Methods of improving photo stability and controlling formation of HMW species in biologic formulations, and biologic formulations produced by the methods

Abstract

Methods of improving photo-stability of proteins in protein-containing aqueous formulations in containers such as those preserved in prefilled syringes (PFS) for therapeutic applications can include reducing the headspace of the closed container, thereby reducing a size of a bubble and the headspace air in the container and overlaying inert gas on to the headspace air in the closed container.

Description

METHODS OF IMPROVING PHOTO STABILITY AND CONTROLLING FORMATION OF HMW SPECIES IN BIOLOGIC FORMULATIONS, AND BIOLOGIC FORMULATIONS PRODUCED BY THE METHODS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 694,378 filed 13 September 2024, the entire disclosure of which is hereby incorporated by reference herein. TECHNICAL FIELD

[0002] The present inventions provide methods of improving photo-stability of proteins in protein-containing aqueous formulations. The inventions also provide methods of preventing photo-degradation of proteins in protein-containing aqueous formulations preserved in prefilled syringe (PFS) for therapeutic applications. Improved protein-containing aqueous formulations also are provided. BACKGROUND

[0003] Liquid formulations containing proteins are photo-sensitive and undergo varying degrees of degradation during storage or transportation. Light-induced oxidation is one of the major degradation pathways that has a destructive effect on protein stability and potency. Light- induced degradation of protein, especially in closed container results formation of acidic species and high molecular weight (HMW) aggregates / species Oxidative reactions cause degradation of amino acid residues, peptide bond hydrolysis, and therefore cause protein instability due to changes in the protein's tertiary structure and the resulting aggregation (Hipper, Elena, et al. "Photo-oxidation of therapeutic protein formulations: from radical formation to analytical techniques." Pharmaceutics 14.1 (2021): 72). Therefore, there is a need for developing a method for improving photo-stability and preventing photo-degradation of protein for enhancing the stability and preventing the aggregation and / or oxidative degradation of proteins in protein-containing aqueous formulations.SUMMARY OF THE DISCLOSURE

[0004] The inventions provide methods of improving photo-stability of a protein, wherein the method comprises the steps of: (a)providing an aqueous solution comprising a protein in a closed container; (b) reducing the headspace size of the closed container, thereby reducing the size of the bubble and the headspace air in the container; and (c) overlaying inert gas (e.g., nitrogen gas) on to the headspace air in the closed container; wherein the inert gas replaces oxygen content in the headspace air to a level that is below 21%, thereby reducing oxygen content in the headspace air, and wherein the reduced oxygen content and the reduced bubble size of the headspace air decreases photo-degradation by: (i) reducing oxidation of the protein, (ii) reducing formation of acidic species, and / or (iii) reducing formation of high molecular weight (HMW) aggregates / species, thereby improving photo-stability of the protein in the closed container. The oxygen content in the headspace air can be about 20.94%, about 20.93%, about 20.92%, about 20.91%, about 20.90%, about 20.85%, about 20.80%, about 20.75%, about 20.70%, about 20.65%, about 20.60%, about 20.55%, about 20.50%, about 20.40%, about 20.30%, about 20.20%, about 20.10%, about 20.00%, about 19.90%, about 19.80%, about 19.70%, about 19.60%, about 19.50%, about 19.40%, about 19.30%, about 19.20%, about 19.10%, about 19.00%, about 18.50%, or about 18.00%. Overlaying of an inert gas can be carried out by any replacement method, including purge, partial vacuum and refill, blow the inert gas or blanket the inert gas, etc. The protein can be selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein. The formation of HMW aggregates decreases with inert gas, such as nitrogen gas, overlaying and decreasing size of the bubble. The formation of acidic species decreases with inert gas overlaying and decreasing size of the bubble. The oxidation of the protein decreases with inert gas overlaying and decreasing size of the bubble. The inert gas can be selected from the group consisting of: argon, nitrogen, helium, and any mixtures thereof. The oxidation of the protein can be resulting from the oxidation of methionine residue of the protein. The photo-stability of the protein can be determined by exposing the aqueous solution under light or photo exposure (e.g., 0.5 ICH Cool White (CW) light) and analysis of: (a) the HMW aggregates by size exclusion ultra performance liquid chromatography (SE- UPLC), (b) acidic species (charge variants) by cation exchange chromatography (CEX), (c) the protein recovery by reversed phase ultra performance liquid chromatography (RP-UPLC), (d)peptide mapping by identifying and quantifying methionine oxidation, (e) optical density (OD), and / or (f) pH. The container can be a vial or a prefilled syringe (PFS). The PFS can be configured to hold an aqueous solution having a volume from about 1.0 ml to 50.0 ml of the aqueous solution. For example, the PFS can be sized to hold a volume of about 1.0 ml, 2.0 ml, 3.0 ml.4.0 ml, 5.0 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml, or any value therebetween. The bubble size (headspace air) can be about 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 5 mm, 1 mm or less, or even as low as 0 mm. The inventions provide methods that control the formation of the HMW species in biological formulations.

[0005] The inventions also provide methods of preventing photo-degradation of a protein in prefilled syringe (PFS), wherein the method comprises the steps of: (a) providing an aqueous solution of a protein in a prefilled syringe (PFS); (b) reducing the headspace size of the PFS, thereby reducing the size of the bubble and the headspace air in the PFS; and (c) overlaying inert gas, such as nitrogen gas, on to the headspace air in the PFS; wherein the inert gas replaces oxygen content in the headspace air to a level that is below 21%, thereby reducing oxygen content in the headspace air, and wherein the reduced oxygen content and the reduced bubble size decreases photo-degradation by: (i) reducing oxidation of the protein, (ii) reducing formation of acidic species, and / or (iii) reducing formation of high molecular weight (HMW) aggregates / species, thereby preventing photo-degradation of the protein in the PFS. The oxygen content in the headspace air can be about 20.94%, about 20.93%, about 20.92%, about 20.91%, about 20.90%, about 20.85%, about 20.80%, about 20.75%, about 20.70%, about 20.65%, about 20.60%, about 20.55%, about 20.50%, about 20.40%, about 20.30%, about 20.20%, about 20.10%, about 20.00%, about 19.90%, about 19.80%, about 19.70%, about 19.60%, about 19.50%, about 19.40%, about 19.30%, about 19.20%, about 19.10%, about 19.00%, about 18.50%, or about 18.00%.

[0006] The protein can be selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein. The formation of HMW aggregates decreases with inert gas, such as nitrogen gas, overlaying and decreasing size of the bubble. The formation of acidic species decreases with inert gas overlaying and decreasing size of the bubble. The inert gas can be selected from the group consisting of: argon, nitrogen, helium, and any mixtures thereof. Overlaying of an inert gas can be carried out by any replacement method, including purge, partial vacuum and refill, blow the inert gas or blanket the inert gas, etc. The oxidation of the protein decreases with inert gas overlayingand decreasing size of the bubble. The oxidation of the protein can be resulting from the oxidation of methionine residue of the protein. The photo-degradation of the protein can be determined by exposing the aqueous solution under 0.5 ICH CW light and analysis of: (a) the HMW aggregates by size exclusion ultra performance liquid chromatography (SE-UPLC), (b) acidic species (charge variants) by cation exchange chromatography (CEX), (c) the protein recovery by reversed phase ultra performance liquid chromatography (RP-UPLC), (d) peptide mapping by identifying and quantifying methionine oxidation, (e) optical density (OD), and / or (f) pH. The PFS can be for about 1.0 ml to 50.0 ml aqueous solution. The size of the PFS can be 1.0 ml, 2.0 ml, 3.0 ml.4.0 ml, 5.0 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml. The bubble size (headspace air) can be about 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 5 mm, 1 mm or less, or 0 mm.

