and methods can be used to prepare parenteral Fc-containing proteins in aqueous solutions. One embodiment provides a parenteral Fc-containing protein preparation in aqueous solution, which is at least one Fc-containing protein. The parenteral Fc-containing protein preparation may also contain phenol and / or soluble phenol derivatives in aqueous solution. Benzyl alcohol and / or its soluble derivatives can also be used.

[0076] The preparation may include one or more of the following exemplary features: The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to about 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%). The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The preparation may contain two or more Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. The Fc-containing protein may also be a trap protein.

[0077] C. Method for stabilizing Fc-containing protein preparations in aqueous solution In one embodiment, a method is provided for stabilizing an Fc-containing protein preparation in an aqueous solution. For example, stabilizing an Fc-containing protein preparation may include the step of providing the Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol. The method for stabilizing an Fc-containing protein preparation may also include the step of filling a container with the Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol.

[0078] One or more of the following exemplary features may be included. Phenol or benzyl alcohol may be added to an aqueous solution containing an Fc-containing protein. The Fc-containing protein may be at a concentration of about 0.1 mg / mL to about 500 mg / mL. The phenol may be at a concentration of about 1 mg / mL to about 10 mg / mL. The phenol may also be at a concentration of about 2 mg / mL to about 5 mg / mL. The phenol may further be at a concentration of, for example, about 3 mg / mL (about 0.3%). The benzyl alcohol may be at a concentration of about 1 mg / mL to about 15 mg / mL. The benzyl alcohol may further be at a concentration of about 3 mg / mL to about 12 mg / mL. The benzyl alcohol may further be at a concentration of, for example, about 10 mg / mL (about 1%). The Fc-containing protein may be a monoclonal antibody. The monoclonal antibody may be a bispecific antibody. The preparation may contain two or more Fc-containing proteins. The Fc-containing protein may be a receptor Fc fusion protein. The Fc-containing protein may also be a trap protein.

[0079] D. Proteins in protein complexes In one embodiment, one of the proteins in the protein complex is either a protein pharmaceutical or a target protein suitable for expression in prokaryotic or eukaryotic cells. For example, the protein in the protein complex may be an antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, an ScFv or its fragment, an Fc fusion protein or its fragment, a growth factor or its fragment, a cytokine or its fragment, or the extracellular domain or fragment of a cell surface receptor. The protein in the complex may be a simple polypeptide consisting of a single subunit, or a complex multi-subunit protein containing two or more subunits. The target protein may be a biopharmaceutical, a food additive or preservative, or any protein product subject to purification and quality standards.

[0080] In some embodiments, the protein in the protein complex is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific antibody, bispecific antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody, or tetrabody, a bispecific tetravalent immunoglobulin G-like molecule called bivariable domain immunoglobulin (DVD-IG), IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody contains a chimeric hinge. In yet another embodiment, the antibody contains a chimeric Fc. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0081] In some embodiments, the antibodies include anti-programmed cell death 1 antibody (e.g., anti-PD1 antibody as described in U.S. Patent Application Publication US2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., anti-PD-L1 antibody as described in U.S. Patent Application Publication US2015 / 0203580A1), anti-Dll4 antibody, anti-angiopoietin-2 antibody (e.g., anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), anti-angiopoietin-like 3 antibody (e.g., anti-AngPtl3 antibody as described in U.S. Patent No. 9,018,356), and anti-platelet-derived growth factor. Receptor antibodies (e.g., anti-PDGFR antibody described in U.S. Patent No. 9,265,827), anti-Erb3 antibody, anti-prolactin receptor antibody (e.g., anti-PRLR antibody described in U.S. Patent No. 9,302,015), anti-complement 5 antibody (e.g., anti-C5 antibody described in U.S. Patent Application Publication US2015 / 0313194A1), anti-TNF antibody, anti-epidermal growth factor receptor antibody (e.g., anti-EGFR antibody described in U.S. Patent No. 9,132,192, or anti-EGFRvIII antibody described in U.S. Patent Application Publication US2015 / 0259423A1), anti-prota Protein-converting enzyme subtilisin kexin-9 antibody (e.g., anti-PCSK9 antibody described in U.S. Patent No. 8,062,640 or U.S. Patent No. 9,540,449), anti-growth and differentiation factor-8 antibody (e.g., anti-GDF8 antibody, also known as anti-myostatin antibody, described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibody described in U.S. Patent Application Publication US2015 / 0337045A1 or U.S.2016 / 0075778A1), anti-VEGF antibody, anti-IL1R antibody , interleukin-4 receptor antibodies (e.g., anti-IL4R antibodies described in U.S. Patent Publication US2014 / 0271681A1 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (e.g., anti-IL6R antibodies described in U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,880), 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., U.S. Patent No. 9,453,Anti-IL33 antibody (as described in U.S. Patent No. 072 or No. 9,637,535), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Application Publication No. 9,447,173), anti-differentiation cluster 3 (e.g., anti-CD3 antibody as described in U.S. Patent Nos. 9,447,173 and 9,447,173, and U.S. Patent Application No. 62 / 222,605), anti-differentiation cluster 20 (e.g., anti-CD20 antibody as described in U.S. Patent Nos. 9,657,102 and US20150266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibody, anti-CD28 antibody, anti-differentiation cluster 48 (e.g., anti-CD48 antibody 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 US2015 / 0337029A1), anti-Ebola virus antibody (e.g., as described in U.S. Patent Publication US2016 / 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 US2016 / 0017029, and U.S. Patents No. 8,309,088 and 9,353,The anti-NGF antibody (as described in Patent No. 176) and the anti-protein Y antibody are selected from the group. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibody (as described in U.S. Patent Application Publications US2014 / 0088295A1 and US20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibody (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibody (e.g., anti-CD3 × anti-PSMA bispecific antibody). In some embodiments, the target protein is absiximab, adalimumab, adalimumab-at, ad-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoximab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, semiprimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, facinumab, golimumab, guselkumab The following group is selected: ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesbakumab, nivolumab, obilutoxaximab, obinutuzumab, ocrelizumab, ofatumumab, oraratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, laxibakumab, reslizumab, linukumab, rituximab, sarilumab, secukinumab, silutuximab, tocilizumab, tocilizumab, trastuzumab, trevoglumab, ustekinumab, and vedolizumab. ,

