Antibody formulations

Abstract

The present invention relates to pharmaceutical formulations of pharmaceutically active antigen binding proteins, such as monoclonal antibodies. The formulations comprise, in addition to the antigen binding protein, a buffer and a tonicity agent.

Description

Background of the Invention

[0002] This invention relates to pharmaceutical formulations of active antigen-binding proteins (e.g., monoclonal antibodies). In addition to the antigen-binding protein, the formulation also contains a buffer and a tensile agent.

[0003] The pharmaceutical use of antibodies has increased in recent years. In many cases, the antibody is administered intravenously (IV). Unfortunately, the amount of antibody that can be administered intravenously is limited by the antibody's physicochemical properties, particularly its solubility and stability in suitable liquid formulations and the volume of the infusion fluid. Other routes of administration are subcutaneous or intramuscular injection, which offer potential benefits in terms of patient compliance and ease of administration. These routes require a high protein concentration in the final solution to be injected.

[0004] Therefore, there is a need for stable subcutaneous formulations with high concentrations of therapeutically active antigen-binding proteins (such as antibodies for subcutaneous injection). The advantage of subcutaneous injection is that it allows for relatively short physician intervention during administration. Furthermore, patients can be trained to administer subcutaneous injections themselves. This self-administration is particularly useful during maintenance therapy, as it eliminates the need for hospitalization (reducing the use of medical resources). Subcutaneous injections are typically limited to approximately 2 mL. For patients requiring multiple doses, several unit doses can be injected at multiple sites on the body surface. Invention Overview

[0006] In one aspect, the present invention provides a pharmaceutical formulation of an antigen-binding protein comprising a buffer and a tensor. More specifically, the present invention provides an antigen-binding protein of about 150 to 250 mg / mL; a buffer of about 1 to 100 mM to provide a pH of about 5.0 to about 7.0; and a tensor of about 70 to 170 mM. In one embodiment, the antigen-binding protein is an anti-BLyS antibody.

[0007] In another aspect, the present invention provides a pharmaceutical formulation of an antigen-binding protein comprising a buffer, a stabilizer, a tensioning agent, and a nonionic surfactant. More specifically, the present invention provides a pharmaceutical formulation comprising an antigen-binding protein, histidine, arginine, NaCl, and polysorbate 80. In one embodiment, the antigen-binding protein is an anti-BLys antibody.

[0008] In another aspect, the present invention provides a method for treating a disease or condition in a subject, said disease or condition being compliant with treatment of an anti-BLyS antibody, said method comprising administering the formulation of the present invention to the subject in an amount effective for treating said disease or condition. In one aspect, said disease or condition is an autoimmune disease or disorder.

[0009] In another aspect, the present invention provides a kit comprising one or more vials containing the formulation of the present invention and instructions for administering the formulation subcutaneously to a patient.

[0010] In another aspect, the present invention provides an injection device comprising the stable anti-BLys antibody formulation described in this application.

[0011] On the other hand, the present invention provides an agent according to the invention for treating diseases selected from: systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis, lupus nephritis, primary Sjögren's syndrome. Syndrome), chronic immune thrombocytopenic purpura, myasthenia gravis, symptomatic Waldenström macroglobulinemia (symptomatic) (macroglobulinaemia), immune desensitization in patients awaiting kidney transplantation, membranous nephropathy, systemic sclerosis, rheumatoid arthritis, multiple myeloma, multiple sclerosis, and kidney failure. Attached Figure Description

[0012] Figure 1 The effect of protein concentration on the aggregation rate of formulation 1 was shown.

[0013] Figure 2 It shows that it should be stored at 2-8℃ for 5 days. 1 / Turbidity of formulations 1 and 5 after 4 months.

[0014] Figure 3 The relationship between viscosity and concentration of belimumab was shown.

[0015] Figure 4 The changes in the percentage of aggregation (%) of different formulations after storage at 2-8°C for 3 months are shown.

[0016] Figure 5 The changes in the percentage of aggregation (%) of different formulations after 3 months of storage at different temperatures are shown.

[0017] Figure 6 It shows that it can be stored at a maximum of 25°C for 5 days. 1 The effect of temperature on the aggregation rate after 4 months was investigated, and it was shown that the arginine formulation (hollow square in the figure) significantly reduced aggregation compared to formulation 1 (filled square).

[0018] Figure 7 Formulation 1 (solid cube; 06-C) and Formulation 5 (hollow cube; 06-D) at concentrations of 125 to 200 mg / mL are shown for storage at -80°C to 40°C. 1The aggregation rate after 4 months, and formulation 5 (dashed line), consistently showed a lower aggregation rate than formulation 1 (solid line).

[0019] Figure 8 Formulations 1 (06-C) and 5 (06-D) with concentrations ranging from 125 to 200 mg / mL were shown for storage at -80°C to 40°C. 1 / The CGE degradation rate decreased after 4 months.

[0020] Figure 9 Formulations 1 (solid cube; 06-C) and 5 (hollow cube; 06-D) with concentrations ranging from 125 to 200 mg / mL were shown for storage at -80°C to 40°C. 1 / Acidic rate after 4 months.

[0021] Figure 10 Formulations 1 and 5, with concentrations ranging from 125 to 200 mg / mL, are shown to be stored at -80°C to 40°C. 1 / Belimumab heavy chain oxidation level 4 months later.

[0022] Figure 11 The 200 mg / mL formulation was shown in storage 5. 1 / Belimab peptide profile 4 months later.

[0023] Figure 12 The 200 mg / mL formulation is shown in Figure 5. Storage 5 1 / Belimab peptide profile 4 months later.

[0024] Figure 13 The peptide profiles of belimumab samples with different arginine levels are shown.

[0025] Figure 14 The HTF pH-buffer screening is shown.

[0026] Figure 15 The interaction between two factors—pH x buffer–SEC monomer—was shown.

[0027] Figure 16 The interaction between two factors—pH x buffer—is shown, along with the cIEF main peak (cIEF main).

[0028] Figure 17 The viscosity of different concentrations of anti-IL13 antibody is shown.

[0029] Figure 18 The results show the viscosity (cP) of the anti-IL13T=0 sample compared to the concentration (mg / mL) in the vibration study.

[0030] Figure 19As shown, the 7-day acetate sample was gelled (left). No gelation was observed in the succinate or histidine samples (middle and right). The 10-day succinate vial was observed to be in a semi-gel state.