[0007] The concentration of the protein in the aqueous solution can be about 10 to about 700 mg / ml and any range thereof, such as from about 100 mg / ml to about 700 mg / ml, 100 mg / ml to about 350 mg / ml and various ranges thereof. In some aspects, the concentration of the protein in the aqueous solution can be about 10 to 50 mg / ml, about 50 to 100 mg / ml, about 100 to 120 mg / ml, about 120 to 150 mg / ml, about 150 to 175 mg / ml, about 175 to 200 mg / ml, about 200 to 250 mg / ml, about 250 to 300 mg / ml, about 300 to 350 mg / ml, about 350 to 400 mg / ml, about 400 to 450 mg / ml, about 450 to 500 mg / ml, about 500 to 550 mg / ml, about 550 to 600 mg / ml, about 600 to 650 mg / ml, or about 650 to 700 mg / ml. The protein in the aqueous formulation can be selected from the group consisting antibodies, antibody fragments or antibody derivatives, wherein the protein can be an Fc-containing protein. The Fc-containing protein can be a receptor-Fc-fusion protein. The protein in the aqueous formulation can be an antibody, a monoclonal antibody or an IgG antibody. The aqueous protein formulation can comprise pharmaceutically acceptable excipients including one or more buffer, salt, surfactant, and sugar.

[0008] In some aspects, improving photo-stability of a protein in a PFS can include (a) providing an aqueous solution comprising a protein in a PFS configured to hold a volume of about 1 ml; (b) reducing the headspace size of the PFS to a low volume, e.g., no more than about 0.1 ml, such as less than 0.05 ml or no head space, thereby reducing the size of the bubble and the headspace in the PFS; and (c) overlaying inert gas (e.g., nitrogen gas) on to the headspace in the PFS, thereby reducing oxygen content in the headspace air, and wherein the reduced oxygen content and the reduced bubble size of the headspace air decreases photo-degradation by: (i)reducing oxidation of the protein, (ii) reducing formation of acidic species, and / or (iii) reducing formation of high molecular weight (HMW) aggregates / species, thereby improving photo-stability of the protein in the closed container. Optionally, the PFS can be filled automatically by a syringe filler and with an inert gas overlay (e.g., a nitrogen gas overlay). BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1A and Figure 1B illustrate graphs showing the effect of 0.5 ICH CW light exposure on 150 mg / ml mAb1 and variations in in the formation of HMW % (Figure 1A) and Charge (Acidic species %) (Figure 1B).

[0010] Figure 2A and Figure 2B illustrate graphs showing the effect of 0.5 ICH CW light exposure on 150 mg / ml mAb1 at different bubble size for DPs in PFS (Headspace Size) and variations in the formation of HMW % (Figure 2A) and Charge (Acidic species %) (Figure 2B).

[0011] Figure 3A and Figure 3B illustrate graphs showing the effect of 0.5 ICH CW light exposure without nitrogen overlay (replacing O2 with N2) on 150 mg / ml mAb1 for DPs in PFS and variations in in the formation of HMW % (Figure 3A) and Charge (Acidic species %) (Figure 3B).

[0012] Figure 4 illustrates the effect of 0.5 ICH CW light exposure on the concentration of 150 mg / ml mAb1 as indicated by total protein recovery.

[0013] Figure 5A and Figure 5B illustrate graphs showing the effect of 0.5 ICH CW light exposure on mAb1 and methionine oxidation in DP containing DTLM[+16]ISR (Figure 5A) and DP containing WQEGNVFSCSVM[+16]HEALHNHYTQK (Figure 5B).

[0014] Figure 6A and Figure 6B illustrate graphs showing the effect of 0.5 ICH CW light exposure on 120 mg / ml mAb2 and variations in the formation of HMW % (Figure 6A) and Charge (Acidic species %) (Figure 6B).

[0015] Figure 7A and Figure 7B illustrate graphs showing the effect of 0.5 ICH CW light exposure on 120 mg / ml mAb2 at different bubble size for DPs in PFS (Headspace Size) and variations in the formation of HMW % (Figure 7A) and Charge (Acidic species %) (Figure 7B).

[0016] Figure 8A and Figure 8B illustrate graphs showing the effect of 0.5 ICH CW light exposure without nitrogen overlay (replacing O2 with N2) on 120 mg / ml mAb2 for DPs in PFS and variations in in the formation of HMW % (Figure 8A) and Charge (Acidic species %) (Figure 8B).

[0017] Figure 9 illustrates the effect of 0.5 ICH CW light exposure on 120 mg / ml mAb2 as indicated by total protein recovery. Graphs showing no significant changes in mAb2 recovery % or protein concentration.

[0018] Figure 10A and Figure 10B illustrate graphs showing the effect of 0.5 ICH CW light exposure on mAb2 and methionine oxidation in DP containing DTLMISR (Figure 10A) and DP containing WQQGNVFSCSVMHEALHNHYTQK (Figure 10B).

[0019] Figure 11 illustrates UV Chromatograms (F8, F9, F10, F11, and F12) of mAb2 exposed under CW light.

[0020] Figure 12 illustrates UV Chromatograms (F13, F14, F15, F17, and F19) of mAb2 exposed under CW light.

[0021] Figure 13 illustrates UV Chromatograms (F8, F9, F10, F11, F12, F13, F14, F15, F17, and F19) of mAb2 exposed under CW light. DETAILED DESCRIPTION OF THE DISCLOSURE

[0022] The present invention is not limited to 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 unless indicated, since the scope of the present invention will be limited only by the appended claims.

[0023] Unless stated otherwise, all technical and scientific terms and phrases used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. 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.DEFINITIONS

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] The term “about” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the invention can perform as intended, such as having a desired rate, amount, density, degree, increase, decrease, percentage, value, purity, pH, concentration, presence of a form or variant, temperature or amount of time, as is apparent from the teachings contained herein. For example, “about” can signify values either above or below the stated value in a range of approx. + / - 10% or more or less depending on the ability to perform. Thus, this term encompasses values beyond those simply resulting from systematic error.

[0026] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” can include one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0027] “Protein”, “Polypeptide” or “peptide” refers to sequence(s) of amino acids covalently joined. Polypeptides include natural, semi-synthetic and synthetic proteins and protein fragments. “Polypeptide” and “protein” can be used interchangeably. Oligopeptides are considered shorter polypeptides.

[0028] “Protein of interest” or “polypeptide of interest” (POI) can have any amino acid sequence, and can include any protein, polypeptide, or peptide that is desired to be expressed, typically for gene therapy purposes. Protein types can include, but are not limited to, receptors, fusion proteins, agonists, antagonists, activators, inhibitors, enzymes (such as those used in enzyme replacement therapy), factors and co-factors, repressors, activators, ligands, protein hormones, structural proteins, storage proteins, transport proteins, signal proteins, neurotransmitters and contractile proteins. Derivatives, components, domains, chains andfragments of the above also are included. The sequences can be natural, semi-synthetic or synthetic.

[0029] “Antibodies” (also referred to as "immunoglobulins") are examples of proteins having multiple polypeptide chains and extensive post-translational modifications. The canonical immunoglobulin protein (for example, IgG) comprises four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine disulfide bond, and the two heavy chains are bound to each other via two cysteine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (for example, at asparagine residues) with various polysaccharides, and can differ from species to species, which may affect antigenicity for therapeutic antibodies. Butler and Spearman, "The choice of mammalian cell host and possibilities for glycosylation engineering", Curr. Opin. Biotech.30:107-112 (2014).

