Stabilized antibody composition and method for producing same

Abstract

To provide a stable liquid antibody composition in which occurrence or influence of deterioration of a medicine caused by oxidation is reduced, and to provide a method of producing the composition.SOLUTION: Provided is a stable liquid pharmaceutical product including a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas contains less than 5% oxygen by volume, the recombinant protein is stable for a period of at least 28 days when stored at 45°C, and being stable for at least 28 days refers to an increase in the percentage of high molecular weight species of 2% or less over the period.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to liquid antibody compositions and methods for producing such compositions by minimizing and / or controlling the level of oxidizing gases in the headspace of containers in which the compositions are filled and stored prior to administration. [Background technology]

[0002] background Antibody therapeutics, including monospecific and bispecific antibodies, continue to be developed for the treatment of various diseases and conditions, including cancer and autoimmune diseases. Antibody efficacy has increased with improvements in the production of antibody molecules directed to specific targets. Antibody molecules, whether stored at high concentrations for small doses or at lower concentrations for high-potency treatments, can be susceptible to oxidation when in liquid formulations filled and stored in pharmaceutical containers such as vials and administered via syringe. The importance of maintaining stable compositions to minimize loss of bioactive agents due to oxidation or other degradation processes has been emphasized by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). According to ICH standards (Q6A and Q6B), product degradation testing may be reduced or eliminated under regulatory approval when the drug substance does not degrade under the specific formulation and storage conditions proposed in the new drug application, as demonstrated through appropriate analytical methods.

[0003] The use of nitrogen or inert gases (e.g., argon) to replace "air" in the headspace of pharmaceutical containers has been discussed in the art. EP 1174148 (Patent Document 1) discusses the preparation of a Fab fragment composition at a concentration of 2 mg / ml, in which the gas headspace of each vial was purged with nitrogen via repeated cycles in a laboratory-scale freeze-drying chamber (i.e., not suitable for Good Manufacturing Practice (GMP) standards). EP 1174148 (Patent Document 1) does not mention the oxygen concentration of the headspace gas after the nitrogen purge, nor does it provide guidance for controlling the oxygen content in the headspace to a predetermined target level, but a significant percentage increase in the presence of high molecular weight (HMW) impurities was observed under accelerated stability storage conditions (e.g., 1 month at 40°C). In some instances, the increase in HMW species after one month at 40°C exceeded 300% (Table 1), while three months of storage at 40°C resulted in a greater than 14-fold increase in HMW species (Table 4). Similarly, US2016 / 0129028 (Patent Document 2) discusses the use of a nitrogen overlay process to maintain polysaccharide stability, and US2012 / 0183531 (Patent Document 3) discusses the reduction or replacement of oxygen in the headspace of protein pharmaceuticals using nitrogen or an inert gas to prevent or inhibit yellow color formation due to oxidation of histidine buffers. There is a need in the pharmaceutical industry for a reliable method to reduce the occurrence or effects of pharmaceutical degradation due to oxidation. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] EP1174148 [Patent Document 2] US2016 / 0129028 [Patent Document 3] US2012 / 0183531 Summary of the Invention

[0005] In a first aspect, the present invention provides a pharmaceutical product comprising a sealed container containing a recombinant protein (e.g., an antigen-binding protein or antibody) in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 5% oxygen by volume, and wherein the recombinant protein is stable for a period of at least 28 days when stored at 45° C. In this context, stability for at least 28 days refers to an increase in the percentage of high molecular weight species of no more than 2% over that period.

[0006] In some cases, the gas contains no more than 2% oxygen by volume, no more than 1% oxygen by volume, less than 1% oxygen by volume, or no more than 0.1% oxygen by volume.

[0007] In certain embodiments, the pharmaceutical liquid formulation contains the recombinant protein (e.g., antigen binding protein) at a concentration of between about 2 mg / ml and 200 mg / ml. In some embodiments, the pharmaceutical liquid formulation contains the recombinant protein (e.g., antigen binding protein) at a concentration of between 150 and 200 mg / ml. In some embodiments, the pharmaceutical liquid formulation contains the recombinant protein (e.g., antigen binding protein) at a concentration of less than 10 mg / ml, less than 5 mg / ml, or less than 2 mg / ml.

[0008] In some embodiments, the pharmaceutical product contains a recombinant protein that is stable for a period of at least 3 months when stored at 45° C., where stability for at least 3 months refers to an increase in the percentage of high molecular weight species of no more than 10% over that period. In some cases, the stability of the recombinant protein refers to an increase in the percentage of high molecular weight species of no more than 5% over that period, or an increase in the percentage of high molecular weight species of less than 1% over that period.

[0009] In another aspect, the present invention provides a pharmaceutical product comprising a sealed container containing a recombinant protein (e.g., an antigen-binding protein or antibody) in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 1% oxygen by volume, and wherein the recombinant protein is stable for a period of at least 31 months when stored at 5° C. In this context, stability for at least 31 months refers to a change in the percentage of the major charge variant of no more than 10% over that period.

[0010] In another aspect, the present invention provides a pharmaceutical product comprising a sealed container containing a recombinant protein (e.g., an antigen binding protein) in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 1% oxygen by volume. In some embodiments, the gas comprises 0.1% oxygen by volume or less. In some embodiments, the recombinant protein is present in the liquid formulation at a concentration of less than 5 mg / ml. In some embodiments, the recombinant protein is present in the liquid formulation at a concentration of about 2 mg / ml. In some embodiments, the recombinant protein is present in the liquid formulation at a concentration of less than 2 mg / ml, about 1.5 mg / ml, or about 1 mg / ml.

[0011] In various embodiments of the medicaments described above or discussed herein, the recombinant protein is an antigen-binding protein. In some cases, the antigen-binding protein is an antibody. In some cases, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody.

[0012] In some cases, the pharmaceutical container is a vial. In certain embodiments, the vial includes a stopper, e.g., a rubber stopper, that includes a vent stem. In some cases, the pharmaceutical is further administered via a syringe or transferred to a syringe or injection device.

[0013] In another aspect, the present invention provides a method for preparing a pharmaceutical product in a sealed container containing a recombinant protein (e.g., an antigen-binding protein or antibody) in a liquid formulation and a headspace comprising a gas with reduced oxygen content, the method comprising the steps of: (a) placing one or more containers containing the liquid formulation of the recombinant protein into a vacuum chamber at atmospheric pressure; (b) evacuating the chamber at a first pressure of 0.05 bar to 0.15 bar; (c) venting the chamber with a non-oxidizing gas at a second pressure of 800 mbar to 1000 mbar; and (d) sealing the one or more containers within the sealed vacuum chamber, wherein the method is carried out at a temperature in the range of 15-25°C, and wherein the sealed container contains a headspace gas comprising less than 5% oxygen by volume.