[0082] In some embodiments, the protein in the complex is a recombinant protein (e.g., an Fc fusion protein) containing an Fc moiety and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor bound to the Fc moiety. In some embodiments, the Fc moiety includes a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains bound to a single ligand or multiple ligands. For example, an Fc fusion protein is a trap protein such as an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding domain fused to an Il-1R1 extracellular domain fused to the Fc of hIgG1, see U.S. Patent No. 6,927,044 (the entire patent is incorporated herein by reference)) or a VEGF trap (e.g., aflibercept or ziv-aflibercept containing an Ig domain 2 of VEGF receptor Flt1 fused to an Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1, see U.S. Patents No. 7,087,411 and 7,279,159). In other embodiments, an Fc fusion protein is an ScFv-Fc fusion protein containing one or more antigen-binding domains, such as variable heavy chain fragments and variable light chain fragments of an antibody conjugated to the Fc portion. [Examples]

[0083] The present invention will be further illustrated by the following embodiments illustrating many aspects of the invention, but will not be limited in any way.

[0084] The effect of typical antimicrobial preservatives on the stability of several mAb liquid formulations was evaluated by adding preservatives to five formulated active pharmaceutical ingredients (FDS) formulations, and the stability of the resulting formulations was evaluated over time at different temperatures. The compatibility of chlorobutanol, m-cresol, phenol, and benzyl alcohol was evaluated using several mAb formulations. Since chlorobutanol and m-cresol were found to be incompatible with the evaluated formulations, only phenol and benzyl alcohol were further tested. The long-term stability of formulations with added preservatives was tested under various conditions, including different temperatures and relative humidity (RH) (e.g., 5°C, 25°C / 60%RH, 40°C / 75%RH, and 45°C). Key quality attributes of the tested substances included solution clarity by UV absorbance at 405 nm, subvisible particle formation by MFI, aggregate formation by SE-UPLC, protein concentration by RP-UPLC, and protein charge variants by CEX. Official assays were used to evaluate the antimicrobial efficacy of the preservatives. See, for example, Chapter 51 of the United States Pharmacopeia (USP). The stability of all mAb formulations in the presence of preservatives was evaluated for up to 24 months at 5°C and 25°C, as determined by the methods listed below. However, under stress conditions (e.g., 40°C and 45°C), the preservatives increased mAb aggregation in some formulations, as detected by SE-UPLC (e.g., Figures 4A–4D). The effect of preservatives on mAb aggregation depended on the properties of the mAb, the preservative, and the mAb concentration. The preservatives did not affect the protein charge or the formation of subvisible particles, even under stress conditions. The effectiveness of preservatives in reducing microbial activity in these formulations was also evaluated.

[0085] Example 1: Effect of preservatives on the turbidity of mAb formulations under identical stress conditions. method Turbidity of formulations under identical stress conditions To determine the turbidity of different mAb formulations, various antibodies were incubated at 45°C for 3 months with or without the test preservative. In the first stage, the mAb formulations included 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, all stored at 45°C. See Figure 1A. In the second stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, all stored at 45°C. See Figure 1B. In the third stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, all stored at 45°C. See Figure 1C. In the fourth stage, the mAb formulations included 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, all stored at 45°C. See Figure 1D.