[0030] Antibodies are often used as therapeutic biomolecules. An antibody can include immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDRl, LCDR2 and LCDR3. The term "high affinity" antibody refers to those antibodies having a binding affinity to their target of at least 10-9 M, at least 10-10 M; at least 10-11 M; or at least 10-12 M, as measured by surface plasmon resonance, for example, BIACORE™ or solution-affinity ELISA.

[0031] The phrase "bispecific antibody" can include an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope -- either on two different molecules(for example, antigens) or on the same molecule (for example, on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two, three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (for example, on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen- binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen- binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.

[0032] The phrase "heavy chain," or "immunoglobulin heavy chain" can include an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified can include a heavy chain variable domain. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain can include a fragment that is capable of specifically recognizing an antigen (for example, recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.

[0033] The phrase "light chain" can include an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified can include human kappa and lambda light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain caninclude, from amino terminus to carboxyl terminus, a VL domain that can include FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used with these inventions include those, for example, that do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening for the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not substantially interfere with the affinity and / or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen- binding regions of the antigen-binding protein.

[0034] The phrase "variable domain" can include an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" can include an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.

[0035] The phrase "complementarity determining region" ("CDR") can include an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally (that is, in a wild-type organism) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (for example, an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. In some circumstances (for example, for a CDR3), CDRs can be encoded by two or more sequences (for example, germline sequences) that are not contiguous (for example, in a nucleic acid sequence that has not been rearranged) but are contiguous in a B cell nucleic acid sequence, for example, as the result of splicing or connecting the sequences (for example, V-D-J recombination to form a heavy chain CDR3).

[0036] “Antibody derivatives and fragments” include, but are not limited to: antibody fragments (for example, ScFv-Fc, dAB-Fc, half antibodies, Fab), multispecifics (for example, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, tri-specific). “Fab” refers to an antibody fragment comprising an antigen binding region. An Fab typically will lack the Fc portion.

[0037] The phrase "Fc-containing protein" can include antibodies, bispecific antibodies, antibody derivatives containing an Fc, antibody fragments containing an Fc, Fc-fusion proteins, receptor Fc-fusion proteins (including trap proteins), immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region. A "functional portion" refers to a CH2 and CH3 region that can bind a Fc receptor (for example, an FcyR; or an FcRn, (neonatal Fc receptor), and / or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and / or insertions or other modifications that render it unable to bind any Fc receptor and also unable to activate complement, the CH2 and CH3 region is not functional. Fc-fusion proteins include, for example, Fc-fusion (N-terminal), Fc-fusion (C-terminal), mono-Fc-fusion and bispecific Fc-fusion proteins.

[0038] “Fc" stands for fragment crystallizable, and is often referred to as a fragment constant. Antibodies contain an Fc region that is made up of two identical protein sequences. IgG has heavy chains known as γ-chains. IgA has heavy chains known as α-chains, IgM has heavy chains known as µ-chains. IgD has heavy chains known as σ-chains. IgE has heavy chains known as ε-chains. In nature, Fc regions are the same in all antibodies of a given class and subclass in the same species. Human IgGs have four subclasses and share about 95% homology amongst the subclasses. In each subclass, the Fc sequences are the same. For example, human IgG1 antibodies will have the same Fc sequences. Likewise, IgG2 antibodies will have the same Fc sequences; IgG3 antibodies will have the same Fc sequences; and IgG4 antibodies will have the same Fc sequences. Alterations in the Fc region create charge variation.

[0039] Fc-containing proteins, such as antibodies, can comprise modifications in immunoglobulin domains, including where the modifications affect one or more effector function of the binding protein (for example, modifications that affect FcyR binding, FcRn binding and thus half-life, and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338,339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0040] For example, and not by way of limitation, the binding protein is an Fc-containing protein (for example, an antibody) and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modification(s)) and have a modification at position 250 (for example, E or Q); 250 and 428 (for example, L or F); 252 (for example, L / Y / F / W or T), 254 (for example, S or T), and 256 (for example, S / R / Q / E / D or T); or a modification at 428 and / or 433 (for example, L / R / SI / P / Q or K) and / or 434 (for example, H / F or Y); or a modification at 250 and / or 428; or a modification at 307 or 308 (for example, 308F, V308F), and 434. In another example, the modification can comprise a 428L (for example, M428L) and 434S (for example, N434S) modification; a 428L, 2591 (for example, V259I), and a 308F (for example, V308F) modification; a 433K (for example, H433K) and a 434 (for example, 434Y) modification; a 252, 254, and 256 (for example, 252Y, 254T, and 256E) modification; a 250Q and 428L modification (for example, T250Q and M428L); a 307 and / or 308 modification (for example, 308F or 308P).

[0041] “Fv” stands for fragment variable, and is primarily responsible for binding to epitopes.

[0042] As used herein, the expression “formulation” means a combination of at least one active ingredient (e.g., a protein, such as polypeptide, antibody, monoclonal antibody, etc. which is capable of exerting a biological effect in a human or non-human animal), and at least one inactive ingredient which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term “formulation”, as used herein, means “pharmaceutical” or “therapeutic” formulation unless specifically indicated otherwise. The present invention provides pharmaceutical formulations comprising at least one therapeutic protein. According to the present invention, the therapeutic protein is an antibody, or an antigen-binding fragment thereof. The term “formulation”, as used herein, also means a solid, semisolid, and / or liquid formulation, such as suitable for oral, intramuscular, subcutaneous and / or intravenous administration.

[0043] The term “excipient” can include a non-therapeutic agent added to a pharmaceutical composition to provide a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0044] The term “headspace”, as described herein, can include an unfilled space adjacent to the formulation in the closed container.

[0045] Various methods of stability testing are known in the art, including: Fleischman et al. Shipping-Induced Aggregation in Therapeutic Antibodies: Utilization of a Scale-Down Model to Assess Degradation in Monoclonal Antibodies. J. Pharm. Sci. (2017) 106: 994-1000; Ghazvini et al. Evaluating the Role of the Air-Solution Interface on the Mechanism of Subvisible Particle Formation Caused by Mechanical Agitation for an IgG1 mAbI. J. Pharm. Sci. (2016) 105: 1643- 1656; and Torisu et al. Synergistic Effect of Cavitation and Agitation on Protein Aggregation. J. Pharm. Sci. (2017) 106: 521-529).

[0046] All numerical limits and ranges set forth herein include all numbers or values thereabout or there between of the numbers of the range or limit. The ranges and limits described herein expressly denominate and set forth all integers, decimals and fractional values defined and encompassed by the range or limit. Thus, a recitation of ranges of values herein are intended to serve as a way of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. DESCRIPTION

[0047] The main degradation pathways for photo exposure are HMW species, acidic charge variants formation and methionine oxidation. The inventions provide that photo- degradation of proteins (such as mAb drug product) in protein-containing aqueous formulations preserved in prefilled syringe (PFS) is mediated through oxidation and can be mitigated by reduce headspace or by removing oxygen in the headspace. It is revealed by the instant inventions that reduced headspace air bubble size can decrease photo degradation. The inventions also provide that replacement of headspace air, that contains about 21% oxygen, with inert gas can mitigate the mAb photo sensitivity. The inert gas can be selected from the group consisting of: argon, nitrogen, helium, and any mixtures thereof. Replacement of the headspace air can be carried out byoverlaying of an inert gas by any replacement method, including purge, partial vacuum and refill, blow the inert gas or blanket the inert gas, etc. The oxygen content in the headspace air can be about 20.94%, about 20.93%, about 20.92%, about 20.91%, about 20.90%, about 20.85%, about 20.80%, about 20.75%, about 20.70%, about 20.65%, about 20.60%, about 20.55%, about 20.50%, about 20.40%, about 20.30%, about 20.20%, about 20.10%, about 20.00%, about 19.90%, about 19.80%, about 19.70%, about 19.60%, about 19.50%, about 19.40%, about 19.30%, about 19.20%, about 19.10%, about 19.00%, about 18.50%, or about 18.00%. Protein Concentration