[0031] Figure 20 The comparison of near-UV circular dichroism spectroscopy of samples with chemical stability after 3 months is shown. Invention Details

[0033] It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Unless otherwise expressly stated, the singular forms "a," "an," and "the" used in this specification and the appended claims include plural references. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides, etc.

[0034] The term “about” as used in this application, when referring to measurable values ​​(such as quantity, duration, etc.), is intended to cover variations of ±20% or ±10% relative to a specific value, including ±5%, ±1%, and ±0.1%, because such variations are suitable for carrying out the disclosed methods.

[0035] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or identical to those disclosed in this application may be used in the practice of testing the invention, preferred materials and methods are described herein. The following terms will be used in describing and claiming the rights to the invention.

[0036] In one aspect, the present invention provides a pharmaceutical formulation of an antigen-binding protein comprising a buffer and a tensile agent. In another aspect, the present invention provides a pharmaceutical formulation of an antigen-binding protein comprising a buffer, a stabilizer, a tensile agent, and a nonionic surfactant. In one embodiment, the formulation is lyophilized or spray-dried. In some embodiments, the formulation is lyophilized or spray-dried and subsequently reconstituted with a dispersant. In one embodiment, the dispersant is sterile water or "water for injection" (WFI). The antigen-binding protein may be further diluted with isotonic saline or other excipients to produce a desired concentration prior to administration. In one embodiment, the formulation is a reconstituted formulation. In another embodiment, the formulation is a liquid pharmaceutical formulation.

[0037] The term "pharmaceutical preparation" or "preparation" refers to a product in a form that enables the bioactivity of an active ingredient to be effective and that contains no other ingredients that would have unacceptable toxicity to a subject to which the preparation is administered. The preparation is sterile.

[0038] "Sterile" preparations are sterile or do not contain any live microorganisms or their spores.

[0039] In an exemplary embodiment of the invention, the liquid formulation exhibits desired properties, such as desired viscosity and surface tension properties.

[0040] The term "surface tension" refers to the attractive force exerted by molecules below a surface on the molecules at the surface / air interface. It arises from the higher molecular concentration of liquids compared to the lower molecular concentration of gases. Liquids with low surface tension values, such as nonpolar liquids, flow more easily than water. Surface tension values ​​are typically expressed in Newtons per meter (N / m) or dynes per centimeter (dynes / cm).

[0041] The "dynamic surface tension" referred to in this application refers to the dynamic interfacial tension at the surface / air interface and at the surface / surface interface. Various methods exist for measuring dynamic surface tension, such as captive bubble surface tensionometry or pulsating bubble surface tensionometry.

[0042] The term "viscosity" refers to the internal resistance a fluid exhibits to flow at a given temperature; it is the ratio of shear stress to shear rate. If a force of 1 dyne / cm² causes two parallel liquid surfaces, each with an area of ​​1 cm² and separated by 1 cm², to move relative to each other at a speed of 1 cm / s, then the liquid has a viscosity of 1 poise. 1 poise equals 100 centipoise.

[0043] In one embodiment, the viscosity of the formulation containing the buffer and stabilizer is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% compared to the viscosity of the formulation without the buffer and stabilizer. In one embodiment, the viscosity of the formulation containing the buffer and stabilizer is less than about 50 cP, less than about 45 cP, less than about 40 cP, less than about 35 cP, less than about 30 cP, less than about 25 cP, less than about 20 cP, less than about 15 cP, or less than about 10 cP.

[0044] When discussing apparent viscosity, it's important to understand that the numerical value depends on the conditions under which the measurement is performed, such as the temperature, shear rate, and shear stress. Apparent viscosity is defined as the ratio of shear stress to the applied shear rate. Several methods exist for measuring apparent viscosity. For example, viscosity can be measured using a suitable cone and plate, parallel plate, or other types of viscometers or rheometers.

[0045] “Gelification” is defined as the process of forming a hard network, presumably resulting from topological overlap between polymeric MAb or filaments, as well as crosslinking and bundling of these filaments. This hard network is characterized by a solution elastic modulus (G′) and an increased intrinsic elastic modulus (G″).

[0046] In one aspect, the present invention relates to a method for reducing or inhibiting gelation of a solution, comprising using the formulation of the present invention. In another aspect, the present invention relates to a method for reducing or inhibiting gelation of a solution containing a therapeutic protein, the method comprising applying histidine and sodium chloride to the solution.

[0047] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. Polypeptides may be derived from natural sources (derived from tissues), recombinant or naturally expressed from prokaryotic or eukaryotic cell preparations, or chemically prepared by synthetic methods. The term applies to amino acid polymers (where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids) as well as to both naturally occurring and non-naturally occurring amino acid polymers. An amino acid mimic is a chemical compound that has a structure different from the general chemical structure of an amino acid but functions similarly to a naturally occurring amino acid. Non-natural residues are described in detail in scientific and patent literature; several exemplary non-natural compositions that can be used as mimics of natural amino acid residues, along with guiding principles, are described below. Aromatic amino acid analogs can be generated by substitution with compounds such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thieneylalanine; D- or L-1,-2,3- or 4-pyreneylalanine; D- or L-3-thieneylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-( (Trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylalanine (ainine), wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid. Aromatic rings of non-natural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrroleyl, and pyridyl aromatic rings.

[0048] As used in this application, "peptide" includes peptides that are conserved variants of those peptides specifically exemplified herein. A "conserved variant" as used herein refers to a peptide in which an amino acid residue is replaced by another biologically similar residue. Examples of conserved variants include, but are not limited to, replacing one hydrophobic residue with a hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, or methionine; or replacing one polar residue with another polar residue, such as replacing lysine with arginine, aspartic acid with glutamic acid, or asparagine with glutamine. Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine, and threonine. "Conserved variants" also include the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that an antibody against the substituted peptide also reacts immunologically with the unsubstituted peptide. Such conserved substitutes fall within the definition of the peptide category of this invention. The biological activity of the peptides can be determined by standard methods known to those skilled in the art and by the methods described herein.

[0049] When the term "recombinant" is used to describe a protein, it means that the protein has been modified by introducing a foreign nucleic acid or protein or by altering the native nucleic acid or protein.