[0086] Analysis of turbidity of formulations under identical stress conditions At the end of the 3-month incubation period, each of the four different mAb formulations containing the preservative benzyl alcohol exhibited relatively higher turbidity than the mAb formulations combined with the preservative phenol or the mAb formulations without any preservatives, when incubated at 45°C. Overall, the mAb formulation containing mAb C with 2 mg / mL benzyl alcohol showed the smallest increase in turbidity, while the mAb formulation containing mAb D with 200 mg / mL benzyl alcohol showed the largest increase in turbidity.

[0087] Example 2: Effect of preservatives on the turbidity of mAb formulations under different stress conditions. method Turbidity of formulations under different stress conditions To determine the turbidity of different mAb formulations, various antibodies were incubated at 25°C for 6 months with and without preservatives, and at 5°C for 24 months with and without the same preservatives. In the first stage, where antibodies were incubated at 25°C for 6 months, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol. See Figure 2A. In the second stage, where antibodies were incubated at 5°C for 24 months, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, respectively; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively. See Figure 2B.

[0088] Analysis of turbidity of formulations under different stress conditions No significant instability was observed when the formulations were incubated with the test preservatives at 25°C for 6 months or at 5°C for 24 months.

[0089] Example 3: Effect of preservatives on high molecular weight (HMW) complex formation when stored at 40°C for 3 months. method HMW complex formation when stored at 40°C To determine the effect of preservatives on high molecular weight (HMW) complex formation for different mAb formulations, various antibodies were incubated at 40°C for 3 months with or without the test preservative. In the first stage, the mAb formulations included 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, stored at 40°C. See Figure 3A. In the second stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, stored at 40°C. See Figure 3B. In the third stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, all stored at 40°C. See Figure 3C. In the fourth stage, the mAb formulations included 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, all stored at 40°C. See Figure 3D.

[0090] Analysis of HMW complex formation when stored at 40°C At the end of the 3-month incubation period, each of the four different mAb formulations containing the preservative benzyl alcohol exhibited a relatively higher destabilization effect than mAb formulations combined with the preservative phenol or mAb formulations without any preservatives, when incubated at 40°C. Overall, the mAb formulation containing 120 mg / mL of mAb A+B showed the least destabilization effect, if any, while the mAb formulation containing mAb D with 200 mg / mL of benzyl alcohol showed the strongest destabilization effect.

[0091] Example 4: Effect of preservatives on high molecular weight (HMW) complex formation when stored at 25°C for 6 months. method HMW complex formation when stored at 25°C To determine the effect of preservatives on high molecular weight (HMW) complex formation for different mAb formulations, various antibodies were incubated at 25°C for 6 months with or without the test preservative. In the first stage, the mAb formulations included 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, stored at 25°C. See Figure 4A. In the second stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, stored at 25°C. See Figure 4B. In the third stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of benzyl alcohol mAb D, all stored at 25°C. See Figure 4C. In the fourth stage, the mAb formulations included 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, all stored at 25°C. See Figure 4D.

[0092] Analysis of HMW complex formation when stored at 25°C At the end of the 6-month incubation period, no significant destabilizing effect of the added preservatives on the percentage of high molecular weight (HMW) complex formation was observed when incubated at 25°C.

[0093] Example 5: Effect of preservatives on the stability of antibodies stored at 5°C for 24 months. method Antibody stability when stored at 5°C with preservatives. To determine the stability of different mAb formulations, various antibodies were incubated at 5°C for 24 months with and without the preservatives under test. The mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, respectively; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively. See Figure 5.

[0094] Analysis of antibody stability when stored at 5°C with preservatives. At the end of the 24-month period, no significant instability was observed when the mAb formulation (pharmaceutical) was incubated with the test preservative at 5°C.

[0095] Example 6: Effect on charge variant formation in antibodies containing preservatives incubated at 40°C. method Charge variant formation of antibodies containing preservatives when stored at 40°C To determine the effect of different mAb formulations on charge variant formation, various antibodies were incubated at 40°C for a period of 2 months with and without the preservatives under test. In the first stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively, stored at 40°C. See Figure 6A. In the second stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively, stored at 40°C. See Figure 6B. In the third stage, the mAb formulations included 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively, stored at 40°C. See Figure 6C.

[0096] Analysis of charge variant formation of antibodies containing preservatives when stored at 40°C At the end of the two-month incubation period, no substantial destabilizing effect of preservatives on antibody charge variants was observed when the mAb formulation was incubated at 40°C.

[0097] Example 7: Effect on charge variant formation in antibodies containing preservatives incubated at 25°C. method Charge variant formation of antibodies containing preservatives when stored at 25°C. To determine the effect of different mAb formulations on charge variant formation, various antibodies were incubated with the test preservatives at 25°C for a period of 6 months. In the first stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, all stored at 25°C. See Figure 7A. In the second stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, all stored at 25°C. See Figure 7B. In the third stage, the mAb formulations included 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, all stored at 25°C. See Figure 7C.