[0014] In some embodiments, the method further comprises repeating steps (b) and (c) one or more additional times before sealing the one or more containers.

[0015] In some cases, the first pressure is about 0.1 bar and / or the second pressure is between 0.2 bar and 0.1 bar or between 0.1 bar and 0.12 bar. In some embodiments, the pressure in the chamber is measured via a Pirani gauge. In some embodiments, the temperature is about 19°C.

[0016] In further embodiments, the stopper may be partially stoppered prior to the inert gas overlay and then fully stoppered and sealed after the inert gas overlay process. In some cases, the sealed container contains a headspace gas containing less than 2% oxygen by volume, less than 1% oxygen by volume, or 0.1% oxygen or less by volume.

[0017] In some embodiments, the liquid pharmaceutical formulation prepared by the methods described above or discussed herein contains the recombinant protein at a concentration of 2 mg / ml to 200 mg / ml. In other embodiments, the liquid pharmaceutical formulation prepared by the methods described above or discussed herein contains the recombinant protein at a concentration of 150 to 200 mg / ml. In some embodiments, the liquid pharmaceutical formulation prepared by the methods described above or discussed herein contains the recombinant protein at a concentration of less than 10 mg / ml, less than 5 mg / ml, or less than 2 mg / ml.

[0018] In various embodiments of the methods described above or discussed herein, the recombinant protein is an antigen-binding protein or antibody. In some cases, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody.

[0019] In some cases, the pharmaceutical container used in the methods of the invention is a vial. In a further embodiment, the vial comprises a rubber stopper for lyophilized products with a vented stem (which may be partially stoppered before the inert gas overlay and then fully stoppered and sealed after the inert gas overlay process).

[0020] In another aspect, the present invention provides a method for controlling oxygen content in the headspace of a closed container containing a liquid pharmaceutical formulation, the method comprising: (a) determining a desired final oxygen content in the headspace of a sealed container; (b) The terminal % oxygen content after the first cycle of oxygen reduction is calculated using the formula (I): TIFF0007824917000001.tif16128, wherein %O 2開始 is the oxygen content % at the start of the first cycle, and P 真空 is the vacuum pressure applied in the first cycle of oxygen reduction, and P 通気 is P真空 Higher but less than 1 bar pressure, %O 2終端 is the % oxygen content at the end of the first cycle; (c) optionally applying formula (I) to further cycles, wherein: 2開始 is the % oxygen content at the end of the previous cycle until the desired final oxygen content is reached; (d)(i) Pressure P between 0.05 bar and 0.15 bar 真空 performing one or more oxygen reduction cycles by evacuating the non-sealed container in a vacuum chamber, and venting the non-sealed container in the vacuum chamber with a non-oxidizing gas at a venting pressure of 800 mbar to 1000 mbar; (ii) sealing the container in a closed freeze-drying chamber; preparing a medicinal product in a sealed container by Includes. When the requirement for % oxygen in the headspace of a pharmaceutical product is low (e.g., less than approximately 2%), multiple oxygen reduction cycles are performed to achieve the target level of oxygen content. The % oxygen content after multiple oxygen reduction cycles using the same vacuum and vent pressures can be calculated using formula (II): TIFF0007824917000002.tif18128, where %O 2開始 is the oxygen content % at the start of the first cycle, and P 真空 is the vacuum pressure applied during the oxygen reduction cycle, and P 通気 is the pressure of the inert gas vent, and %O 2最終 is the % oxygen content at the end of the cycles, and n is the total number of oxygen reduction cycles applied to the product. Therefore, the number of oxygen reduction cycles required to achieve the final oxygen level in the vial headspace is obtained by solving equation (II):

[0021] In various embodiments of the methods described above or discussed herein, the non-oxidizing gas is selected from the group consisting of nitrogen, argon, helium, xenon, neon, krypton, and radon. In one embodiment, the non-oxidizing gas is nitrogen. In one embodiment, the non-oxidizing gas is argon.