[0050] As used herein, “therapeutic protein” refers to any protein and / or polypeptide that, for example, is sought by researchers or clinicians and can be administered to mammals to elicit a biological or medical response in tissues, systems, animals, or humans. Therapeutic proteins can elicit more than one biological or medical response. Furthermore, the term “therapeuticly effective amount” refers to any amount that, compared to a corresponding subject who did not receive that amount, results in (but is not limited to) cure, prevention, or reduction of disease, disorder, or side effects, or a slower rate of disease or disorder progression. Within its scope, the term also includes amounts that effectively enhance normal physiological function and amounts that effectively induce physiological functions in patients that enhance or contribute to the therapeutic effect of a second drug.

[0051] All amino acid residues identified in this application are in the native L-configuration. In accordance with standard polypeptide nomenclature, the abbreviations of the amino acid residues are shown in the table below.

[0052] Table 1. Abbreviations for amino acids.

[0053]

[0054]

[0055] It should be noted that all amino acid residue sequences are represented by a general formula in this application, and their direction from left to right is consistent with the conventional direction from the amino terminus to the carboxyl terminus.

[0056] In another embodiment, the polypeptide is an antigen-binding protein. In one embodiment, the antigen-binding protein is selected from soluble receptors, antibodies, antibody fragments, immunoglobulin single variable domains, Fab, F(ab')2, Fv, disulfide-linked Fv, scFv, closed conformation multispecific antibodies, disulfide-linked scFv, or bispecific antibodies.

[0057] As used in this application, the term "antigen-binding protein" refers to antibodies, antibody fragments, and other protein structures that can bind to antigens.

[0058] The terms Fv, Fc, Fd, Fab, or F(ab)2 are used with their standard meanings (see, for example, Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).

[0059] "Chimeric antibody" refers to a type of engineered antibody that contains naturally occurring variable regions (light and heavy chains) derived from the donor antibody and constant regions of the light and heavy chains derived from the recipient antibody.

[0060] "Humanized antibody" refers to a type of engineered antibody whose CDR is derived from a non-human donor immunoglobulin, and whose other immunoglobulin-derived portions are derived from one (or more) human immunoglobulins. Furthermore, framework support residues can be modified to maintain binding affinity (see, for example, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio / Technology, 9:421 (1991)). Suitable human receptor antibodies can be selected from a standard database, such as KABAT, based on homology to the donor antibody's nucleotide and amino acid sequences. TM Databases, the Los Alamos database, and the Swiss Protein database. Human antibodies characterized (based on amino acid homology with the framework region of the donor antibody) are suitable for providing heavy chain constant regions and / or heavy chain variable framework regions for insertion into the donor CDR. Suitable receptor antibodies capable of contributing light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the receptor antibody heavy chain and receptor antibody light chain do not necessarily originate from the same receptor antibody. Several methods for generating this humanized antibody are described in the prior art—see, for example, EP-A-0239400 and EP-A-054951.

[0061] The term "donor antibody" refers to a (monoclonal and / or recombinant) antibody that provides the amino acid sequence of its variable region, CDR, or other functional fragment or analogue to a first immunoglobulin chaperone, thereby providing an altered immunoglobulin coding region and thus enabling the expression of an altered antibody with antigen specificity and the neutralizing activity characteristics of the donor antibody.

[0062] The term "receptor antibody" refers to a (monoclonal and / or recombinant) antibody that is heterologous to a donor antibody, providing the first immunoglobulin chaperone with the complete amino acid sequence (or any portion thereof, but in some embodiments, the complete amino acid sequence) encoding its heavy and / or light chain framework region and / or its heavy and / or light chain constant region. In some embodiments, the human antibody is the receptor antibody.

[0063] "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody, which is a hypervariable region of the heavy and light chains of an immunoglobulin. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable region of an immunoglobulin. Therefore, as used herein, "CDR" refers to all three heavy chain CDRs or all three light chain CDRs (or all heavy chain CDRs and all light chain CDRs, if appropriate). The structure and protein folding of an antibody may imply that other residues are considered part of the antigen-binding region, and this will be understood by those skilled in the art. See, for example, Chothia et al., (1989) Conformations of Immunoglobulin Hypervariable Regions; Nature 342, pp. 877-883.

[0064] As used in this application, the term "domain" refers to a folded protein structure whose tertiary structure is independent of the rest of the protein. Typically, domains are responsible for different functional properties of a protein and can be added, removed, or transferred to other proteins in many cases without loss of function of the protein and / or other parts of the domain. "Antibody single variable domain" is a folded polypeptide domain containing the characteristic sequence of an antibody variable domain. Therefore, it includes complete antibody variable domains and modified variable domains (e.g., where one or more loop regions are replaced with sequences that do not possess antibody variable domain characteristics) or includes truncated antibody variable domains or those containing N-terminal or C-terminal extensions, as well as folded fragments including variable domains that at least retain the binding activity and specificity of the full-length domain.

[0065] The phrase "immunoglobulin single variable domain" refers to an antibody variable domain (V domain) that binds specifically to an antigen or epitope, independent of its respective V region or V domain. H V HH V L Immunoglobulin single variable domains can exist in the form of other different variable regions or domains (e.g., homo- or hetero-multimers) where the other regions or domains are not essential for antigen binding to a single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds the antigen independently of additional variable domains). The terms “domain antibody” or “dAb” as used herein are the same as “immunoglobulin single variable domain” capable of binding to an antigen. Immunoglobulin single variable domains can be human antibody variable domains, but also include single antibody variable domains from other species, such as rodents (e.g., those disclosed in WO00 / 29004), wrasses, and camelids. HH dAb (nanobody). Camelidae V HH This is an immunoglobulin monovariable domain polypeptide derived from species including camels, llamas, alpacas, dromedary camels, and guanacos, which produces naturally occurring heavy chain antibodies that do not contain light chains. This V HH The domain may be humanized according to existing techniques in the art, and the domain is still considered a "domain antibody" of this invention. The "V" used in this application... H "Including Camelidae V" HH Domains. NARV is another type of immunoglobulin single variable domain, which has been identified in cartilaginous fish, including the harpooned shark. These domains are also known as neoantigen receptor variable regions (often abbreviated as V(NAR) or NARV). For further details, see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.

[0066] The term "epitope-binding domain" refers to a structural domain that specifically binds to an antigen or epitope, independent of different V regions or V domains. It can be a domain antibody (dAb), such as a single variable domain of human, camel, or shark immunoglobulins.

[0067] As used in this application, the term "antigen binding site" refers to a site on a protein that can specifically bind an antigen. This site can be a single domain, such as an epitope binding domain, or a paired V-shaped domain. H / V L Structural domains, as can be found on standard antibodies. In some aspects of the invention, a single-chain Fv (ScFv) domain can provide an antigen-binding site.