[0098] Analysis of charge variant formation of antibodies containing preservatives when stored at 25°C. At the end of the 6-month period, no substantial instability was observed in the antibody charge variants when the mAb formulations were incubated with the test preservative at 25°C.

[0099] Example 8: Effect of preservatives on charge variant formation in antibodies incubated at 5°C. method The effect of different antibody formulations containing preservatives on charge variant formation when stored at 5°C for 12 and 24 months. Various antibodies were incubated at 5°C for 12 months with and without the preservatives under test. In the first stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of phenol-containing mAb D, and 200 mg / mL of benzyl alcohol-containing mAb D, respectively, stored at 5°C. See Figure 8A.

[0100] Furthermore, various antibodies were incubated at 5°C for 24 months with and without the preservatives under test. In the first stage, the mAb formulations included 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, all stored at 5°C. In the second stage, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, all stored at 5°C, as well as 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, all stored at 5°C. See Figure 8B.

[0101] Analysis of charge variant formation of antibodies containing preservatives when stored at 25°C. At the end of both 12-month and 24-month incubation periods, no significant instability was observed in antibody charge variants when the mAb formulations were incubated with the test preservatives at 5°C.

[0102] Example 9: Effect on subvisible particle formation of mAb formulations with and without preservatives incubated at 5°C for 24 months, 25°C for 6 months, and 40°C for 3 months. method Subvisible particle formation in mAb formulations when incubated at 5°C, 25°C, and 40°C. To determine the effect of different mAb formulations on charge variant formation, various antibodies were incubated with or without the preservative under test at 5°C for 24 months, at 25°C for 6 months, and at 40°C for 3 months. In the first stage, in which antibodies were incubated at 5°C for 24 months, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, respectively; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively. See Figure 9A.

[0103] In the second stage, where antibodies were incubated at 25°C for 6 months, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, respectively; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively. See Figure 9B.

[0104] In the third stage, where antibodies were incubated at 40°C for 3 months, the mAb formulations included 2 mg / mL of preservative-free mAb C, 2 mg / mL of mAb C with phenol, and 2 mg / mL of mAb C with benzyl alcohol, respectively; 100 mg / mL of preservative-free mAb C, 100 mg / mL of mAb C with phenol, and 100 mg / mL of mAb C with benzyl alcohol, respectively; 120 mg / mL of preservative-free mAb A+B, 120 mg / mL of mAb A+B with phenol, and 120 mg / mL of mAb A+B with benzyl alcohol, respectively; and 200 mg / mL of preservative-free mAb D, 200 mg / mL of mAb D with phenol, and 200 mg / mL of mAb D with benzyl alcohol, respectively. See Figure 9C.

[0105] Analysis of subvisible particle formation in mAb formulations when incubated at 5°C, 25°C, and 40°C. When incubated at 5°C, 25°C, and 40°C, no subvisible particle formation was observed for the mAb formulations with or without the preservatives under test.

[0106] Overall, incubation at 5°C for 24 months with or without the preservatives tested showed no significant destabilizing effect on mAb formulations. Phenol demonstrated the least destabilizing effect and was therefore presented as a more promising preservative for use in stabilizing mAb formulations. Of the four common preservatives tested, cresol and chlorobutanol caused turbidity and were therefore unsuitable for common mAb formulations. Benzyl alcohol under stress conditions, where the degradation pathway was demonstrated, showed higher mAb aggregation, as evidenced by the HMW species, turbidity, and subvisible particle count detected by SE-UPLC. Phenol and benzyl alcohol showed only minimal destabilizing effects on mAbs.

[0107] Preservatives can be concluded to potentially destabilize antibodies, especially for high-concentration protein formulations under high temperatures, especially in terms of the turbidity of the formulation and the percentage of high-molecular-weight formation. Both phenol and benzyl alcohol can be used as potential preservatives in antibody formulations (such as mAb liquid formulations). The preservative effect under stress conditions depends on the properties of the preservative, the properties of the mAb, and the mAb concentration. The preservative did not affect the charge of the protein or the formation of subvisible particles even under stress conditions. Thus, prior to preservative selection, it is necessary to evaluate the stability of the mAb under stress conditions for each individual formulation.

[0108] Example 10: Testing of antibacterial antibody formulations containing phenol or benzyl alcohol. Method Antibacterial antibody formulations containing phenol or benzyl alcohol were tested against Escherichia coli or Staphylococcus aureus. Refer to Table 2 below. [Table 2]

[0109] Verification of method qualification For each formulation containing a preservative (phenol or benzyl alcohol), 2 mL was taken from the stock and 1 mL was transferred to two separate tubes containing 10 mL of tryptic soy broth (TSB). Next, one of the two types of bacteria was inoculated into the tubes at 1×10 6 ~1×10 7 CFU / mL. After vortexing each tube for 10 seconds to ensure homogeneity, 1 mL was taken out of the tube and plated onto two empty Petri dishes. The same procedure was repeated with phosphate-buffered saline (PBS) instead of the formulation. Next, melted and tempered tryptic soy agar (TSA) was poured onto the samples and gently stirred. The agar was allowed to solidify and the plates were placed in an incubator at 30 - 35 °C.