[0022] The various embodiments described above or discussed herein may be combined in any manner consistent with the present invention. Other embodiments will become apparent from review of the detailed description that follows. [The present invention 1001] 1. A stable liquid pharmaceutical product comprising a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas contains less than 5% by volume of oxygen, and wherein the recombinant protein is stable for a period of at least 28 days when stored at 45°C, wherein stability for at least 28 days refers to an increase in the percentage of high molecular weight species of 2% or less over said period. [The present invention 1002] 1001. The pharmaceutical of the present invention, wherein the gas contains 2% by volume or less of oxygen. [The present invention 1003] 1002. The pharmaceutical of claim 1002, wherein the gas contains 1% by volume or less of oxygen. [The present invention 1004] 1003. The pharmaceutical product of claim 1003, wherein the gas contains less than 1% by volume of oxygen. [The present invention 1005] 1004. The pharmaceutical of claim 1004, wherein the gas contains 0.1% by volume or less of oxygen. [The present invention 1006] 1006. The pharmaceutical of any one of claims 1001 to 1005, wherein the liquid formulation contains the recombinant protein at a concentration of less than 10 mg / ml. [The present invention 1007] 1006. The pharmaceutical of claim 1006, wherein the concentration of the recombinant protein is less than 5 mg / ml. [The present invention 1008] 1007. The pharmaceutical of claim 1007, wherein the concentration of the recombinant protein is less than 2 mg / ml. [The present invention 1009] 1006. The pharmaceutical of any one of claims 1001 to 1005, wherein the liquid formulation contains the recombinant protein at a concentration of 1 mg / ml to 200 mg / ml. [The present invention 1010] Any of the pharmaceuticals of present inventions 1001 to 1009, wherein the recombinant protein is stable for a period of at least 3 months when stored at 45°C, and stability for at least 3 months means that the percentage of high molecular weight species increases by 10% or less over said period. [The present invention 1011] The pharmaceutical of the present invention 1010, wherein being stable for at least 3 months means that the percentage of high molecular weight species increases by no more than 5% over said period. [The present invention 1012] The pharmaceutical product of the present invention, wherein being stable for at least 3 months means that the percentage of high molecular weight species increases by less than 1% over said period. [The present invention 1013] 1. A stable liquid pharmaceutical product comprising a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 5% oxygen by volume, and wherein the recombinant protein is stable for a period of at least 28 days when stored at 45°C, wherein stability refers to a change in at least one product CQA above or below a predetermined threshold. [The present invention 1014] 1. A pharmaceutical product comprising a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 1% oxygen by volume, and wherein the antigen binding protein is stable for a period of at least 31 months when stored at 5° C., wherein stability for at least 31 months refers to a change in the percentage of major charge variants of no more than 10% over said period. [The present invention 1015] 1. A pharmaceutical product comprising a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas comprises less than 1% oxygen by volume. [The present invention 1016] 1015. The pharmaceutical of claim 1015, wherein the gas contains 0.1% by volume or less of oxygen. [The present invention 1017] The pharmaceutical of any one of claims 1001 to 1016, wherein the recombinant protein is an antigen-binding protein. [The present invention 1018] The pharmaceutical of the present invention, wherein said antigen-binding protein is a monospecific antibody. [The present invention 1019] The pharmaceutical of the present invention, wherein said antigen-binding protein is a bispecific antibody. [The present invention 1020] The pharmaceutical product of any one of claims 1001 to 1019, wherein the container is a vial. [The present invention 1021] 1020. The pharmaceutical product of claim 1020, wherein the vial comprises a stopper having one or more vent legs. [The present invention 1022] 1. A method for preparing a pharmaceutical product in a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas having a reduced oxygen content, comprising: (a) placing one or more containers containing the liquid formulation of the recombinant protein into a vacuum chamber at atmospheric pressure; (b) evacuating the chamber to a first pressure of 0.05 bar to 0.15 bar; (c) venting the chamber with a non-oxidizing gas at a second pressure greater than the first pressure but less than 1 bar; (d) sealing said one or more containers; Including, The method is carried out at a temperature in the range of 5 to 45°C, and the sealed container contains a headspace gas containing less than 5% by volume of oxygen. The method. [The present invention 1023] 1023. The method of claim 1022, further comprising repeating steps (b) and (c) one or more additional times before sealing said one or more containers. [The present invention 1024] 1024. The method of claim 1022 or 1023, wherein said first pressure is about 0.1 bar. [The present invention 1025] The method of any one of claims 1022 to 1024, wherein the second pressure is from about 800 mbar to about 1000 mbar. [The present invention 1026] 1026. The method of any one of claims 1022 to 1025, wherein the temperature is in the range of about 15 to 25°C. [The present invention 1027] 1026. The method of claim 1026, wherein the temperature is about 19°C. [The present invention 1028] 1028. The method of any one of claims 1022 to 1027, wherein the sealed container contains a headspace gas containing less than 2% by volume of oxygen. [The present invention 1029] 1028. The method of claim 1028, wherein the sealed container contains a headspace gas containing less than 1% by volume of oxygen. [The present invention 1030] 1029. The method of claim 1029, wherein the sealed container contains a headspace gas containing 0.1% by volume or less of oxygen. [The present invention 1031] 1031. The method of any of claims 1022 to 1030, wherein the liquid formulation contains the recombinant protein at a concentration of 1 mg / ml to 200 mg / ml. [The present invention 1032] 1031. The method of any of claims 1022 to 1030, wherein the liquid formulation contains the recombinant protein at a concentration of less than 10 mg / ml. [The present invention 1033] 1033. The method of claim 1032, wherein the concentration of the recombinant protein is less than 5 mg / ml. [The present invention 1034] 1034. The method of claim 1033, wherein the concentration of the recombinant protein is less than 2 mg / ml. [This invention 1035] The method of any of claims 1022 to 1034, wherein the recombinant protein is an antigen-binding protein. [The present invention 1036] 1035. The method of claim 1035, wherein said antigen-binding protein is a monospecific antibody. [This invention 1037] 1035. The method of claim 1035, wherein said antigen-binding protein is a bispecific antibody. [The present invention 1038] The method of any one of claims 1022 to 1037, wherein the container is a vial. [This invention 1039] The method of claim 1038, wherein the vial comprises a stopper having one or more vent legs. [The present invention 1040] 1039. The method of claim 1039, wherein the one or more vent legs are partially closed during step (b) and / or step (c). [The present invention 1041] The method of any one of claims 1039 and 1040, wherein the one or more vent legs are completely closed during step (d). [The present invention 1042] The method of claim 1022, wherein the pressure in the chamber is measured via a Pirani gauge. [This invention 1043] The method according to any one of claims 1022 to 1042, wherein the non-oxidizing gas is nitrogen. [This invention 1044] The method according to any one of claims 1022 to 1042, wherein the non-oxidizing gas is argon. [This invention 1045] 1042. The method of any one of claims 1022 to 1042, wherein the non-oxidizing gas is selected from the group consisting of helium, xenon, neon, krypton, and radon. [The present invention 1046] 1. A method for controlling oxygen content in the headspace of a closed container containing a liquid pharmaceutical formulation, comprising: (a) determining a desired final oxygen content in the headspace of the sealed container; (b) The terminal % oxygen content after the first cycle of oxygen reduction is calculated using the formula (I): TIFF0007824917000003.tif16128, wherein %O 2開始 is the oxygen content % at the start of the first cycle, and P真空 is the vacuum pressure applied in the first cycle of the oxygen reduction, and P 通気 is P 真空 Higher but less than 1 bar pressure, %O 2終端 is the % oxygen content at the end of the first cycle; (c) optionally applying said formula (I) to further cycles, wherein: 2開始 is the % oxygen content at the end of the previous cycle until the desired final oxygen content is reached; (d)(i) Pressure P between 0.05 bar and 0.15 bar 真空 performing one or more oxygen reduction cycles by evacuating a non-sealed container in a vacuum chamber and venting the non-sealed container in the vacuum chamber with a non-oxidizing gas at a venting pressure of 800 mbar to 1000 mbar; (ii) sealing said container; preparing a pharmaceutical product in the sealed container by The method comprising: [This invention 1047] 1. A pharmaceutical product comprising a sealed container containing a recombinant protein in a liquid formulation and a headspace comprising a gas, wherein the gas comprises a controlled, predetermined level of oxygen, and wherein the container comprises a stopper having one or more vent legs. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the change (%) of the major charge variant by CEX-UPLC as a function of oxygen headspace content for a liquid formulation of a bispecific antibody after 31 months of storage at 5° C. [Figure 2] FIG. 2 shows the increase in high molecular weight species (%) as a function of oxygen headspace content for a liquid formulation of a bispecific antibody after 28 days of storage at 45° C. using a nitrogen overlay according to the methods discussed herein. [Figure 3]FIG. 3 shows the increase in high molecular weight species (%) as a function of oxygen headspace content for a liquid formulation of a bispecific antibody after 3 months of storage at 45° C. using a nitrogen overlay according to the methods discussed herein. [Figure 4] FIG. 4 shows the increase in high molecular weight species (%) as a function of oxygen headspace content for a liquid formulation of a bispecific antibody after 3 months of storage at 45° C. with an argon overlay according to the methods discussed herein. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description Before describing the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about," when used in reference to a specific recited numerical value, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0026] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0027] definition The term "recombinant protein," as used herein, is intended to include all proteins that are prepared, expressed, produced or isolated by recombinant means, for example, proteins expressed using a recombinant expression vector that has been transfected into a host cell.