[0068] This application uses the terms "mAbdAb" and "dAbmAb" to refer to the antigen-binding protein of the present invention. These two terms are used interchangeably and are intended to have the same meaning as used in this application.

[0069] The pharmaceutical formulation of the present invention provides: about 150 to 250 mg / mL of antigen-binding protein; about 1 to 100 mM of buffer for providing a pH of about 5.0 to about 7.0; and about 70 to 170 mM of tensioning agent. Alternatively, the pharmaceutical formulation of the present invention provides: about 150 to 250 mg / mL of antigen-binding protein; about 1 to 100 mM of buffer for providing a pH of 6.0 ± 0.5; about 1 to 100 mM of stabilizer; about 90 to 150 mM of tensioning agent; and about 0.005 to 0.015% (w / v) of nonionic surfactant. In one embodiment, the antigen-binding protein is an anti-B lymphocyte stimulator (anti-BLyS) protein antibody.

[0070] The pharmaceutical formulation is also described as comprising: about 150 to 250 mg / mL of an antigen-binding protein; about 1 to 100 mM of histidine at a pH of 6.0 ± 0.5; and about 70 to 170 mM of NaCl. In one embodiment, the formulation further comprises about 0.005 to 0.03% (w / v) of a nonionic surfactant. In one embodiment, the formulation further comprises about 0.01 to about 0.1 mM of a metal chelating agent. In one embodiment, the antigen-binding protein is an anti-IL-13 antibody.

[0071] The pharmaceutical formulation of the present invention may be provided in liquid form or in lyophilized form.

[0072] The pharmaceutical formulation of the present invention comprises a buffer. The buffer includes, but is not limited to, citric acid, HEPES, histidine, potassium acetate, potassium citrate, potassium phosphate (KH₂PO₄), sodium acetate, sodium bicarbonate, sodium citrate, sodium phosphate (NaH₂PO₄), Tris base, and Tris–HCl. In one embodiment, the buffer is histidine. In embodiments, the histidine concentration is about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In one embodiment, the histidine concentration is 10 ± 5 mM. In one embodiment, the histidine concentration is 10 ± 2 mM. In one embodiment, the histidine concentration is about 10 mM. In one embodiment, the histidine concentration is about 15 mM.

[0073] As used herein, the term "buffer providing a pH of about 5.0 to about 7.0" refers to a reagent that, through the action of its acid / base conjugate components, enables a solution containing the reagent to resist pH changes. The buffer used in the formulations of this invention may have a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.8 to about 6.2. In one embodiment, the pH is about 6.0. In another embodiment, the pH is about 6.250. Examples of buffers that control the pH within this range include acetates, succinates, gluconates, histidine, citrates, diglycinate, and other organic acid buffers. According to the invention, the most suitable buffer is a histidine (e.g., L-histidine) buffer.

[0074] "Histidine buffer" is a buffer containing the amino acid histidine. Examples of histidine buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate. The most suitable histidine formulation identified in the examples is a histidine buffer made from 0.65 mg / mL L-histidine and 1.2 mg / mL L-histidine monohydrochloride.

[0075] The pharmaceutical formulation of the present invention comprises a tensile agent. The tensile agent includes, but is not limited to, dextran, glycerol, mannitol, potassium chloride, and sodium chloride. In one embodiment, the tensile agent is sodium chloride. In one embodiment, the sodium chloride concentration is about 70 to 170 mM; about 90-150 mM; or about 115 ± 10 mM. In some embodiments, the sodium chloride concentration is about 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, or 175 mM. In one embodiment, the sodium chloride concentration is about 115 mM. In another embodiment, the sodium chloride concentration is 150 ± 10 mM. In one embodiment, the sodium chloride concentration is approximately 150 mM.

[0076] "Isotonic" means that the preparation has an osmotic pressure that is essentially the same as that of human blood. Isotonic preparations generally have an osmotic pressure of approximately 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmoremeter.

[0077] In some embodiments, the pharmaceutical formulation of the present invention comprises a stabilizer. Stabilizers include, but are not limited to, human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Stabilizers also include amino acids and their metabolites, such as glycine, alanine (α-alanine, β-alanine), arginine, betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines (alanine, strombine, and trimethylamine N-oxide (TMAO)). In one embodiment, the stabilizer is an amino acid. In one embodiment, the amino acid is arginine. In one embodiment, the arginine concentration is about 20 to 30 mM. In one embodiment, the arginine concentration is about 25 ± 2 mM.

[0078] In some embodiments, the pharmaceutical formulation of the present invention comprises a nonionic surfactant. Nonionic surfactants include, but are not limited to, polyoxyethylene dehydrated sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). In one embodiment, the nonionic surfactant is polysorbate 80. In one embodiment, the concentration of polysorbate 80 is about 0.005 to 0.02% (w / v). In one embodiment, the concentration of polysorbate 80 is about 0.01% (w / v). In one embodiment, the concentration of polysorbate 80 is about 0.02% (w / v).

[0079] In some embodiments, the pharmaceutical formulation of the present invention comprises a metal chelating agent. The metal chelating agent includes, but is not limited to, EDTA and EGTA. In one embodiment, the metal chelating agent is EDTA. In one embodiment, the concentration of EDTA is about 0.01 to about 0.02 mM. In one embodiment, the concentration of EDTA is about 0.05 mM.

[0080] In one embodiment, the antigen-binding protein is a monoclonal antibody or a fragment thereof. In one embodiment, the monoclonal antibody or fragment thereof is mouse, chimeric, humanized, or wholly human. In one embodiment, the monoclonal antibody or fragment thereof binds to BLys or IL-13.

[0081] In one aspect, the formulation comprises an antigen-binding protein, histidine, arginine, NaCl, and polysorbate 80. In another aspect, the formulation comprises: approximately 200 mg / mL of antigen-binding protein, approximately 10 mM of histidine, approximately 25 mM of arginine, approximately 115 mM of NaCl, and approximately 0.01% of polysorbate 80, at a pH of approximately 6.0. In one embodiment, the antigen-binding protein binds to BLys.

[0082] In one embodiment, the pharmaceutical formulation of the present invention provides: about 200 mg / mL of antigen-binding protein; about 15 mM of histidine at a pH of about 6.25; about 150 mM of NaCl; about 0.02% (w / v) polysorbate 80; and about 0.05 mM EDTA. In one embodiment, the antigen-binding protein binds to IL-13.