[0110] Plates were counted after 5 days of incubation, and the average was calculated for the final CFU count. When total CFUs were compared between PBS controls and formulation samples, the acceptable recovery rate was 50–200% of the control.

[0111] Vaccination and incubation Transfer 10 mL of each formulation of the drug into separate 10 mL Falcon tubes, and raise each challenge bacterium to the target final countable concentration of 1 × 10⁻¹⁶. 6 ~1 × 10 7 Inoculation was continued until the CFU / mL level was reached. After inoculation, 50 mL tubes were stored at 20-25°C for up to 28 days.

[0112] sampling To evaluate microbial growth, samples were taken at specified time points and tested for bioburden determination using serial dilutions required for sample analysis. For E. coli, at each time point, prior to sampling, 50 mL conical tubes were vortexed for at least 10 seconds. Using a 200 μl pipette, 100 μl was taken from each tube and diluted in 10 mL of PBS. After vortexing the diluent for an additional 10 seconds, 3 mL was taken from the tube. 1 mL was further diluted in an additional 9 mL of phosphate-buffered saline, and 1 mL was plated into two empty petri dishes. The second diluent was vortexed for an additional 10 seconds, then 2 mL was taken, and 1 mL was plated into two empty petri dishes.

[0113] For Staphylococcus aureus, samples of all formulations were diluted to 1:1,000 and 1:10,000 at the initial time point. At all other time points, samples were diluted in a manner similar to that described for Escherichia coli. Tempered TSA was poured over the samples, allowed to solidify on the benchtop, and then placed in the appropriate incubator based on the microorganism. Plates were counted after 5 days of incubation. Table 3 summarizes the details of sampling time points at the corresponding temperatures. [Table 3]

[0114] Data Analysis After 5 days of incubation, the plates were removed from the incubator, and the colonies were counted using an automated plate reader or, if necessary, manually. The number of colonies between the two plates was averaged with respect to the final number of colony-forming units.

[0115] The logarithmic change was calculated based on Equation 1. In the equation, T x T0 represents the microbial recovery rate of the sample at the specified time point, while T0 represents the microbial recovery rate of the sample at time 0. [Mathematics 1] Equation 1: Logarithmic change = Log(Sample T) x )-Log(T0)

[0116] Results and Discussion Formulations 1 through 8, which were tested for method qualification, achieved target recovery rates of 50% to 200% for both tested bacteria (Escherichia coli and Staphylococcus aureus), demonstrating no interference between the formulations in the growth and recovery of the two bacteria. Table 4 summarizes the results of method qualification for formulations 1 through 8. [Table 4]

[0117] Figure 10 illustrates the bioburden count (CFU / mL) of formulation 1. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 5 summarizes the data points collected as shown in Figure 10. [Table 5]

[0118] Figure 11 illustrates the bioburden count (CFU / mL) of formulation 2. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 6 summarizes the data points collected as shown in Figure 11. [Table 6]

[0119] Figure 12 illustrates the bioburden count (CFU / mL) of formulation 3. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 7 summarizes the data points collected as shown in Figure 12. [Table 7]

[0120] Figure 13 illustrates the bioburden count (CFU / mL) of formulation 4. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 8 summarizes the data points collected as shown in Figure 13. [Table 8]

[0121] Figure 14 illustrates the bioburden count (CFU / mL) of formulation 5. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 9 summarizes the data points collected as shown in Figure 14. [Table 9]

[0122] Figure 15 illustrates the bioburden count (CFU / mL) of formulation 6. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 10 summarizes the data points collected as shown in Figure 15. [Table 10]

[0123] Figure 16 illustrates the bioburden count (CFU / mL) of formulation 7. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 11 summarizes the data points collected as shown in Figure 16. [Table 11]

[0124] Figure 17 illustrates the bioburden count (CFU / mL) of formulation 8. The graph shows the logarithmic change in bacteria (Escherichia coli and Staphylococcus aureus) in the formulation over a 28-day period at 20-25°C. Table 12 summarizes the data points collected as shown in Figure 17. [Table 12]

[0125] Deviation Initial experiments with E. coli did not yield high CFU values ​​that indicated a 3-log decrease. This was thought to be partly due to the difficulty in reading the initial plates based on the amount of growth. The experiment was repeated to reach a higher initial CFU. Furthermore, 1 mL of the sample was divided into 10 0.1 mL aliquots, which were plate-scattered into 10 separate Petri dishes for plate counting purposes. Tempered agar was then poured over these samples, and the initial CFU was calculated using a total of 10 plates.