[0028] The term "antigen-binding molecule" or "antigen-binding protein" includes, for example, antibodies and antigen-binding fragments of antibodies, including monospecific and bispecific antibodies.

[0029] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules containing four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V). H The heavy chain constant region comprises three domains: C H 1. C H 2, and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H and V L The region can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of an antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0030] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived, for example, from intact antibody molecules using any suitable standard technique, such as proteolysis or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the variable and, optionally, constant domains of an antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0031] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0032] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR that is contiguous or in-frame with one or more framework sequences. L V related to domain H In an antigen-binding fragment having a domain, V H and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0033] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly attached to one another or may be connected by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to one another and / or to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above covalently associated with the domains (e.g., by disulfide bond(s)).

[0034] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0035] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, and particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences.

[0036] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutation (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutation) so that the V of the recombinant antibody is H and V L The amino acid sequence of the region is human germline V H and V LThese are sequences that are derived from and related to sequences, but may not naturally exist within the human antibody germline repertoire in vivo.

[0037] Human antibodies can exist in two forms, heterogeneously associated at the hinge. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, forming a molecule of approximately 75-80 kDa composed of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0038] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for modifying the hinge, C ... H 2 or C H Antibodies with one or more mutations in three regions are included.

[0039] The antibody of the present invention may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or recovered from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0040] The present invention also includes one-arm antibodies that bind to a specific antigen. As used herein, "one-arm antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain.

[0041] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Therefore, different antibodies may bind to different regions of the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include polysaccharide, phosphoryl, or sulfonyl groups on the antigen.

[0042] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned, with appropriate nucleotide insertions or deletions, with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, e.g., FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0043] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned, for example, by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference.A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0044] Sequence similarity, also referred to as sequence identity, for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, e.g., homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0045] Pharmaceuticals and compositions containing recombinant proteins The pharmaceutical product of the present invention comprises a sealed container (e.g., a vial) in which the headspace gas has a reduced concentration of oxidizing gas (e.g., oxygen) compared to the atmospheric concentration of the oxidizing gas. The pharmaceutical product of the present invention is based on the inventors' discovery of a method for reducing the level of oxidizing gas in the headspace of a pharmaceutical container containing a liquid pharmaceutical formulation of a recombinant protein (e.g., an antigen-binding protein or antibody). In contrast to standard lyophilization techniques, drawing a vacuum and venting the headspace gas above the liquid composition requires fine adjustment of the pressure in the vacuum chamber to minimize or eliminate bubbling or splashing of the liquid composition, which can cause material loss or evaporation and result in changes in the concentration of the active agent (e.g., antibody). Material loss or concentration changes are particularly problematic for high-potency compositions in which the active agent is present at low concentrations (e.g., approximately 2 mg / ml). Changes in the stability of high-concentration formulations are problematic because oxidative degradation products can result in changes in the purity / impurity profile of the conjugate, potentially leading to immunogenicity concerns.

[0046] In some embodiments, the pharmaceutical product of the present invention may contain a headspace gas having less than 5% oxidizing gas (e.g., oxygen) by volume. In various embodiments, the concentration of oxidizing gas (e.g., oxygen) in the headspace of the pharmaceutical container may be less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, or less than 1.5%. In one embodiment, the concentration of oxidizing gas (e.g., oxygen) in the headspace is less than about 1%. In one embodiment, the concentration of oxidizing gas (e.g., oxygen) in the headspace is less than about 0.5%. In one embodiment, the concentration of oxidizing gas (e.g., oxygen) in the headspace is less than about 0.1%. In various embodiments, the concentration of oxidizing gas (e.g., oxygen) in the headspace of the pharmaceutical container is less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%. In some cases, the concentration of oxygen in the headspace gas is about 0.01% to about 1.5%, in some cases, the concentration of oxygen in the headspace gas is about 0.75% to about 1.25%, and in some cases, the concentration of oxygen in the headspace gas is about 0.05% to about 0.15%.

[0047] The containers described herein can be vials, flasks, etc., having a volume sufficient to accommodate the desired amount of pharmaceutical formulation and headspace. The containers can be formed from a variety of suitable materials that exhibit inert characteristics with respect to the pharmaceutical formulation contained therein and are sufficiently impermeable to prevent leakage of the pharmaceutical formulation or intrusion of ambient air. Exemplary materials include glass (e.g., polycarbonate polystyrene polypropylene glass), polymers (e.g., plastic, platinum-cured silicone tubing), and metals (e.g., stainless steel 316L). In some embodiments, the containers are Type 1 glass vials. Additionally, the containers can be configured as reusable or single-use disposable components, as desired. Several designs for rubber stoppers with vent leg(s) are available and are suitable for use in conjunction with lyophilization vials adapted for use in the methods described herein. Stoppers containing one vent leg (single vent), "two leg" (two vent points), "three leg" (three vent points), and cross-shaped (four vent points) or even more vents are commercially available. Stoppers for use with vials in lyophilization chambers may be partially stoppered with the vent(s) open to the outside space during the gas overlay / oxygen reduction process, and then fully stoppered and sealed in connection with the container after one or more oxygen reduction cycles.

[0048] Medicaments of the present invention include liquid pharmaceutical compositions comprising antigen-binding molecules (e.g., antibodies) of the present invention. Pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that confer improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. For example, the excipient may include a stabilizer, a buffer, a tonicity agent, a surfactant, an organic solvent, a salt, or a combination thereof. In some embodiments, the stabilizer is selected from the group consisting of polyols, sugars, amino acids, non-ionic surfactants, and combinations thereof. In some embodiments, the tonicity agent is selected from the group consisting of sugars, amino acids, salts, and combinations thereof. In some embodiments, the buffer is selected from the group consisting of histidine, phosphate, citrate, succinate, acetate, carbonate, and combinations thereof.