[0083] In one aspect, the pharmaceutical formulation of the present invention is stable during freezing and thawing. A "stable" formulation is one in which virtually all proteins retain their physical and / or chemical stability and / or biological activity when stored at the intended storage temperature (e.g., 2-8°C). It is desirable for the formulation to substantially retain its physical and chemical stability and its biological activity during storage. The storage time is typically selected based on the intended shelf life of the formulation. Furthermore, the formulation should be stable after freezing (e.g., to -70°C) and thawing, for example, after 1, 2, or 3 freeze-thaw cycles. Various analytical techniques for measuring protein stability exist in the art, which are reviewed in references such as: Peptide and Protein Drug Delivery, 247-301; Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991); and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at selected temperatures and within selected time periods. Stability can be assessed qualitatively and / or quantitatively in various ways, including: assessing aggregation formation (e.g., using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography or capillary band electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis for comparing degraded and intact antibodies; peptide profiling (e.g., trypsin or LYS-C) analysis; assessing antibody bioactivity or antigen-binding function; etc.

[0084] In one embodiment, the pharmaceutical formulation of the present invention is suitable for subcutaneous or intramuscular administration.

[0085] The "identity percentage" between the query amino acid sequence and the subject amino acid sequence is a numerical value calculated by the BLASTP algorithm after a pairwise BLASTP alignment, where the subject amino acid sequence has 100% query coverage with the query amino acid sequence, expressed as a percentage. This pairwise BLASTP alignment between the query and subject amino acid sequences is performed using the default settings of the BLASTP algorithm, which is available from the website of the National Biotechnology Center, with filtering for low-complexity regions disabled. Importantly, the query amino acid sequence can be described by the amino acid sequence identified in one or more claims of this application.

[0086] The query sequence may have 100% identity with the subject sequence, or compared to the subject sequence, it may include up to an integer number of amino acid variations, such that the percentage of identity (%) is less than 100%. For example, the query sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the subject sequence. This variation includes the deletion, substitution (including conserved and non-conserved substitutions), or insertion of at least one amino acid, and said variation may occur at the amino or carboxyl terminus of the query sequence, or at any position between these terminus positions, and such variation may be independently dispersed among the amino acids of the query sequence, or dispersed within the query sequence in the form of one or more consecutive groups.

[0087] The identity percentage can be determined over the entire length of the query sequence (including the CDR). Alternatively, the identity percentage may exclude the CDR; for example, the CDR may have 100% identity with the topic sequence, and the identity percentage variation may exist in the remainder of the query sequence, thus making the CDR sequence immutable / complete.

[0088] In one embodiment, the antigen-binding protein is a monoclonal antibody or a fragment thereof. In one embodiment, the monoclonal antibody or a fragment thereof is mouse, chimeric, humanized, or wholly human. In one embodiment, the monoclonal antibody or a fragment thereof binds to BLyS (SEQ ID NO:1) or a heterotrimeric or homotrimeric form of BLyS, for example, the monoclonal antibody or a fragment thereof binds to a soluble form of BLyS (SEQ ID NO:10). In one embodiment, the monoclonal antibody comprises heavy chain and light chain variable regions, the heavy chain and light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2; and an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3; or the heavy chain and light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3; or the heavy chain and light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3; or the heavy chain and light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2; or the heavy chain and light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3 ... light chain variable regions respectively comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 9 NO:4 has an amino acid sequence with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity; and an amino acid sequence with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with SEQ ID NO:5. In one embodiment, the monoclonal antibody comprises heavy and light chain variable regions, each comprising an amino acid sequence having 95% identity with SEQ ID NO:2 and 95% identity with SEQ ID NO:3, respectively; or the heavy and light chain variable regions each comprising an amino acid sequence having 95% identity with SEQ ID NO:4 and 95% identity with SEQ ID NO:5, respectively. In another embodiment, the monoclonal antibody comprises heavy and light chain variable regions, each comprising an amino acid sequence having 90% identity with SEQ ID NO:2 and 90% identity with SEQ ID NO:3, respectively; or the heavy and light chain variable regions each comprising an amino acid sequence having 90% identity with SEQ ID NO:4 and 90% identity with SEQ ID NO:5, respectively.In one embodiment, the monoclonal antibody comprises heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NO:2 and 3, respectively, or the amino acid sequences shown in SEQ ID NO:4 and 5, respectively. In one embodiment, the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain each comprising: an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:6; and an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7; or the heavy chain and the light chain each comprising: ...2%, 93%, 94%, 9 NO:8 has an amino acid sequence with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity; and an amino acid sequence with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with SEQ ID NO:9. In one embodiment, the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain each comprising an amino acid sequence having 95% identity with SEQ ID NO:6 and an amino acid sequence having 95% identity with SEQ ID NO:7, respectively, or each comprising an amino acid sequence having 95% identity with SEQ ID NO:8 and an amino acid sequence having 95% identity with SEQ ID NO:9, respectively. In one embodiment, the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain each comprising an amino acid sequence having 90% identity with SEQ ID NO:6 and an amino acid sequence having 90% identity with SEQ ID NO:7, respectively, or each comprising an amino acid sequence having 90% identity with SEQ ID NO:8 and an amino acid sequence having 90% identity with SEQ ID NO:9, respectively. In one embodiment, the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain each comprising the amino acid sequences shown in SEQ ID NO:6 and 7, respectively, or each comprising the amino acid sequences shown in SEQ ID NO:8 and 9, respectively.In one embodiment, the monoclonal antibody comprises a CDR containing the amino acid sequences shown in SEQ ID NO: 11, 12, 13, 14, 15, and 16. In one embodiment, the anti-BLyS antibody is selected from belimumab, tabalumab, and mixtures thereof. In one embodiment, the anti-BLyS antibody comprises the heavy chain and light chain sequences shown in SEQ ID NO: 6 and 7, respectively.

[0089] In one embodiment, the pharmaceutical formulation of the present invention comprises a monoclonal antibody concentration of 200 ± 20 mg / mL. In one embodiment, the antibody concentration is about 200 mg / mL. In one embodiment, the anti-BLyS antibody is administered concurrently or sequentially with a corticosteroid. In one embodiment, the corticosteroid is selected from prednisone, prednisolone, hydrocortisone, methylprednisolone, and dexamethasone. In one embodiment, the corticosteroid is prednisone.