[0048] The aqueous protein formulation or the aqueous solution of protein of the present disclosure can comprise a protein concentration from about 10±0.75 mg / mL to about 700±50.0 mg / mL, about 50 to 500±37.5 mg / mL, or 50 to 200±37.5 mg / mL any ranges of the foregoing. The aqueous protein formulation of the present disclosure can comprise a protein concentration from about 10 to 50 mg / ml, about 50 to 100 mg / ml, about 100 to 120 mg / ml, about 120 to 150 mg / ml, about 150 to 175 mg / ml, about 175 to 200 mg / ml, about 100 to 150 mg / ml, about 150 to 200 mg / ml, about 200 to 250 mg / ml, about 250 to 300 mg / ml, about 300 to 350 mg / ml, about 350 to 400 mg / ml, about 400 to 450 mg / ml, about 450 to 500 mg / ml, about 500 to 550 mg / ml, about 550 to 600 mg / ml, about 600 to 650 mg / ml, or about 650 to 700 mg / ml. Proteins can include antibodies such as monoclonal antibodies, and the like. For example, the antibody concentration in the aqueous protein formulation of the present disclosure can range from about 10 mg / ml to about 700 mg / ml, such as from about 100 to about 700 mg / ml, about 100 mg / ml to about 350 mg / ml and any range or value thereof. Several examples include about 10 mg / mL±2.5 mg / mL, about 50 mg / mL±7.5 mg / mL, about 50 mg / mL, about 100 mg / mL±15 mg / mL, about 100 mg / mL, about 150 mg / mL±22.5 mg / mL, about 150 mg / mL, about 175 mg / mL±26.25 mg / mL, about 175 mg / mL, about 200 mg / mL±30 mg / mL, about 200 mg / mL, about 250±37.5 mg / mL, about 250 mg / mL, about 300±50.0 mg / mL, or about 300 mg / mL.

[0049] The inventions are amenable to use with a wide variety of Fc-containing proteins and other proteins. The inventions can be employed in the production of biological and pharmaceutical products. For example, for antibodies, the inventions are amendable for research and production use for diagnostics and therapeutics based upon all major antibody classes, namely IgG, IgA, IgM, IgD and IgE. IgG is a preferred class, such as IgG1 (including IgG1λ and IgG1κ),IgG2 and IgG4. Exemplary antibodies to be produced according to the inventions include Alirocumab, Atoltivimab, Maftivimab, Odesivimab, Odesivivmab-ebgn, Casirivimab, Imdevimab, Cemiplimab, Cemplimab-rwlc, Dupilumab, Evinacumab, Evinacumab-dgnb, Fasimumab, Nesvacumab, Trevogrumab, Rinucumab and Sarilumab. Antibodies can include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecifϊc antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. The antibody can be an IgG1 antibody. The antibody can be an IgG2 antibody. The antibody can be an IgG3 antibody. The antibody can be an IgG4 antibody. The antibody can be a chimeric IgG2 / IgG4 antibody. The antibody can be a chimeric IgG2 / IgG1 antibody. The antibody can be a chimeric IgG2 / IgG1 / IgG4 antibody.

[0050] In addition to the antibodies described in the Examples and Figures, the antibody can be selected from the group consisting of an anti-Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0203579A1), an anti- Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody as described in in U.S. Pat. Appln. Pub. No. US2015 / 0203580A1), an anti-Dll4 antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti- Angiopoetin-Like 3 antibody (e.g. an antiAngPtl3 antibody as described in U.S. Pat. No. 9,018,356), an anti-platelet derived growth factor receptor antibody (e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti- Prolactin Receptor antibody (e.g. anti-PRLR antibody as described in U.S. Pat. No.9,302,015), an anti-Complement 5 antibody (e.g. an 25 anti- C5 antibody as described in U.S. Pat. Appln. Pub. No US2015 / 0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g. an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g. an anti- PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. Appln. Pub. No. US2014 / 0044730A1), an anti-Growth And Differentiation Factor-8 antibody (e.g. an anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g. anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015 / 0337045A1 or US2016 / 0075778A1), an anti-VEGF antibody, an anti-IL1Rantibody, an interleukin 4 receptor antibody (e.g an antiIL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014 / 0271681A1 or U.S. Pat Nos.8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g. an anti-IL6R antibody as described in U.S. Pat. Nos.7,582,298, 8,043,617 or 9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g. anti- IL33 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014 / 0271658A1 or US2014 / 0271642A1), an anti-Respiratory syncytial virus antibody (e.g. anti-RSV antibody as described in U.S. Pat. Appln. Pub. No. US2014 / 0271653A1), an anti-Cluster of differentiation 3 (e.g. an anti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos. US2014 / 0088295A1 and US20150266966A1, and in U.S. Application No. 62 / 222,605), an anti- Cluster of differentiation 20 (e.g. an anti-CD20 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014 / 0088295A1 and US20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti- Cluster of Differentiation48 (e.g. anti-CD48 antibody as described in U.S. Pat. No. 9,228,014), an anti-Fel d1 antibody (e.g. as described in U.S. Pat. No. 9,079,948), an anti- Middle East Respiratory Syndrome virus (e.g. an anti-MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0337029A1), an anti-Ebola virus antibody (e.g. as described in U.S. Pat. Appln. Pub. No. US2016 / 0215040), an anti-Zika virus antibody, an anti-Lymphocyte Activation Gene 3 antibody (e.g. an anti-LAG3 antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g. an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016 / 0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody. The bispecific antibody can be selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos. US2014 / 0088295A1 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 x anti- Muc16 bispecific antibody), and an anti-CD3 x anti- Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody). See also U.S. Patent Publication No. US 2019 / 0285580 A1.

[0051] The inventions also are amenable to the production of other molecules, including fusion proteins. Preferred fusion proteins include Receptor-Fc-fusion proteins, such as Trap proteins. the protein of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein). The Fc-fusion protein can be a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety. The Fcmoiety can comprise a hinge region followed by a CH2 and CH3 domain of an IgG. The receptor Fc-fusion protein can contain two or more distinct receptor chains that bind to either a single ligand or multiple ligands. For example, an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular region fused to Fc of hIgG1; see U.S. Pat. No. 6,927,044, or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hIgG1; see U.S. Pat. Nos.7,087,411 and 7,279,159). The Fc-fusion protein can be a ScFv-Fc-fusion protein, which contains one or more of one or more antigen binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.

[0052] Other proteins lacking Fc portions, such as recombinantly produced enzymes and mini-traps, also can be made according to the inventions. Mini-traps are trap proteins that use a multimerizing component (MC) instead of an Fc portion, and are disclosed in U.S. Patent Nos. 7,279,159 and 7,087,411. Derivatives, components, domains, chains and fragments of the above also are included. Buffers and pH

[0053] The aqueous protein formulations of the present disclosure can include pharmaceutically acceptable buffering agents. The buffering agents can include, without limitation, phosphate buffers, histidine buffers, sodium citrate buffers, HEPES buffers, Tris buffers, Bicine buffers, glycine buffers, N-glycylglycine buffers, sodium acetate buffers, sodium carbonate buffers, glycyl glycine buffers, lysine buffers, arginine buffers, sodium phosphate buffers, and / or mixtures thereof. The buffering agent can be a Histidine buffer, a Phosphate buffer (e.g., a sodium phosphate buffer) or a Tris buffer.