[0049] The concentration of the antigen-binding molecule (e.g., antibody) in the liquid composition may vary depending on the potency of the molecule and the dose administered from the pharmaceutical container. In some cases, the concentration may be in the range of about 1 mg / ml to about 200 mg / ml. In some cases, the concentration may be in the range of about 1 mg / ml to about 10 mg / ml. In some cases, the concentration may be in the range of about 1 mg / ml to about 5 mg / ml. In some cases, the concentration may be in the range of about 0.1 mg / ml to about 2 mg / ml. In various embodiments, the concentration is less than about 25 mg / ml, less than about 20 mg / ml, less than about 15 mg / ml, less than about 10 mg / ml, or less than about 5 mg / ml. In various embodiments, the concentration of the antigen-binding molecule in the liquid composition is about 5 mg / ml or less, about 4 mg / ml or less, about 3 mg / ml or less, about 2 mg / ml or less, or about 1 mg / ml or less. In one embodiment, the concentration is less than about 2 mg / ml. In one embodiment, the concentration is less than about 1 mg / ml.

[0050] The concentration of the antigen-binding molecule (e.g., antibody) in the liquid composition may vary depending on the volume and dose administered from the pharmaceutical container. In some cases, the concentration may be in the range of about 1 mg / ml to about 200 mg / ml. In some cases, the concentration may be in the range of about 10 mg / ml to about 200 mg / ml. In some cases, the concentration may be in the range of about 50 mg / ml to about 100 mg / ml. In some cases, the concentration may be in the range of about 100 mg / ml to about 150 mg / ml. In some cases, the concentration may be in the range of about 150 mg / ml to about 200 mg / ml. In one embodiment, the concentration is greater than about 10 mg / ml. In another embodiment, the concentration is greater than about 50 mg / ml. In another embodiment, the concentration is less than about 100 mg / ml. In one embodiment, the concentration is greater than about 150 mg / ml.

[0051] The dose of an antigen-binding molecule administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. When used for therapeutic purposes in adult patients, the antigen-binding molecule of the present invention may be advantageously administered intravenously at a single dose of typically about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering antigen-binding molecules may be determined empirically; for example, the patient's progress may be monitored by periodic assessment and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages may be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0052] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. For subcutaneous delivery, pen delivery devices also readily find application in delivering the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device comes pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0053] Injectable liquid preparations may include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared in a sterile aqueous medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. The injections prepared in this manner can be filled or transferred into appropriate containers or devices (e.g., ampoules, syringes, injection devices, or pens).

[0054] Stability of antigen-binding molecules Reducing the amount of oxidizing gas (e.g., oxygen) in the headspace of the pharmaceutical container of the present invention favorably affects the stability of the formulated antigen-binding molecule in the liquid composition within the container. Oxidation is the primary degradation pathway for protein therapeutics, including antibodies and bispecific antigen-binding molecules. The effects of such degradation are pronounced at low concentrations, when any loss of active agent disproportionately affects the amount of active agent remaining in the composition after a given storage period. Manifestations of such degradation include an increase in high molecular weight (HMW) species and a change in the percentage of charge variants. Changes in the amount and percentage of HMW species can be detected using standard size-exclusion chromatography techniques known in the art (e.g., Lu et al., MAbs, 5(1):102-113, 2013). Changes in the amount and percentage of charge variants can be detected using standard cation-exchange chromatography techniques known in the art (e.g., Chumsae, et al., Journal of Chromatography B, 850:285-294, 2007). The stability of the antigen-binding molecules in the pharmaceutical preparations of the present invention can be determined by measuring the change in the amount or percentage of HMW or charge variants as a function of time and temperature parameters corresponding to specific storage conditions. In some cases, the storage conditions can be comparable to those under which the pharmaceutical preparation would normally be maintained during manufacture and use. In other cases, the storage conditions can be accelerated conditions intended to provide an indication of longer-term stability in a shorter period of time.

[0055] In various embodiments of the compositions of the present invention, the antigen-binding molecule (e.g., an antibody) may remain stable for a period of at least 28 days when stored at 45°C. Stability, in this context, may refer, for example, to an increase in the percentage of HMW species of about 2% or less over the storage period. In some cases, the percentage increase in HMW species is about 1.5% or less or about 1% or less over the storage period. In some cases, the antigen-binding molecule remains stable for a period of at least 3 months when stored at 45°C. Under these longer storage conditions, stability may refer, for example, to an increase in HMW species of about 10% or less over the storage period. In some cases, stability under these longer storage conditions may refer to an increase in HMW species of about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less over the storage period. In other embodiments, the antigen-binding molecule (e.g., an antibody) remains stable for a period of at least 31 months when stored at 5°C. Stability in this context can refer, for example, to a change in the percentage of charge variants of 10% or less over a period of storage, which in some cases can be 12, 18, 24, 30, or 36 months.

[0056] Throughout the manufacturing process of a particular therapeutic protein product, certain product quality attributes can be identified based on their potential clinical impact. Relevant quality attributes can be considered critical quality attributes (CQAs) depending on their potential impact on purity, safety, and / or efficacy. High molecular weight (HMW) species and charge variations are just two of many product CQAs that can be altered during the manufacturing process. Proteins are monitored for changes in these quality attributes during the manufacturing process, including after the product is filled into containers and during container storage. Susceptibility of a drug product to oxidation refers to changes in a product's CQA above or below a threshold for a particular CQA due to increased oxidation levels, which can affect the purity, safety, and / or efficacy of the product. Susceptibility of a drug product to oxidation also refers to changes in a product due to increased oxidation levels, which can affect the purity, safety, and / or efficacy of the product. In some cases, stability can refer to changes in a product's CQAs above or below a given threshold that can affect the purity, safety, and / or efficacy of the product.

[0057] Binding properties of antigen-binding molecules As used herein, the term "binding" in the context of the binding of an antigen-binding molecule, antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to, for example, a given antigen, e.g., a cell surface protein or any of its fragments, typically refers to the interaction or association between the minimal or molecular structures of two entities, e.g., an antibody-antigen interaction.

[0058] For example, binding affinities are typically about 10 when determined by surface plasmon resonance (SPR) techniques on a BIAcore 3000 instrument, e.g., using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0059] Thus, the antibodies or antigen-binding proteins of the invention have a K that is at least 10-fold lower than the affinity that they bind to a non-specific antigen (e.g., BSA, casein). D According to the present invention, the antibody binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is at least 10 times lower than that of a non-specific antigen. DAntibody affinities corresponding to the values ​​may be considered as non-detectable binding, however such antibodies may be paired with a second antigen-binding arm to generate bispecific antibodies of the invention.