[0090] In one aspect, the present invention provides the aforementioned pharmaceutical formulation for treating diseases or disorders that are compliant with treatment using anti-BLyS antibodies. In one embodiment, the present invention relates to a method of treating a disease or condition in a subject who is compliant with treatment using anti-BLyS antibodies, the method comprising administering the formulation of the present invention to the subject in an amount effective for treating the disease or condition. In one embodiment, the disease or condition is selected from systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis, lupus nephritis, primary Sjögren's syndrome, chronic immune thrombocytopenic purpura, myasthenia gravis, symptomatic Waldenström macroglobulinemia, immune desensitization in patients awaiting kidney transplantation, membranous nephropathy, systemic sclerosis, rheumatoid arthritis, multiple myeloma, multiple sclerosis, and renal failure. In another embodiment, the disease or condition is systemic lupus erythematosus. In another aspect, the present invention provides formulations for treating diseases selected from: systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis, lupus nephritis, primary Sjögren's syndrome, chronic immune thrombocytopenic purpura, myasthenia gravis, symptomatic Waldenström macroglobulinemia, immune desensitization in patients awaiting kidney transplantation, membranous nephropathy, systemic sclerosis, rheumatoid arthritis, multiple myeloma, multiple sclerosis, and renal failure. In another aspect, the present invention provides formulations for treating systemic lupus erythematosus. In another aspect, the present invention provides the use of the formulation in the preparation of a medicament for treating diseases selected from: systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis, lupus nephritis, primary Sjögren's syndrome, chronic immune thrombocytopenic purpura, myasthenia gravis, symptomatic Waldenström macroglobulinemia, immune desensitization in patients awaiting kidney transplantation, membranous nephropathy, systemic sclerosis, rheumatoid arthritis, multiple myeloma, multiple sclerosis, and renal failure. In another aspect, the present invention provides the use of the formulation in the preparation of a medicament for treating systemic lupus erythematosus.

[0091] In one aspect, the present invention provides a kit comprising: one or more vials containing the formulation of the present invention; and instructions for subcutaneous administration of said formulation to a patient. In one embodiment, the kit further comprises an injection device for subcutaneous administration of said formulation to a patient.

[0092] In one embodiment, the present invention relates to an injection device comprising the stable anti-BLyS antibody formulation described in this application. For subcutaneous delivery, the formulation can be administered via a suitable device, such as (but not limited to): a syringe; an injection device (e.g., INJECT-EASE). TM and GENJECT TM Devices); infusion pumps (e.g., Accu-Chek)TM ); Injection pens (e.g., GENPEN) TM ); or needle-free devices (e.g., MEDDECTOR) TM and BIOJECTOR TM ).

[0093] The pharmaceutical formulation of this invention is substantially free of visible particles (observed by the human eye). The invisible particles (measured by opacity) should meet the following criteria: maximum number of particles ≥10μm per vial -> 6000; maximum number of particles ≥25μm per vial -> 600.

[0094] The pharmaceutically active anti-BLyS antibody formulation of the present invention can be administered by subcutaneous injection, wherein the administration is repeated multiple times at intervals of 1, 2, 3, or 4 weeks. In one embodiment, the pharmaceutically active anti-BLyS antibody formulation is administered once weekly or once every two weeks. In most cases, the entire volume of the injection fluid is administered over a time period of 1 to 10 minutes, preferably over 2 to 6 minutes, and most preferably over 3 ± 1 minutes.

[0095] For the prevention or treatment of a disease, the appropriate dosage of an antibody depends on the following factors: the type of disease to be treated (as defined above); the severity and duration of the disease; whether the antibody is being administered for preventative or therapeutic purposes; prior treatment; the patient's clinical history and their response to the antibody; and the judgment of the attending physician. The antibody is suitable for single-dose administration or administration over a series of treatments. Depending on the type or severity of the disease, an antibody dose of approximately 1 μg / kg body weight to 50 mg / kg body weight (or more specifically, approximately 0.1 mg / kg body weight to 20 mg / kg body weight) is an alternative starting dose for administration to the patient, whether, for example, by single or multiple administrations alone, or by continuous infusion. More specifically, the antibody dose range is from approximately 0.05 mg / kg body weight to approximately 10 mg / kg body weight.

[0096] In another embodiment of the invention, an article is provided containing the pharmaceutical formulation of the invention and providing instructions for use thereof. The article comprises a container. Suitable containers include, for example, bottles, vials (e.g., multi-chamber or dual-chamber vials), syringes (e.g., multi-chamber or dual-chamber syringes), and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container holds the formulation, and instructions for use may be indicated on the container or on a label attached to the container. The container holding the formulation may be a multi-purpose vial that allows for repeated administration of the reconstituted formulation (e.g., 2 to 6 doses). The article may further include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and packaging instructions with usage instructions.

[0097] The antibodies formulated according to the present invention are preferably substantially pure and, preferably, substantially homogeneous. "Substantially pure" antibody means that, based on the total weight of the composition, the composition contains at least about 90% antibody by weight, preferably at least about 95% by weight. "Substantially homogeneous" antibody means that, based on the total weight of the composition, the composition contains at least about 99% antibody by weight.

[0098] The invention will be more fully understood through the reference to the following embodiments. However, these embodiments are merely exemplary and should not be construed as limiting the scope of the invention. Minor operational variations, such as minor changes in time, temperature, amount, concentration, scale, etc., are not expected to affect the results of the experiments. All references to literature and patents are incorporated herein by reference.

[0099] Through the attached Figure 1-20 The embodiments are further described below. Example

[0100] Example 1: Belimumab formulation

[0101] Container encapsulation

[0102] Unless otherwise stated, all studies used Daikyo D21-7S. A Type I Schott vial with a stopper and easy-open aluminum seal. This vial and stopper combination is recommended for Phase 1. Use a Gerresheimer 1.0mL long, 29G, thin-walled, stalled, pre-filled syringe (with a Stelmi 4800 needle cap and Daikyo W4023). The piston is used to store long-term stable samples at >2-8℃ and then evacuate and purge with nitrogen. The samples are then filled into low-temperature vials at <2-8℃.

[0103] Product processing procedures

[0104] Prior to all experiments, belimumab was aseptically filtered using a 0.22 μm filter and aseptically packed into the selected containers. All stability samples were protected from light during storage.

[0105] Selection of excipients

[0106] In screening studies, when possible, a variety of pharmacopoeia-grade excipients specified in GMP BDS and manufactured by FDP were used and applied to long-term stability studies of all formulations.