[0126] Positive control group: Four preservative-free formulations (i.e., positive control formulations 1-4) were used as controls and showed continuous growth for at least one time point after complete sterilization occurred in the same formulations containing any of the preservatives. Tables 13-17 summarize the collected data points for positive control formulations 1-4 that do not contain preservatives.

[0127] Positive control formulation 1 is summarized in Table 13.

[0128] Table 13 summarizes the collected data for positive control formulation 1, which was stored without preservatives at 20-25°C for a period of 28 days. [Table 13]

[0129] Table 14 summarizes the collected data for positive control formulation 2, which was stored without preservatives at 20-25°C for a period of 28 days. [Table 14]

[0130] Table 15 summarizes the collected data for positive control formulation 3, which was stored without preservatives at 20-25°C for a period of 28 days. [Table 15]

[0131] Table 16 summarizes the collected data for positive control formulation 4, which was stored without preservatives at 20-25°C for a period of 28 days. [Table 16]

[0132] conclusion For Staphylococcus aureus, a representative Gram-positive bacterium, all drug formulations achieved a reduction of more than 1 log by day 7 of the study, and the remaining studies achieved a reduction of more than 3 log.

[0133] For Escherichia coli, a representative Gram-negative bacterium, all drug formulations achieved a reduction of more than 1 log by day 7 of the study, and the remaining studies achieved a reduction of more than 3 log.

[0134] The data in Figures 10-17 demonstrate that for Staphylococcus aureus, a representative Gram-positive bacterium, all drug formulations achieved a reduction of more than 1 log by day 7 of the study, and a reduction of more than 3 log in the remaining studies. For Escherichia coli, a representative Gram-negative bacterium, all drug formulations achieved a reduction of more than 1 log by day 7 of the study, and a reduction of more than 3 log in the remaining studies.

[0135] Additional exam Tables 17–40 summarize data points collected from additional studies on the effects of preservatives on pharmaceutical formulations, including turbidity, HMW complex formation, charge variant formation, and subvisible particle formation under various stress conditions. The studies were conducted on eight target formulations (i.e., target formulations 1–8) containing preservatives (phenol or benzyl alcohol).

[0136] Targeted drug 1 is summarized in Tables 17 to 19.

[0137] Table 17 summarizes the collected data for Target Formulation 1, which was stored with 0.3% phenol at 5°C for 24 months. [Table 17]

[0138] Table 18 summarizes the collected data for Target Formulation 1, which was stored with 0.3% phenol at 25°C for 6 months. [Table 18]

[0139] Table 19 summarizes the collected data for Target Formulation 1, which was stored with 0.3% phenol at 40°C and 45°C for 3 months. [Table 19]

[0140] Targeted therapies 2 are summarized in Tables 20 to 22.

[0141] Table 20 summarizes the collected data for Target Formulation 2, which was stored with 1% benzyl alcohol at 5°C for 24 months. [Table 20]

[0142] Table 21 summarizes the collected data for Target Formulation 2, which was stored with 1% benzyl alcohol at 25°C for 6 months. [Table 21]

[0143] Table 22 summarizes the collected data for Target Formulation 2, which was stored with 1% benzyl alcohol at 40°C and 45°C for 3 months. [Table 22]

[0144] Targeted drug 3 is summarized in Tables 23 to 25.

[0145] Table 23 summarizes the collected data for Target Formulation 3, which was stored with 0.3% phenol at 5°C for 24 months. [Table 23]

[0146] Table 24 summarizes the collected data for Target Formulation 3, which was stored with 0.3% phenol at 25°C for 6 months. [Table 24]

[0147] Table 25 summarizes the collected data for Target Formulation 3, which was stored with 0.3% phenol at 40°C and 45°C for 3 months. [Table 25]

[0148] Targeted therapies 4 are summarized in Tables 26 to 28.

[0149] Table 26 summarizes the collected data for Target Formulation 4, which was stored with 1% benzyl alcohol at 5°C for 24 months. [Table 26]

[0150] Table 27 summarizes the collected data for Target Formulation 4, which was stored with 1% benzyl alcohol at 25°C for 6 months. [Table 27]

[0151] Table 28 summarizes the collected data for Target Formulation 4, which was stored with 1% benzyl alcohol at 40°C and 45°C for 3 months. [Table 28] TIFF2026514305000030.tif72170

[0152] Targeted therapies 5 are summarized in Tables 29 to 31.

[0153] Table 29 summarizes the collected data for Targeted Formulation 5, which was stored with 0.3% phenol at 5°C for 24 months. [Table 29]

[0154] Table 30 summarizes the collected data for Target Formulation 5, which was stored with 0.3% phenol at 25°C for 6 months. [Table 30]

[0155] Table 31 summarizes the collected data for targeted formulation 5 stored with 0.3% phenol at 40°C and 45°C for 3 months. [Table 31]

[0156] The six targeted therapies are summarized in Tables 32 to 34.