[0054] The aqueous protein formulation of the present disclosure can comprise about 1 to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 25 mM, about 5 mM to about 15 mM, about 7 mM to about 13 mM, or about 8 mM to about 12 mM of a buffering agent. The aqueous protein formulation can comprise about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of a buffering agent.

[0055] The aqueous protein formulations of the present disclosure can comprise about 1 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or 10 mM±1 mM Histidine buffer. The formulation can comprise about 1 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or 10 mM±1 mM Histidine buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM Histidine buffer.

[0056] The aqueous protein formulations of the present disclosure can comprise about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM±1 mM Tris buffer. The formulation can comprise about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM±1 mM sodium phosphate buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM Tris buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM sodium phosphate buffer. The formulation also can comprise 10 mM±2 mM Tris buffer. The formulation can comprise 10 mM±2 mM sodium phosphate buffer.

[0057] The aqueous protein formulation of the present disclosure has a physiologically compatible pH. The aqueous protein formulations are provided that contain a buffering agent suitable to maintain the formulation at pH between about 5.0 and about 8.0. The pH of the aqueous protein formulation of the present disclosure can be about 5.0 to about 7.0, about 5.5 to about 6.5, about 5.6 to about 7.0, about 5.7 to about 7.5, about 5.8 to about 7.0, about 5.9 to about 7.0, about 5.0 to about 7.0, about 6.5 to about 7.0, about 6.9 to about 7.0, about 7.0 to about 7.2, about 7.2 to about 7.3, about 7.3 to about 7.5, about 7.5 to about 7.7, about 7.7 to about 7.8, or about 7.8 to about 8.0. The pH of the formulation can be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2,about 7.3, about 7.4, about 7.5, about 7.7, about 7.8, about 7.8, or about 8.0. The pH of the aqueous protein formulation of the present disclosure can be about 7.5±0.1. The pH of the aqueous protein formulations of the present disclosure can be about 7.3±0.05. The pH of the aqueous protein formulation of the present disclosure can be about 7.2±0.1. The pH of the aqueous protein formulations of the present disclosure can be about 7.0±0.05. The pH of the aqueous protein formulation of the present disclosure can be about 6.0±0.1. The pH of the aqueous protein formulations of the present disclosure can be about 6.0±0.05. The pH of the aqueous protein formulation of the present disclosure can be about 5.9±0.1. The pH of the aqueous protein formulations of the present disclosure can be about 5.9±0.05. Sugars

[0058] The aqueous protein formulation of the present disclosure can include one or more sugars.

[0059] Inclusion of one of more sugars (e.g., at between about 1% to about 10%) improves the stability of the liquid formulations of the present disclosure. The aqueous protein formulation of the present disclosure can contain from about 1% to about 10% of one or more sugars. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethylcellulose can be used in the formulation. The sugar can be sucrose, trehalose, or a combination thereof.

[0060] The sugars can be used individually or in combination. The sugar, or a combination thereof, can be present in the aqueous protein formulation or composition at a concentration of about 0.10% to about 1.0% (w / v), about 0.20% to about 1.0% (w / v), about 0.30% to about 1.0% (w / v), about 0.40% to about 1.0% (w / v), about 0.50% to about 1.0% (w / v), about 0.60% to about 1.0% (w / v), about 0.70% to about 1.0% (w / v), about 0.80% to about 1.0% (w / v), about 0.90% to about 1.0% (w / v), about 1.0% to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.5% to about 7.5% (w / v), about 3% (w / v) to about 7% (w / v), or about 4% to about 6% (w / v). The formulation of the present disclosure can comprise about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), about 1.7% (w / v), about 1.8% (w / v), about 1.9% (w / v), about 2.0% (w / v), about 2.1% (w / v), about 2.2% (w / v), about 2.3% (w / v), about 2.4% (w / v), about 2.5% (w / v), about 2.6% (w / v), about 2.7% (w / v), about 2.8%(w / v), about 2.9% (w / v), about 3.0% (w / v), about 3.1% (w / v), about 3.2% (w / v), about 3.3% (w / v), about 3.4% (w / v), about 3.5% (w / v), about 3.6% (w / v), about 3.7% (w / v), about 3.8% (w / v), about 3.9% (w / v), about 4.0% (w / v), about 4.1% (w / v), about 4.2% (w / v), about 4.3% (w / v), about 4.4% (w / v), about 4.5% (w / v), about 4.6% (w / v), about 4.7% (w / v), about 4.8% (w / v), about 4.9% (w / v), about 5.0% (w / v), about 5.1% (w / v), about 5.2% (w / v), about 5.3% (w / v), about 5.4% (w / v), about 5.5% (w / v), about 5.6% (w / v), about 5.7% (w / v), about 5.8% (w / v), about 5.9% (w / v), about 6.0% (w / v), about 6.1% (w / v), about 6.2% (w / v), about 6.3% (w / v), about 6.4% (w / v), about 6.5% (w / v), about 6.6% (w / v), about 6.7% (w / v), about 6.8% (w / v), about 6.9% (w / v), about 7.0% (w / v), about 7.1% (w / v), about 7.2% (w / v), about 7.3% (w / v), about 7.4% (w / v), about 7.5% (w / v), about 7.6% (w / v), about 7.7% (w / v), about 7.8% (w / v), about 7.9% (w / v), about 8.0% (w / v), about 8.1% (w / v), about 8.2% (w / v), about 8.3% (w / v), about 8.4% (w / v), about 8.5% (w / v), about 8.6% (w / v), about 8.7% (w / v), about 8.8% (w / v), about 8.9% (w / v), about 9.0% (w / v), about 9.1% (w / v), about 9.2% (w / v), about 9.3% (w / v), about 9.4% (w / v), about 9.5% (w / v), about 9.6% (w / v), about 9.7% (w / v), about 9.8% (w / v), about 19.9% (w / v), or about 10% (w / v) sugar.

[0061] The aqueous protein formulation of the present disclosure can include about 0.10% to about 1.0% (w / v), about 0.20% to about 1.0% (w / v), about 0.30% to about 1.0% (w / v), about 0.40% to about 1.0% (w / v), about 0.50% to about 1.0% (w / v), about 0.60% to about 1.0% (w / v), about 0.70% to about 1.0% (w / v), about 0.80% to about 1.0% (w / v), about 0.90% to about 1.0% (w / v), about 1.0% to about 10% (w / v), sucrose. The aqueous protein formulation can contain about 0.5% w / v±0.1% or about 1.0% w / v±0.1% w / v sucrose. Salts

[0062] The aqueous protein formulation of the present disclosure can include one or more pharmaceutically acceptable salts.

[0063] The pharmaceutically acceptable salts can include, but are not limited to, metal salts such as sodium, potassium and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine and N-substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N′-dibenzylethylenediamine salts. Pharmaceutically acceptable salts (of basic nitrogen centers) can include, but are not limited to inorganic acid salts such as the hydrochloride, hydrobromide, sulfate, phosphate; organic acid salts such astrifluoroacetate and maleate salts; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphor sulfonate and naphthalenesulfonate; amino acid salts such as arginate, alaninate, asparginate and glutamate; and carbohydrate salts such as gluconate and galacturonate. Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sodium salts, ammonium salts, potassium salts, calcium salts, and magnesium salts (e.g., sodium, ammonium, potassium, calcium, and magnesium chloride; sodium, ammonium, potassium, calcium and magnesium acetate; sodium, ammonium, potassium, calcium and magnesium citrate; sodium, ammonium, potassium, calcium and magnesium phosphate; sodium, ammonium, potassium, calcium and magnesium fluoride; sodium, ammonium, potassium, calcium and magnesium bromide; and sodium, ammonium, potassium, calcium and magnesium iodide). The pharmaceutically acceptable salt can be sodium chloride or arginine hydrochloride (L-arginine hydrochloride).