[0107] Table 2 provides a list of the tested formulations.

[0108] Table 2

[0109]

[0110] Long-term stability

[0111] Concentration-dependent aggregation in Formulation 1

[0112] As expected, aggregation increased with increasing protein concentration (Table 3). Figure 1 At concentrations of 100 mg / mL to 260 mg / mL, the aggregation rate roughly doubled, but compared to 200 mg / mL belimumab, even at 260 mg / mL, aggregation increased by only about 1% over a 3-year storage period at 2–8°C. Note that the initial amount of aggregation observed by SEC-HPLC increased with increasing protein concentration, although by only about 0.1% (the 0-month row in Table 3).

[0113] Table 3 Effect of protein concentration on aggregation %

[0114]

[0115] Screening of alternative long-term formulations

[0116] Based on the following results, after evaluating 3 months of data, the candidate formulations were narrowed down to formulations 1 and 5, and then 5... 1 / Four months later, formulation 5 was selected as the final formulation.

[0117] Appearance, pH and osmotic pressure

[0118] In a long period of 5 1 At all time points over 4 months, all samples were opalescent, pale yellow, and free of visible particulate matter. When tested colorimetrically at the initial time points, the final drug products of all 8 formulations at all 3 concentrations were closest to the Y5 color standard. Storage at 2-8°C for 3 months and 5 months... 1 Four months later, all FDP samples in the histidine / NaCl (referred to as Formulation 1 in this application) and histidine / NaCl / arginine (referred to as Formulation 5 in this application) formulations also met the Y5 standard. The turbidity of the samples containing sugar stabilizers (sucrose and sorbitol) was significantly lower than all other samples, ranging from 29 to 38 NTU at both the initial and 3-month time points. In the NaCl-containing samples, turbidity also increased with decreasing protein concentration. After storage at 2-8°C for 5 months... 1 Four months later, only formulations 1 and 5 were tested, and they showed no response to formulation, concentration, or time. Figure 2 ).

[0119] Table 4. Turbidity of samples after long-term stability at 2-8℃ for 3 months.

[0120]

[0121] At the initial time point, the pH range of all samples was 6.1 to 6.3, and at 5... 1 Four months later, the pH of formulations 1 and 5 remained unchanged (data for formulation 5 are shown in Table 13). Osmolarity was tested only at the initial time point; all samples were 299 ± 17 mOsm / kg.

[0122] Viscosity and injectability

[0123] Sugar-containing (sucrose, sorbitol) formulations showed the highest viscosity, followed by succinate / sodium chloride formulations (Table 5). The remaining salt-containing samples had comparable viscosities. In formulations 1 and 5, viscosity increased exponentially with increasing protein concentration. Figure 3 ).

[0124] Table 5 shows the viscosity of samples with long-term stability at T0.

[0125]

[0126] Injectability was measured as the force required to deliver 1 mL through a thin-walled 29G needle within 10 seconds; injectability showed a similar trend at the initiation time point. At 5 1 Four months later, only formulations 1 and 5 were tested, and no significant increase in injectability over time was observed at 2–8°C. In formulation 5... 1 At a timeframe of 4 months, injectability within 20 seconds was also tested on various syringes, and it was shown that delivery force decreased by up to 40% when delivery time was doubled. Although not tested, delivery force could also be reduced by increasing needle size.

[0127] Table 6 shows the T0 and 5... 1 Injectability of samples with long-term stability over 4 months

[0128]

[0129] The force required to administer the drug using seven commercially available pen syringes (which are more similar to pre-filled syringes as they all require manual actuation force) is similar to that required for 200 mg / mL belimumab (Table 7). Injection times varied with different volumes and container diameters, which are listed in Table 7 for comparison. Finally, a study commissioned by the UK Department for Trade and Industry and conducted by the University of Nottingham showed that 59 women aged 16 to 90 years were able to apply a static force of 53.7 to 237.7 N downwards at hip level with their thumbs while seated. Although neither the data from pen syringes nor the study on force is entirely relevant to the use of pre-filled syringes, both data confirm that manual administration is feasible for the viscosity and injectability of 200 mg / mL belimumab. The force required to deliver 200 mg / mL belimumab from a 1 mL long pre-filled syringe via a 29G thin-walled needle reaches or approaches the limit of what is expected with manual injection, thus a thicker needle is preferred.

[0130] Table 7. Injection force of commercially available pen injectors at 80 mm / min

[0131]

[0132] *Delivery using Ypsomed Penfine 29Gx12.7mm needles.

[0133] Size variants

[0134] 3-month SEC-HPLC data

[0135] For belimumab in all formulations, aggregation, as observed by SEC-HPLC, was the dominant concentration-dependent pathway. The fragmentation percentage (observed as the back shoulder) varied between 0.1% and 0.2%, but did not change over time (obtained 5). 1 (Supported by 4 months of data).

[0136] After storage at 2-8°C for 3 months, significant differences in aggregation rate were observed in belimumab formulated in 8 different formulations. Figure 4 Formulation 5 (histidine / NaCl / arginine) showed the lowest rate of reaction over a 3-month period, especially at 200 mg / mL. Figure 4 (Medium blue). This is supported by the accelerating trend at 200 mg / mL. Figure 5 Succinates are the worst stabilizers at low temperatures but the best at elevated temperatures. Several formulations of other salts and sugars (including formulation 1 (histidine / NaCl)) show similar absolute aggregation percentages and aggregation rates.

[0137] 51 / 4 months of SEC-HPLC data

[0138] In 5 1 Belimumab in formulations 1 and 5 was evaluated at 4 months. The trend observed at 3 months continued, with the arginine-containing formulations showing lower aggregation rates, especially at the highest concentration of 200 mg / mL. Figure 6 It shows that it can be stored at up to 25°C for 5 days. 1 The aggregation rate after 4 months was analyzed, and it was shown that the arginine formulation (hollow cube in the figure) significantly reduced aggregation compared to formulation 1 (solid cube). Figure 7 Further analysis of the aggregation rate at different temperatures showed that formulation 5 (dashed line) consistently exhibited a lower aggregation rate than formulation 1 (solid line). If at a temperature up to 5... 1 If the aggregation rate observed at 2-8°C within 4 months remains constant for 3 years, then FDP will increase by only about 1.2%.

[0139] CGE

[0140] 5 stored at different temperatures 1 Four months later, capillary gel electrophoresis of formulations 1 and 5 showed no decreasing trend (rates shown in...). Figure 8 (in the middle). Therefore, cross-linking and clipping are not dependent on concentration or formulation.