[0157] Table 32 summarizes the collected data for targeted formulation 6 stored with 1% benzyl alcohol at 5°C for 24 months. [Table 32]

[0158] Table 33 summarizes the collected data for targeted formulation 6 stored with 1% benzyl alcohol at 25°C for 6 months. [Table 33]

[0159] Table 34 summarizes the collected data for targeted formulation 6 stored with 1% benzyl alcohol at 40°C and 45°C for 3 months. [Table 34]

[0160] Targeted therapies 7 are summarized in Tables 35 to 37.

[0161] Table 35 summarizes the collected data for targeted formulation 7 stored with 0.3% phenol at 5°C for 24 months. [Table 35]

[0162] Table 36 summarizes the collected data for targeted formulation 7 stored with 0.3% phenol at 25°C for 6 months. [Table 36]

[0163] Table 37 summarizes the collected data for targeted formulation 7 stored with 0.3% phenol at 40°C and 45°C for 3 months. [Table 37]

[0164] The eight targeted therapies are summarized in Tables 38 to 40.

[0165] Table 38 summarizes the collected data for targeted formulation 8 stored with 1% benzyl alcohol at 5°C for 24 months. [Table 38]

[0166] Table 39 summarizes the collected data for targeted formulation 8 stored with 1% benzyl alcohol at 25°C for 6 months. [Table 39]

[0167] Table 40 summarizes the collected data for targeted formulation 8 stored with 1% benzyl alcohol at 40°C and 45°C for 3 months. [Table 40]

[0168] Control group: Additional tests of four preservative-free formulations (i.e., control formulations 1-4) were used as controls and showed continuous growth for at least one time point after complete sterilization occurred in the same formulations containing any of the preservatives. Tables 41-52 summarize the collected data points for control formulations 1-4 that do not contain preservatives.

[0169] Control formulation 1 is summarized in Tables 41 to 43.

[0170] Table 41 summarizes the collected data for control formulation 1, which was stored at 5°C for 24 months without preservatives. [Table 41]

[0171] Table 42 summarizes the collected data for control formulation 1, which was stored at 25°C for 6 months without preservatives. [Table 42]

[0172] Table 43 summarizes the collected data for control formulation 1, which was stored for 3 months at 40°C and 45°C without preservatives. [Table 43]

[0173] Control formulation 2 is summarized in Tables 44 to 46.

[0174] Table 44 summarizes the collected data of Control Preparation 2 stored at 5°C for 24 months without preservatives.

Table 44

[0175] Table 45 summarizes the collected data of Control Preparation 2 stored at 25°C for 6 months without preservatives.

Table 45

[0182] Table 50 summarizes the collected data of Control formulation 4 stored at 5°C for 24 months without preservatives.

Table 50

[0183] Table 51 summarizes the collected data of Control formulation 4 stored at 25°C for 6 months without preservatives.

Table 51

[0184] Table 52 summarizes the collected data of Control formulation 4 stored at 40°C and 45°C for 3 months without preservatives.

Table 52

[0185] Conclusion of additional tests The effect of preservatives on the stability of proteins was evaluated by comparing sample formulations with preservatives and control formulations without preservatives.

[0186] Under stress conditions (40°C and 45°C), compared to the control formulation, the preservatives increased the aggregation of mAb detected by SE-UPLC in some sample formulations. The preservatives did not affect the charge of the protein or the formation of subvisible particles even under stress conditions. However, the stability of the mAb formulations in the presence of preservatives was equivalent to the control at 5°C for at least 24 months and at 25°C for at least 6 months. Overall, the formulations with preservatives were stable under the proposed storage conditions at 5°C for at least 24 months and at 25°C for at least 6 months.

[0187] In the above specification, the present invention is described in relation to certain aspects thereof, and many details are given for illustrative purposes. However, it will be apparent to those skilled in the art that the present invention is susceptible to further embodiments, and that certain details described herein can be significantly modified without departing from the basic principles of the present invention.

Claims

1. A container for multiple doses containing a parenteral Fc-containing protein preparation, wherein the preparation is in an aqueous solution. (a) at least one Fc-containing protein, and (b) A multi-dose container containing phenol or benzyl alcohol.

2. The container for multiple doses according to claim 1, wherein the Fc-containing protein is concentrated at a concentration of 0.1 mg / mL to 500 mg / mL.

3. The container for multiple doses according to claim 1, wherein the preparation contains phenol at a concentration of 1 mg / mL to 10 mg / mL.

4. The container for multiple doses according to claim 3, wherein the preparation contains phenol at a concentration of 2 mg / mL to 5 mg / mL.

5. The container for multiple doses according to claim 4, wherein the preparation contains phenol at a concentration of 3 mg / mL.

6. The container for multiple doses according to claim 1, wherein the preparation contains benzyl alcohol at a concentration of 1 mg / mL to 15 mg / mL.