[0060] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D Because of the inverse relationship between K and binding affinity, D The smaller the value, the higher, i.e., the stronger, the affinity. Therefore, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and hence a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form interactions and therefore a larger K D In some situations, a higher binding affinity (or K ) of a particular molecule (e.g., antibody) to an interaction partner molecule (e.g., antigen X) compared to the binding affinity of that molecule to another interaction partner molecule (e.g., antigen Y) is referred to as a K . D ) is larger than K D A smaller K value (lower or weaker affinity) D The binding affinity may be expressed as a binding ratio determined by dividing by the binding affinity (higher or stronger affinity), for example, 5-fold or 10-fold higher than the binding affinity in some cases.

[0061] "k d The term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. The above value is also referred to as k off Also called the value.

[0062] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0063] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d is obtained by dividing by

[0064] "EC50" or "EC 50 The term "EC" refers to half the maximal effective concentration, including the concentration of antibody that induces a response halfway between baseline and maximum after a specific exposure time. 50 The EC essentially represents the concentration of antibody at which 50% of the maximal effect is observed. In certain embodiments, 50 The value is equal to the concentration of an antibody of the invention that gives half-maximal binding to cells expressing CD3 or a tumor-associated antigen, as determined, for example, by a FACS binding assay. Thus, reduced or weaker binding is associated with an increased EC 50 or observed at half the maximum effective concentration.

[0065] In one embodiment, decreased binding is an increased EC2 that can bind half-maximal amounts of target cells. 50 It can be defined as the antibody concentration.

[0066] In another embodiment, EC 50 The values ​​represent the concentration of an antibody of the invention that causes maximal half-deletion of target cells by T cell cytotoxicity. Therefore, increased cytotoxicity (e.g., T cell-mediated tumor cell killing) is correlated with decreased EC 50 or observed by the value of the half-maximal effective concentration.

[0067] Bispecific antigen binding molecules Antigen-binding molecules of the present invention, such as antibodies, can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of a single target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Antibodies of the present invention can be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with second or additional binding specificities.

[0068] As used herein, the term "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity-determining region (CDR), alone or in combination with one or more additional CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. In certain embodiments, the antigen-binding molecule is an antibody or antibody fragment, as these terms are defined elsewhere herein.

[0069] As used herein, the term "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs.

[0070] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), the CDRs of the first antigen-binding domain may be designated by the prefix "A1", and the CDRs of the second antigen-binding domain may be designated by the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0071] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or configuration. For example, a multimerization domain can be connected to a second multimerization domain of the same or similar structure or configuration. H A non-limiting example of a multimerization component is an immunoglobulin (C H 2-C H The Fc portion of an IgG antibody may comprise an Fc domain (comprising three domains), for example, an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0072] The bispecific antigen-binding molecules of the present invention typically comprise two multimerization domains, e.g., two Fc domains, each of which is an individual portion of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0073] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0074] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity may be operably linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce the bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in connection with the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2Bispecific formats are included (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the above formats).

[0075] In the context of the bispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., improved or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C H The FcRn-containing FcRn-binding domain contains modifications in three regions, which increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P).

[0076] The present invention also provides a first C H 3 domain and second Ig C H The present invention also includes a bispecific antigen-binding molecule comprising three domains, a first and a second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The 3 domain contains mutations that reduce or disrupt protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (by IMGT; Y436F by EU). See, e.g., U.S. Patent No. 8,586,713. The second C H Additional modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I in the case of IgG1 antibodies (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU), N44S, K52N, and V82I in the case of IgG2 antibodies (N384S, K392N, and V422I by IMGT; EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I in the case of IgG4 antibodies (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

[0077] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain may be a chimeric Fc domain of human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H Part or all of the 2 sequences, and C derived from human IgG1, human IgG2 or human IgG4 HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein is, from the N to C terminus: [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein includes, from the N to C terminus: [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the invention are described in U.S. Patent Publication No. 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains having these general structures, configurations, and variants thereof, may have altered Fc receptor binding, which in turn affects Fc effector function.

[0078] pH dependent binding The present invention includes antibodies and bispecific antigen-binding molecules with pH-dependent binding properties. For example, antibodies of the present invention may exhibit reduced binding to antigens at acidic pH compared to neutral pH. Alternatively, antibodies of the present invention may exhibit improved binding to antigens at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, and 5.0. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0079] In certain instances, "reduced binding at acidic pH compared to neutral pH" refers to the K D K value for antibody binding to its antigen at acidic pH D For example, an antibody or antigen-binding fragment thereof may be used where the antibody or antigen-binding fragment thereof has an acidic / neutral K of about 3.0 or greater. D When a ratio is presented, it can be considered for purposes of the present invention to indicate "reduced binding to MUC16 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0080] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or improved) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody with reduced antigen binding at acidic pH compared to neutral pH can be obtained.

[0081] Antibodies containing Fc variants According to certain embodiments of the invention, antibodies and bispecific antigen-binding molecules comprise an Fc domain comprising one or more mutations that improve or reduce antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides antibodies and bispecific antigen-binding molecules comprising one or more mutations that improve or reduce antibody binding to the FcRn receptor at acidic pH compared to neutral pH. H 2 or C HThe antibodies include those containing mutations in three regions, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 and (e.g., S or T), and 256 and (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P).

[0082] For example, the present invention includes antibodies and bispecific antigen-binding molecules comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the above Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the present invention.

[0083] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific to a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific to two different antigens can be appropriately arranged relative to each other to produce a bispecific antigen-binding molecule of the present invention using routine methods. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of a multispecific antigen-binding molecule of the present invention are derived from a chimeric, humanized, or fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of a bispecific antigen-binding molecule of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), a high-affinity chimeric antibody against a particular antigen is first isolated, having a human variable region and a mouse constant region. The antibody is characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the invention.