[0064] The aqueous protein formulation of the present disclosure can comprise about 10 mM to about 300 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 100 mM to about 200 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, 150 mM to about 200 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, about 250 mM to about 300 mM, about 75 mM to about 100 mM, about 175 mM to about 200 mM, about 175 mM to about 225 mM, about 200 mM to about 225 mM, about 225 mM to about 275 mM, about 275 mM to about 300 mM, or about 175 mM to about 275 mM of a pharmaceutically acceptable salt. The protein formulation of the present disclosure can comprise about 0 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, or about 150 mM of a pharmaceutically acceptable salt. The aqueous protein formulation of the present disclosure can comprise about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM,about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, 106 mM, about 107 mM, about 108 mM, about 109 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, or about 300 mM, of a pharmaceutically acceptable salt.

[0065] The aqueous protein formulation of the present disclosure can comprise about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM of arginine hydrochloride. The aqueous protein formulation of the present disclosure can comprise 100 mM±5 mM, 110 mM±5 mM, 120 mM±5 mM, 130 mM±5 mM, 140 mM±5 mM, 150 mM±5 mM, 160 mM±5 mM, 170 mM±5 mM, 180 mM±5 mM, 190 mM±5 mM, 200 mM±5 mM, or 210 mM±5 mM arginine hydrochloride. Surfactants

[0066] The aqueous protein formulations of the present disclosure can include one or more surfactants.

[0067] The aqueous protein formulations of the present disclosure can contain a stabilizing concentration of a pharmaceutically acceptable non-ionic surfactant. Pharmaceutically acceptable non-ionic surfactants that can be used in the aqueous protein formulations discussed herein can include, without limitation, polysorbate 80 (Tween 80; PS80), polysorbate 81 (Tween 81; PS81), polysorbate 82 (Tween 82; PS82), polysorbate 20 (Tween 20; PS20), and various poloxamers (e.g., poloxamer 188), or mixtures thereof.

[0068] The aqueous protein formulations of the present disclosure can comprise from about 0.05% (w / v) to about 0.30% (w / v) non-ionic surfactant. The aqueous protein formulations can comprise about 0.05% to 0.30% (w / v), about 0.05% to 0.10% (w / v), about 0.10% to 0.15% (w / v), about 0.15% to 0.20% (w / v), about 0.20% to 0.25% (w / v), or about 0.25% to 0.30% (w / v) non-ionic surfactant. The aqueous protein formulations of the present disclosure can comprise about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.10% (w / v), about 0.11% (w / v), about 0.12% (w / v), about 0.13% (w / v), about 0.14% (w / v), about 0.15% (w / v), about 0.16% (w / v), about 0.17% (w / v), about 0.18% (w / v), about 0.19% (w / v), about 0.20% (w / v), about 0.21% (w / v), about 0.22% (w / v), about 0.23% (w / v), about 0.24% (w / v), about 0.25% (w / v), about 0.26% (w / v), about 0.27% (w / v), about 0.28% (w / v), about 0.29% (w / v), or about 0.30% (w / v) non-ionic surfactant. The aqueous protein formulations and / or the reconstitution liquid of the present disclosure can comprise 0.20% w / v±0.01% w / v non-ionic surfactant.

[0069] The aqueous protein formulations and / or the reconstitution liquid of the present disclosure can comprise about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.10% (w / v), about 0.11% (w / v), about 0.12% (w / v), about 0.13% (w / v), about 0.14% (w / v), about 0.15% (w / v), about 0.16% (w / v), about 0.17% (w / v), about 0.18% (w / v), about 0.19% (w / v), about 0.20% (w / v), about 0.21% (w / v), about 0.22% (w / v), about 0.23% (w / v), about 0.24% (w / v), about 0.25% (w / v), about 0.26% (w / v), about 0.27% (w / v), about 0.28% (w / v), about 0.29% (w / v), or about 0.30% (w / v) polysorbate 80 (PS80), polysorbate 81 (PS81), polysorbate 82 (PS82), or polysorbate 20 (PS20). The aqueous protein formulations and / or the reconstitution liquid of the present disclosure can comprise 0.20% w / v±0.01% w / v polysorbate 80 (PS80), polysorbate 81 (PS81), polysorbate 82 (PS82), or polysorbate 20 (PS20).

[0070] The aqueous protein formulations of the present disclosure can comprise about 0.05% to 0.30% (w / v), about 0.05% to 0.10% (w / v), about 0.10% to 0.15% (w / v), about 0.15% to 0.20% (w / v), about 0.20% to 0.25% (w / v), or about 0.25% to 0.30% (w / v) polysorbate 80. The pharmaceutical compositions and / or the reconstitution liquid of the present disclosure can comprise about 0.001% (w / v) to about 0.01% (w / v) poloxamer 188. The aqueous protein formulations of the present disclosure can comprise about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.10% (w / v), about 0.11% (w / v), about 0.12% (w / v), about 0.13% (w / v), about 0.14% (w / v), about 0.15% (w / v), about 0.16% (w / v), about0.17% (w / v), about 0.18% (w / v), about 0.19% (w / v), about 0.20% (w / v), about 0.21% (w / v), about 0.22% (w / v), about 0.23% (w / v), about 0.24% (w / v), about 0.25% (w / v), about 0.26% (w / v), about 0.27% (w / v), about 0.28% (w / v), about 0.29% (w / v), or about 0.30% (w / v) polysorbate 80. The aqueous protein formulations of the present disclosure can comprise about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), or about 0.01% (w / v) poloxamer 188. The aqueous protein formulations of the present disclosure can comprise about 0.005% w / v±0.001% w / v polysorbate 80 or about 0.005% w / v±0.001% w / v poloxamer 188.

[0071] The inventions are further described by the following examples, which do not limit the inventions in any manner. The order of performance of the below experiments and / or examples or example steps can be altered or combined as determined by the person of skill in the art in view of the teachings and data contained herein. EXAMPLES Experimental Approaches for developing and verifying procedures to improve mAb1 and mAb2 drug products (DP) photostability:

[0072] Headspace air bubble size in prefilled syringes (PFS) and in glass vials were measured.

[0073] Nitrogen overlay of PFS was used to replace oxygen with nitrogen in the headspace of the PFS.

[0074] Syringe filler with nitrogen gas overlay was compared with conventional filler with no nitrogen overlay. Materials:

[0075] mAb1 and mAb2 were used as representative model proteins. mAb1 was used at a concentration of 150mg / mL and mAb2 was used at a concentration of 120mg / mL Formulated Drug Substance (FDS).Methods:

[0076] mAbs were filled into 1mL long syringes / PFS (for example, 150mg / mL mAb1, 0.7mL fill and mAb2120mg / mL 1 mL fill).

[0077] The stopper locations were set to obtain headspace of 0 mm, 5 mm, 10 mm, and a maximum of about 30 mm e.g., about 0 ml, 0.1, 0.2 ml, 0.6 ml, respectively, in a 1 mL syringe.

[0078] Control vial contained 0.7mL fill in 2mL glass vial (2R vial). Only 2R vial samples were in glass vials, the rest samples were in prefilled syringes (PFS).

[0079] Nitrogen gas was overlaid (for example, on 5mm headspace in PFS) using Lyo (2 needles) and / or syringe filler.

[0080] PFS and control glass vials were exposed to 0.5 ICH CW light (represents indoor light).

[0081] Protein aggregation or high molecular weight (% HMW) species variations in the aqueous protein solution was measured using size exclusion ultra performance liquid chromatography (SE-UPLC).