7. The container for multiple doses according to claim 3, wherein the preparation contains benzyl alcohol at a concentration of 3 mg / mL to 12 mg / mL.

8. The container for multiple doses according to claim 4, wherein the preparation contains benzyl alcohol at a concentration of 10 mg / mL.

9. The container for multiple doses according to claim 1, wherein the container is a container for a single patient.

10. The container for multiple doses according to claim 1, wherein the container has a capacity of 1 mL to 100 mL.

11. The container for multiple doses according to claim 1, wherein the container has a capacity of 5 mL to 100 mL.

12. The container for multiple doses according to claim 1, wherein the container has a capacity of 10 mL to 50 mL.

13. The container for multiple doses according to claim 1, wherein the container has a capacity of 20 mL to 40 mL.

14. The container for multiple doses according to claim 1, wherein the container has a capacity of 30 mL.

15. The container for multiple doses according to claim 1, wherein the Fc-containing protein is a monoclonal antibody.

16. The container for multiple doses according to claim 12, wherein the monoclonal antibody is a bispecific antibody.

17. The container for multiple doses according to claim 1, wherein the container contains two or more types of Fc-containing proteins.

18. The container for multiple doses according to claim 1, wherein the Fc-containing protein is a receptor Fc fusion protein.

19. The container for multiple doses according to claim 1, wherein the Fc-containing protein is a trap protein.

20. The container for multiple doses according to claim 1, wherein the preparation contains phenol.

21. The container for multiple doses according to claim 1, wherein the preparation contains benzyl alcohol.

22. In aqueous solution, (a) at least one Fc-containing protein, and (b) Parenteral Fc-containing protein preparation comprising phenol or benzyl alcohol.

23. The parenteral Fc-containing protein preparation according to claim 22, wherein the Fc-containing protein is concentrated at a concentration of 0.1 mg / mL to 500 mg / mL.

24. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains phenol at a concentration of 1 mg / mL to 10 mg / mL.

25. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains phenol at a concentration of 2 mg / mL to 5 mg / mL.

26. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains phenol at a concentration of 3 mg / mL.

27. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains benzyl alcohol at a concentration of 1 mg / mL to 15 mg / mL.

28. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains benzyl alcohol at a concentration of 3 mg / mL to 12 mg / mL.

29. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains benzyl alcohol at a concentration of 10 mg / mL.

30. The parenteral Fc-containing protein preparation according to claim 22, wherein the Fc-containing protein is a monoclonal antibody.

31. The parenteral Fc-containing protein preparation according to claim 30, wherein the monoclonal antibody is a bispecific antibody.

32. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation comprises two types of Fc-containing proteins.

33. The parenteral Fc-containing protein preparation according to claim 22, wherein the Fc-containing protein is a receptor Fc fusion protein.

34. The parenteral Fc-containing protein preparation according to claim 22, wherein the Fc-containing protein is a trap protein.

35. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation contains phenol.

36. The parenteral Fc-containing protein preparation according to claim 22, wherein the preparation comprises benzyl alcohol.

37. A method for stabilizing an Fc-containing protein preparation, A step of providing an Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol, and A method comprising the step of filling a container with the Fc-containing protein in an aqueous solution containing phenol or benzyl alcohol.

38. The method according to claim 37, wherein the phenol or benzyl alcohol is added to the aqueous solution containing the Fc-containing protein.

39. The method according to claim 37, wherein the concentration of the Fc-containing protein is 0.1 mg / mL to 500 mg / mL.

40. The method according to claim 37, wherein the aqueous solution contains phenol at a concentration of 1 mg / mL to 10 mg / mL.

41. The method according to claim 37, wherein the aqueous solution contains phenol at a concentration of 2 mg / mL to 5 mg / mL.

42. The method according to claim 37, wherein the aqueous solution contains phenol at a concentration of 3 mg / mL.

43. The method according to claim 37, wherein the aqueous solution contains benzyl alcohol at a concentration of 1 mg / mL to 15 mg / mL.

44. The method according to claim 37, wherein the aqueous solution contains benzyl alcohol at a concentration of 3 mg / mL to 12 mg / mL.

45. The method according to claim 37, wherein the aqueous solution contains benzyl alcohol at a concentration of 10 mg / mL.

46. The method according to claim 37, wherein the Fc-containing protein is a monoclonal antibody.

47. The method according to claim 46, wherein the monoclonal antibody is a bispecific antibody.

48. The method according to claim 37, wherein the aqueous solution contains two or more Fc-containing proteins.

49. The method according to claim 37, wherein the Fc-containing protein is a receptor Fc fusion protein.

50. The method according to claim 37, wherein the Fc-containing protein is a trap protein.

51. The method according to claim 37, wherein the aqueous solution contains phenol.

52. The method according to claim 37, wherein the aqueous solution contains benzyl alcohol.

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