[0084] Genetically engineered animals can be used to produce human bispecific antigen-binding molecules. For example, genetically engineered mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to a mouse κ constant gene at the endogenous mouse κ locus. Such genetically engineered mice can be used to produce fully human bispecific antigen-binding molecules containing two different heavy chains associated with the same light chain, which contains variable domains derived from one of two different human light chain variable region gene segments (see, for example, US2011 / 0195454). Fully human refers to an antibody, or antigen-binding fragment thereof, or immunoglobulin domain that contains amino acid sequences encoded by DNA derived from human sequences throughout the entire length of each polypeptide of the antibody, or antigen-binding fragment thereof, or immunoglobulin domain. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, a fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any one theory, chimeric proteins or sequences are generally designed to minimize the creation of immunogenic epitopes at the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0085] Method for reducing oxidizing gases in the headspace of pharmaceutical containers The methods of the present invention provide pharmaceutical products with increased stability and shelf life by minimizing charge mutations and / or aggregates caused by oxidative degradation. The methods of the present invention involve evacuation of gas in the headspace of a pharmaceutical container and subsequent venting of the headspace with a non-oxidizing gas (e.g., nitrogen or argon) to reduce the concentration of oxygen and / or other oxidizing gases, such as ozone, peroxides, chlorine, fluorine, nitric oxide, nitrogen dioxide, nitrous oxide, or a combination thereof. The methods can be performed in a vacuum chamber (e.g., a lyophilization chamber). In one embodiment, the vacuum chamber is equipped with a Pirani gauge (thermal conduction gauge) to accurately measure and control the pressure within the ranges identified herein. In various embodiments, the methods are performed under sterile conditions and / or under conditions that meet Good Manufacturing Practice (GMP) standards for the production of pharmaceutical preparations.

[0086] The methods of the present invention can be used to prepare pharmaceutical products in sealed containers containing recombinant proteins or antigen-binding proteins (e.g., antibodies or bispecific antigen-binding molecules) in a liquid formulation. The pharmaceutical products are formulated to contain reduced concentrations of oxygen and / or other oxidizing gases in the headspace of the pharmaceutical containers. A method for preparing pharmaceutical products in sealed containers according to the present invention includes the steps of: (a) placing one or more containers containing a liquid formulation of a recombinant protein or antigen-binding protein (e.g., an antibody) into a vacuum chamber at atmospheric pressure; (b) evacuating the chamber at a first pressure of about 0.05 bar to about 0.15 bar; (c) venting the chamber with a non-oxidizing gas at a second pressure of about 800 mbar to about 1000 mbar; and (d) sealing the one or more containers in the sealed vacuum chamber. In some embodiments, process steps (b) and (c) are repeated one or more times before sealing the container(s) to further reduce the concentration of oxidizing gases in the headspace. In various embodiments, the methods of the present invention can be used to produce pharmaceutical products having less than 5% oxygen (or other oxidizing gas) by volume in the container headspace. In some cases, the oxidizing gas concentration is reduced to less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the oxidizing gas (e.g., oxygen) concentration is 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0087] In some embodiments, a final desired concentration of oxygen (or other oxidizing gas) can be predetermined, and the number of cycles of the evacuation / venting process contemplated above can be adjusted accordingly to achieve the desired final concentration. For example, in one embodiment, a method for controlling the oxygen content in the headspace of a sealed pharmaceutical container includes the steps of: (a) determining the desired final oxygen content in the headspace of the sealed container; and (b) calculating the terminal % oxygen content after the first cycle of oxygen reduction using formula (I): TIFF0007824917000004.tif16128, wherein %O 2開始is the oxygen content % at the start of the first cycle, and P 真空 is the vacuum pressure applied in the first cycle of oxygen reduction, and P 通気 is P 真空 Higher but less than 1 bar pressure, %O 2終端 is the % oxygen content at the end of the first cycle; and (c) optionally applying formula (I) above to further cycles, wherein %O 2開始 is the oxygen content % at the end of the previous cycle until the desired final oxygen content is reached; and (d) (i) P is a pressure between 0.05 bar and 0.15 bar. 真空 (ii) preparing a pharmaceutical product in a sealed container by performing one or more oxygen reduction cycles by evacuating the non-sealed container in a vacuum chamber and venting the non-sealed container in the vacuum chamber with a non-oxidizing gas at an aeration pressure of 800 mbar to 1000 mbar, and (ii) sealing the container.

[0088] In various embodiments, the pressure is maintained above the water vapor pressure to avoid evaporation of the liquid formulation containing the recombinant protein or antigen-binding protein. In various embodiments, evacuation and / or venting of the vacuum chamber is performed at a pressure of about 0.02 bar to about 0.2 bar. In one embodiment, evacuation is performed at a pressure of about 0.1 bar, and venting is performed at a pressure of about 800 mbar to about 1000 mbar. The non-oxidizing gas used in venting the vacuum chamber can be selected from, for example, nitrogen, argon, helium, xenon, neon, krypton, and radon. In one embodiment, the non-oxidizing gas is nitrogen. In another embodiment, the non-oxidizing gas is argon. In various embodiments, the method is performed at a temperature in the range of about 5 to 45°C or about 10 to 37°C. In various embodiments, the method is performed at a temperature in the range of about 15 to 25°C. In some cases, the temperature is about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25° C. In one embodiment, the temperature for all cycles is maintained at about 19° C.

[0089] The recombinant protein or antigen-binding protein composition sealed within a pharmaceutical container by the methods of the present invention can be any of the various compositions described above or discussed herein. For example, liquid compositions of antigen-binding proteins (e.g., antibodies) can be formulated with various excipients, including buffers, tonicity adjusters, stabilizers, surfactants, etc., and the protein can be present at a concentration ranging from about 0.1 mg / ml to about 200 mg / ml. In some embodiments, the concentration of the antibody or other antigen-binding protein is from about 1 mg / ml to about 25 mg / ml, from 1 mg / ml to about 15 mg / ml, or from about 1 mg / ml to about 10 mg / ml. In some cases, the concentration is less than 10 mg / ml, less than 5 mg / ml, less than 2 mg / ml, or less than 1 mg / ml.

[0090] As discussed above, after reducing the oxidizing gas content of the headspace gas within the container(s), the container(s) are fully closed / stoppered and finally sealed. The stopper can be made of a variety of materials (e.g., polymer, rubber) and can exhibit resilient properties (e.g., sufficient rigidity, malleability) as desired for engagement with the container. In some embodiments, the stopper is formed from synthetic rubber, and in some embodiments, the stopper is formed from butyl rubber. The stopper can include one or more vents or vent legs. The stopper can be adapted to form and maintain a resilient seal and, in some embodiments, can be a stopper compatible with conventional lyophilization procedures. Thus, stoppers for use with vials in a lyophilization chamber can be partially stoppered, with the vent(s) open to the outside space, during the gas overlay / oxygen reduction process, and then fully stoppered and sealed in connection with the container after one or more oxygen reduction cycles. Examples of lyophilization systems, closure caps, and stopper configurations are provided in the FDA Guide "Lyophilization of Parenteral (7 / 93)" and Bhambhani and Medi, "Selection of Containers / Closures for Use in Lyophilization Applications: Possibilities and Limitations," American Pharmaceutical Review, May 1, 2010, the contents of which are incorporated herein by reference in their entireties. [Example]

[0091] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, but are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0092] Example 1: Reducing oxygen in pharmaceutical headspace In this example, a standard GMP lyophilization chamber equipped with a Pirani gauge to measure pressure and a needle valve to control pressure was used as the vacuum chamber. The bispecific antibody at a concentration of 2 mg / ml in a liquid formulation was packaged in vials equipped with vented rubber stoppers, and the presence of headspace oxygen was reduced from about 21% to about 0.25% by nitrogen overlay in a two-cycle process.