[0082] Acidic species (% charge variants) were identified by cation exchange chromatography (CEX).

[0083] Protein recovery % was measured by reversed phase ultra performance liquid chromatography (RP-UPLC).

[0084] Peptide mapping (oxidation) was done by identifying and quantifying methionine oxidation.

[0085] Other physical and chemical properties of the aqueous protein solutions were determined by visual inspection, and by measuring optical density (OD) and pH. Results and Analyses:

[0086] Figures 1A and 1B are graphs showing the effect of 0.5 ICH CW light exposure on 150 mg / ml mAb1 and variations in in the formation of HMW % (Figure 1A) and Charge (acidic species %) (Figure 1B). Regarding the baseline of Figure 1B, it is notable that there were about 30% acidic species in the aqueous solution prior to the light exposure. Results show consistent trending for protein degradation for HMW formation and acidic charge variant increase. Control2R vial data shows considerable protein degradation (ΔHMW% over 2.5% in Figure 1A) for mAb1 DP. This is attributable to considerable headspace volume in the glass vial containing the mAb1 DP. For PFS, maximum bubble size (30 mm) sample showed maximum degradations and minimum bubble size (min bubble, less than 5 mm) sample showed least degradations (Figure 1B). This observation is consistent with the headspace volume variations. Results also show that nitrogen overlay further improved the photo-stability by reducing protein degradation (ΔHMW% is less than 1.5%), as evident from the graphs showing the results of comparative photo-stability of 5 mm bubble sample (with no N2 overlayed) vs N25 mm bubble (N2 overlayed) and Auto (N2 overlayed) vs N2auto (N2overlayed). “Auto” refers to the PFS being filled automatically by the syringe filler and without a nitrogen overlay. “N2 auto” refers to the PFS being filled automatically by the syringe filler and with a nitrogen overlay. “N2 Manual” refers to the PFS being filled manually with a nitrogen overlay.

[0087] Figures 2A and 2B are graphs showing the effect of 0.5 ICH CW light exposure (ICH guideline defines the dose of the light exposure. One ICH CW dose is 1.2 million lux.hour) on 150 mg / ml mAb1 at different bubble sizes (headspace size) for DPs in PFS and variations in the formation of HMW % (Figure 2A) and charge (acidic species %) (Figure 2B). Regarding the baseline of Figure 2B, it is notable that there were about 30% acidic species in the aqueous solution prior to the light exposure. For PFS, maximum bubble (30 mm) sample showed maximum degradations and minimum bubble sample (“min bubble”, less than 5 mm) showed least degradations. This observation is consistent with headspace volume variations.

[0088] Figures 3A and 3B are graphs showing the effect of 0.5 ICH CW light exposure without nitrogen overlay (replacing O2with N2) on 150 mg / ml mAb1 for DPs in PFS and variations in the formation of HMW % (Figure 3A) and charge (acidic species %) (Figure 3B). Regarding the baseline of Figure 3B, it is notable that there were about 30% acidic species in the aqueous solution prior to the light exposure. Results indicate that the replacement of headspace oxygen with nitrogen improved photo-stability of the mAb1 DP in PFS with less HMW and acidic species formation.

[0089] Figure 4 illustrates the effect of 0.5 ICH CW light exposure on the concentration of 150 mg / ml mAb1 as determined by various assays. Also, no significant changes in mAb1 protein concentration % or OD.

[0090] Figures 5A and 5B are graphs showing the effect of 0.5 ICH CW light exposure on mAb1 and methionine oxidation in DP containing DTLM[+16]ISR (Figure 5A) and DP containing WQEGNVFSCSVM[+16]HEALHNHYTQK (Figure 5B). Results show that maximum bubble size sample (30 mm bubble) showed maximum oxidation and minimum bubble sample (min bubble) showed least methionine oxidation. Results also show that nitrogen overlay further improved the photo-stability, as evident from the graphs showing the results of comparative photo-stability of N25 mm bubble (nitrogen overlayed) vs 5 mm bubble (no N2overlayed), and N2 auto (N2 overlayed) vs auto (no N2 overlayed). Formulated Drug Substance (FDS) control sample shows comparable level of oxidation compared to dark control (Figures 5A and 5B).

[0091] Figures 6A and 6B are graphs showing the effect of 0.5 ICH CW light exposure on 120 mg / ml mAb2 and variations in the formation of HMW % (Figure 6A) and charge (acidic species %) (Figure 6B). Results show consistent trending for protein degradation for HMW formation and acidic charge variant increase. Control 2R vial data shows considerable protein degradation (ΔHMW% over 9% in Figure 6A) for mAb2 DP. Results also show less protein degradation for DP in PFS than DP in glass vials. This is attributable to considerable headspace volume in the glass vial containing the mAb2 DP. In general, there are more headspace in vials than in PFS. For PFS, maximum bubble size (30 mm) sample showed maximum degradations and minimum bubble size (min bubble, no bubble) sample showed least degradations (Figure 6B). This observation is consistent with the headspace volume variations. Results also show that nitrogen overlay further improved the photo-stability, as evident from the graphs showing the results of comparative photo-stability of 5 mm bubble sample (N2 overlayed) vs N2 manual (5 mm bubble), N2auto (N2overlayed) vs auto.

[0092] Figures 7A and 7B are graphs showing the effect of 0.5 ICH CW light exposure on 120 mg / ml mAb2 at different bubble size (headspace size) for DPs in PFS and variations in the formation of HMW % (Figure 7A) and charge (acidic species %) (Figure 7B). For PFS, maximum bubble (30 mm) sample showed maximum degradations and minimum bubble sample (“min bubble”, less than 5 mm) showed least degradations. This observation is consistent with headspace volume variations.

[0093] Figures 8A and 8B are graphs showing the effect of 0.5 ICH CW light exposure without nitrogen overlay (replacing headspace O2with N2) on 120 mg / ml mAb2 for DPs in PFSand variations in the formation of HMW % (Figure 8A) and charge (acidic species %) (Figure 8B). Results indicate that the replacement of headspace oxygen with nitrogen improved photo- stability of the mAb2 DP in PFS with less HMW and acidic species formation, , as evident from the graphs showing the results of comparative photo-stability of degradation levels 5 mm bubble sample (N2 overlayed) vs N2 manual (5 mm bubble), N2 auto (N2 overlayed) vs auto.

[0094] Figure 9 illustrates the effect of 0.5 ICH CW light exposure on the recovery % of 120 mg / ml mAb2 as determined by various assays. Also, no significant changes in mAb2 protein concentration % or OD.

[0095] Figures 10A and 10B are graphs showing the effect of 0.5 ICH CW light exposure on mAb2 and methionine oxidation in DP containing DTLMISR (Figure 10A) and DP containing WQQGNVFSCSVMHEALHNHYTQK (Figure 10B). Oxidation of methionine was observed for photo exposed samples. Control 2R vial data shows considerable protein oxidation (over 40% in Figures 10A and 10B) for mAb2 DP. For PFS, results show that maximum bubble size sample (30 mm bubble) showed maximum oxidation and minimum bubble sample (no bubble) showed least methionine oxidation. Results also show that nitrogen overlay further improved the photo- stability, as evident from the graphs showing the results of comparative photo-stability of N2manual (nitrogen overlayed) vs 5 mm bubble sample (no N2overlayed), and N2auto (N2overlayed) vs auto (no N2 overlayed) (Figures 10A and 10B).

[0096] Overall, it is evident from the inventions that proteins are less light sensitivities in PFS than in glass vials. Photo-stability of DPs can be improved by reducing the headspace volume in PFS. Further, replacement of the headspace air (oxygen) with nitrogen can improve photo- stability of the DP in PFS.

[0097] Figure 11 is illustrating UV Chromatograms (F8, F9, F10, F11, and F12) of mAb2 exposed under CW light.

[0098] Figure 12 is illustrating UV Chromatograms (F13, F14, F15, F17, and F19) of mAb2 exposed under CW light.

[0099] Figure 13 is illustrating UV Chromatograms (F8, F9, F10, F11, F12, F13, F14, F15, F17, and F19) of mAb2 exposed under CW light. Post Translational Modification (PTM):

[0100] Increased levels of oxidation were observed on HC-Met256 and HC- Met432 after photo stress compared to the Formulated Drug Substance (FDS) and dark control samples (Table 1). All other PTMs, including Asn deamidation, Asp isomerization and cyclization, HC N-term pyroglutamate, Lys glycation, HC C-term Lys and amidation did not display significant changes before and after photo stress.

[0101] The Met oxidation level increased with increase of the size of air bubble and decreased when the sample was purged with nitrogen or automated filling / stoppering systems were used either with or without nitrogen purge. The overall trend of oxidation level change is comparable to the trends from SEC and CEX chromatograms.Table 1. Summary of the site specific PTMs of mAb2 with CW light. SiteaPTM percentagebPTM Name Location Peptide Sequence F8 F9 F10 F11 F12 F13 F14 F15 F17 F19 % .5% .0% % .0% .0% .0% .0% .0% % .0% .0% % .0% %peptide. F8: 2R vial; F9: no bubble; F10: 5mm bubble; F11: 10mm bubble; F12: max bubble; F13: N2 manual; F14: Auto; F15: N2 Auto; F17: FDS; F19: dark control.Table 2. Oxidation of mAb1 with CW light. DTLM[+16]ISR WQEGNVFSCSVM[+16]HEALHNHYTQKL23-007325_F1_min bubble15.8% 7.0%

[0102] Table 2 data indicates maximum bubble sample (F4) resulting in maximum oxidation and min bubble sample showing the least oxidation. Sample were digested 3 times and ran on multiple LC-MS instruments and the results consistently showed around 17-18% for oxidation of HC Met257 in DTLMISR peptide. Nitrogen overlay further improved the photo- stability, as evident from the data showing the oxidation level close to F5 (N2) vs F2, F7 (N2) vs F6.

[0103] Formulated Drug Substance (FDS) control sample (F16) showing comparable level of oxidation compared to dark control (F18).

[0104] It is to be understood that the description, specific examples and data are given by way of illustration and are not intended to limit the present inventions. Various changes and modifications within the present inventions, including combining teaching in whole and in part, will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the inventions.

Claims

WHAT IS CLAIMED IS 1. A method of improving photo-stability of a protein, wherein the method comprises the steps of a) providing an aqueous solution comprising a protein in a closed container, wherein the closed container further comprises a bubble; b) reducing the headspace of the closed container, thereby reducing the size of the bubble and the headspace air in the container to 0.6 ml or less; and c) overlaying inert gas on to the headspace air in the closed container; wherein the inert gas replaces the bubble in the headspace to a level to provide an oxygen below 21%, thereby reducing oxygen content in the headspace air, and thereby decrease photo-degradation by at least one selected from the group consisting of: i) reducing oxidation of the protein, ii) reducing formation of acidic species, and / or iii) reducing formation of high molecular weight (HMW) aggregates / species.

2. The method according to claim 1, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

3. The method according to claim 1, wherein the formation of HMW aggregates decreases with inert gas overlaying and decreasing size of the bubble.

4. The method according to claim 1, wherein the formation of acidic species decreases with inert gas overlaying and decreasing size of the bubble.

5. The method according to claim 1, wherein the oxidation of the protein decreases with inert gas overlaying and decreasing size of the bubble.

6. The method according to claim 5, wherein the oxidation of the protein is resulting from the oxidation of methionine residue of the protein.

7. The method according to claim 1, wherein the photo-stability of the protein is determined by exposing the aqueous solution under 0.5 ICH CW light and analysis of: (a) the HMW aggregates by size exclusion ultra performance liquid chromatography (SE-UPLC), (b) acidic species (charge variants) by cation exchange chromatography (CEX), (c) the protein recovery by reversed phase ultra performance liquid chromatography (RP-UPLC), (d) peptide mapping by identifying and quantifying methionine oxidation, (e) optical density (OD), and / or (f) pH.

8. The method according to claim 1, wherein the container is a vial or a prefilled syringe (PFS).

9. The method according to claim 8, wherein the PFS is configured to hold a volume of from about 1.0 ml to 50.0 ml of the aqueous solution.

10. The method according to claim 8, wherein the PFS holds a volume of about 1.0 ml, wherein the bubble size is about 5 mm, or less.

11. The method according to claim 1, wherein the method controls the formation of the HMW species.

12. The method according to claim 1, wherein the inert gas is selected from the group consisting of: argon, nitrogen, helium, and any mixtures thereof.

13. The method according to claim 1, wherein a concentration of the protein in the aqueous solution is from 100 mg / ml to about 700 mg / ml.

14. The method according to claim 1, wherein the overlaying of the inert gas is carried out by a replacement method, including purge, partial vacuum and refill, blow the inert gas or blanket the inert gas.

15. A method of preventing photo-degradation of a protein in prefilled syringe (PFS), wherein the method comprises the steps of (a) providing an aqueous solution of a protein in a prefilled syringe (PFS); (b) reducing the headspace size of the PFS, thereby reducing the size of the bubble and the headspace air in the PFS to 30 mm or less; and(c) overlaying inert gas on to the headspace air in the PFS; wherein the inert gas replaces oxygen content in the headspace air to a level that is below 21%, thereby reducing oxygen content in the headspace air, and wherein the reduced oxygen content and the reduced bubble size decreases photo-degradation by: i) reducing oxidation of the protein, ii) reducing formation of acidic species, and / or iii) reducing formation of high molecular weight (HMW) aggregates / species, thereby preventing photo-degradation of the protein in the PFS.

16. The method according to claim 15, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

17. The method according to claim 15, wherein the formation of HMW aggregates decreases with inert gas overlaying and decreasing size of the bubble.

18. The method according to claim 15, wherein the formation of acidic species decreases with inert gas overlaying and decreasing size of the bubble.

19. The method according to claim 15, wherein the oxidation of the protein decreases with inert gas overlaying and decreasing size of the bubble.

20. The method according to claim 19, wherein the oxidation of the protein is resulting from the oxidation of methionine residue of the protein.

21. The method according to claim 15, wherein the photo-degradation of the protein is determined by exposing the aqueous solution under 0.5 ICH CW light and analysis of: (a) the HMW aggregates by size exclusion ultra performance liquid chromatography (SE-UPLC), (b) acidic species (charge variants) by cation exchange chromatography (CEX), (c) the protein recovery by reversed phase ultra performance liquid chromatography (RP-UPLC), (d) peptide mapping by identifying and quantifying methionine oxidation, (e) optical density (OD), and / or(f) pH.

22. The method according to claim 15, wherein the PFS is configured to hold a volume of from about 1.0 ml to 50.0 ml of the aqueous solution.

23. The method according to claim 22, wherein the PFS has a volume of about 1.0 ml, wherein the bubble size is about 5 mm, or less.

24. The method according to claim 15, wherein the inert gas is selected from the group consisting of: argon, nitrogen, helium, and any mixtures thereof.

25. The method according to claim 15, wherein a concentration of the protein in the aqueous solution is from 100 mg / ml to about 350 mg / ml.

26. The method according to claim 15, wherein the overlaying of the inert gas is carried out by a replacement method, including purge, partial vacuum and refill, blow the inert gas or blanket the inert gas.

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