[0093] (Table 1) Non-oxidizing gas overlay process TIFF0007824917000005.tif64152

[0094] Once the vials were placed in the chamber, a vacuum was applied (step 6) to remove the gas (which to start with is air containing approximately 21% oxygen) from the chamber. The pressure (100,000 μbar) is higher than the water vapor pressure to avoid evaporation, foaming, and possible splashing, and is measured by a Pirani gauge. Under normal lyophilization conditions, pressures much lower than vacuum (as low as 150 μbar) exist, so the pressure is controlled using a capacitance manometer. However, capacitance manometers are only accurate at pressures below approximately 2000 μbar and cannot be used to control pressures at 100,000 μbar.

[0095] Once a pressure of 100,000 μbar was reached, nitrogen was filled into the chamber to replace the evacuated air.

[0096] The process was repeated twice to further reduce the oxygen level (second cycle steps 3-4 below). Once the desired oxygen level was reached, the vial was stoppered (second cycle step 5 below).

[0097] Table 2: Non-oxidizing Gas Overlay Process (continued) TIFF0007824917000006.tif64149

[0098] Oxygen content formula: TIFF0007824917000007.tif8128P=Pressure

[0099] Exemplary calculations from the process discussed above in Example 1: Cycle 1 TIFF0007824917000008.tif26128 Cycle 2 TIFF0007824917000009.tif26128

[0100] Example 2: Stability testing of pharmaceuticals Stability analyses were performed on various pharmaceutical products prepared using the process discussed in Example 1 with varying concentrations of headspace oxygen and compared to a control in which headspace oxygen was at or near atmospheric levels (approximately 21%). In some cases (described herein), nitrogen was replaced with argon in the vent section of the process. High molecular weight (HMW) species were detected using size-exclusion ultra-performance liquid chromatography (SE-UPLC), and charge variants were detected using cation-exchange ultra-performance liquid chromatography (CEX-UPLC).

[0101] As shown in Figure 1, reducing the headspace oxygen content from 21% to less than 1% via nitrogen overlay reduced the degradation of the bispecific antibody observed by CEX-UPLC after 31 months of storage at 5°C. At 21% oxygen, the percentage change of the major charge variant was approximately 46.25%, while at <1% oxygen, the percentage change was reduced to approximately 8.75% over the storage period.

[0102] As shown in Figure 2, reducing the headspace oxygen content from 21% to 0.1% via nitrogen overlay increased the stability of the second bispecific antibody, as indicated by the reduced percentage increase in the presence of HMW species after 28 days of storage at 45°C. At 21% oxygen, the percentage of HMW species increased by approximately 8.62% over the storage period. At 15% oxygen, the percentage of HMW species increased by approximately 8.53% over the storage period. At 10% oxygen, the percentage of HMW species increased by approximately 4.74% over the storage period. At 5% oxygen, the percentage of HMW species increased by approximately 1.42% over the storage period, and at 0.1% oxygen, the percentage of HMW species increased by approximately 0.24% over the storage period.

[0103] Figure 3 shows the reduction in percentage increase in the presence of HMW species observed for a second bispecific antibody after 3 months of storage at 45°C following a nitrogen overlay process to reduce headspace oxygen content. At 5% oxygen, the percentage of HMW species increased by approximately 9.66% over the storage period. At 2% oxygen, the percentage of HMW species increased by approximately 7.27% over the storage period. At 1% oxygen, the percentage of HMW species increased by approximately 4.54% over the storage period, and at less than 1% oxygen, the percentage of HMW species increased by approximately 0.34% over the storage period.

[0104] Figure 4 shows the same headspace oxygen content for a second bispecific antibody stored for 3 months at 45°C as in Figure 3, except that nitrogen was replaced with argon in the overlay process. At 5% oxygen, the percentage of HMW species increased by approximately 16.72% over the storage period. At 2% oxygen, the percentage of HMW species increased by approximately 13.05% over the storage period. At 1% oxygen, the percentage of HMW species increased by approximately 7.68% over the storage period, while below 1% oxygen, there was no detectable increase in HMW species over the storage period.

[0105] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing detailed description. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A method for controlling the oxygen content in the headspace of a sealed container containing a recombinant protein, comprising: (a) A non-sealed container containing a recombinant protein in a liquid pharmaceutical formulation containing water is placed in a vacuum chamber. 真空 and drawing a vacuum at P 真空 is a pressure of about 0.05 bar to about 0.15 bar; and Venting the non-sealed container in the vacuum chamber with a non-oxidizing gas at a venting pressure of 800 mbar to 1000 mbar. performing a cycle of oxygen reduction by (b) The terminal oxygen content % after step (a) is calculated using formula (I): wherein % O ２開始 is the oxygen content % at the start of the cycle, and P 真空 is the vacuum pressure applied in the oxygen reduction cycle, and P 通気 Is, P 真空 higher but less than 1 bar pressure, % O ２終端 is the % oxygen content at the end of the cycle; (c) repeating steps (a) and (b) until the terminal oxygen content % is below the desired oxygen content %; (d) sealing the container; Including, The method is carried out above the vapor pressure of water in the liquid pharmaceutical formulation.

2. 10. The method of claim 1, wherein the desired % oxygen content is less than about 5%, less than about 2%, or about 0.1% oxygen.

3. 3. The method of claim 1 or 2, wherein the desired % oxygen content is less than about 1% oxygen.

4. 4. The method of claim 3, wherein the number of cycles of oxygen reduction performed is two.

5. 5. The method of any one of claims 1 to 4, wherein the evacuation and venting of the chamber is controlled to eliminate evaporation of the liquid pharmaceutical formulation, which would result in changes in the concentration of the recombinant protein.

6. 6. The method of claim 5, wherein the desired % oxygen content is less than about 0.1% oxygen.

7. P 真空 The method according to any one of claims 1 to 6, wherein the pressure is about 0.1 bar.

8. P 通気 8. The method according to claim 1, wherein the pressure is about 0.9 bar or about 912 mbar.

9. 9. The method of any one of claims 1 to 8, carried out at about 19°C.

10. The method of any one of claims 1 to 9, carried out at a temperature.

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