[0141] Charge heterogeneity

[0142] Ion exchange showed that neither the concentration of arginine nor the addition of arginine to histidine buffer preparations affected the charge change. Figure 9 Although the acid variants increase with increasing temperature over time, at 5... 1 / After 4 months, few or no variant changes were observed.

[0143] Oxidation

[0144] After storage at 2-8°C for 3 months, no significant changes in oxidation were observed in any of the 8 formulations (data not shown). In 5 1 Four months later, when comparing data at -80°C and 15°C, no differences in oxidation were observed between formulations 1 and 5, or between the three concentrations of any one formulation. Figure 10 Store at 25°C for 5 days. 1 After 4 months, approximately 1.0% additional oxidation was observed in all samples, and approximately 4.5% additional oxidation was observed at 40°C.

[0145] Peptide profile

[0146] In 5 1Four months later, no difference was observed between the 200 mg / mL formulation 1 samples at -80°C and 2-8°C or the control standard. Figure 11 As expected, the 25°C sample showed a slight increase in T4 deamidation at elevated temperatures. Formulation 5 samples showed a similar T4 deamidation only at 25°C, but also exhibited inconsistent peak heights in some other peptide peaks. Figure 12 In the middle, the heavy chain peaks are T33, T34, and T5, and the light chain peak is T3. These peak heights do not show a trend with temperature change, so it is suspected that they are due to arginine digestion interference.

[0147] To determine whether arginine interference was the cause of the change, 0 mM, 25 mM, and 50 mM arginine were added to Formulation 1 sample (which had already undergone the desalting step of this method). The remaining steps, including trypsin digestion, were then performed on all three samples. The same peptide peaks showing changes in the stability samples exhibited a response related to arginine concentration. Figure 13 This indicates that the desalting step does not always completely remove arginine and explains why the changes observed in the peptide profiles of formulation 5 were independent of temperature. Since no other changes were observed in the peptide profiles, it can be assumed that, despite the differences in the profiles, formulations 1 and 5 were stored at 2–8°C. 1 No degradation was observed after 4 months, and it was only observed after storage at 25°C for 5 months. 1 It exhibits minimal degradation after 4 months.

[0148] efficacy

[0149] Formulation 1 or Formulation 5 with a concentration between 125 mg / mL and 200 mg / mL, after being stored at 2-8°C for 3 months, or Formulation 5 with a concentration of 200 mg / mL, after being stored at 2-8°C for 5 months. 1 Four months later, belimumab still maintained its biological activity (Table 8).

[0150] Table 8. Relative efficacy after stability testing at 2-8℃

[0151]

[0152] Assessment of freezing / thawing

[0153] Samples exposed to five rapid freeze / thaw cycles between -40°C and 2-8°C showed similar aggregation levels to the control sample at -40°C, indicating that rapid freeze / thaw was not an issue in either formulation 1 or formulation 5 (Table 9).

[0154] Table 9. SEC-HPLC results of belimumab exposed to rapid freezing / thawing.

[0155]

[0156] Compared to the liquid control, samples exposed to three slow freeze / thaw cycles showed a 0.2% increase in aggregation levels (Table 10).

[0157] Table 10. SEC-HPLC results of belimumab exposed to slow freezing / thawing.

[0158]

[0159] DSC

[0160] Calorimetry was used to assess the glass transition (Tg') below the freezing point of each formulation and to determine whether eutectic formation below the freezing point occurred. Sodium chloride-water eutectic can form below approximately -21°C, and the eutectic crystallization of excipients can affect product quality by introducing crystal surface interactions and altering the local chemical environment in frozen concentrates containing proteins. Storage at temperatures below Tg' can improve stability by increasing relaxation time and reducing associated degradation.

[0161] Regarding transformations below the freezing point, formulations 1 and 5 of the high-concentration belimumab exhibit similar behavior (Table 11). For formulation 1, Tg' ranges from -23°C (fastest freezing) to -33°C (slowest freezing). For formulation 5, Tg' ranges from -22°C (fastest freezing) to -32°C (slowest freezing). For both formulations, endothermic eutectic was only observed after thermal cycling with multiple annealing steps at -23°C. This eutectic is most likely sodium chloride-water.

[0162] These results indicate that the thermal transformation of these formulations below the freezing point is sensitive to the thermal history of the samples. This is due to the high content of soluble solids and the presence of sodium chloride, which affects the Tg' in the protein / amorphous phase. These results, combined with the stability data at -80°C and -40°C in Section 5.2, also suggest that storage at <-40°C and protected from light in BDS is sufficient for belimumab in formulation 5.

[0163] Table 11 DSC results of belimumab in formulations 5 and 1

[0164]

[0165] Oscillation assessment

[0166] After shaking at 250 rpm for 48 hours, no significant changes in purity (measured by SEC-HPLC) or turbidity were observed within the studied polysorbate concentration range, whether in vials or syringes (Table 12). In both vials and syringes, 0.01% polysorbate 80 demonstrated to be an effective and robust anti-shake protectant in Formulation 5.

[0167] Table 12 shows the SEC-HPLC results and turbidity of belimumab in formulation 5 after shaking at 250 rpm.

[0168]

[0169]

[0170] in conclusion

[0171] Based on its ability to minimize the rate of the main degradation pathway, a subcutaneous formulation of belimumab at 200 mg / mL was selected (Formulation 5; 0.65 mg / mL L-histidine, 1.2 mg / mL L-histidine monohydrochloride, 6.7–7.3 mg / mL sodium chloride, 5.3 mg / mL L-arginine hydrochloride, 0.1 mg / mL polysorbate 80, pH 6.0; or 10 mM histidine, 115 mM sodium chloride, 25 mM L-arginine hydrochloride, 0.01% (w / v) polysorbate 80, pH 6.0). The aggregation rate (~0.03% / month, 2–8 °C) increased with increasing belimumab concentration but was inhibited by the use of 25 mM arginine. At 2–8 °C, the deamide rate was approximately 0.2% / month. For delivery by manual or automated syringe using a 1 mL long syringe and a 29 G thin-walled or thicker needle, the 200 mg / mL formulation has acceptable delivery force. Freezing / thawing properties and storage at -80°C and -40°C have been shown to be acceptable, and the product is not easily affected by vibration stress.

[0172] Long-term GMP stability studies were conducted on formulation 5 (1.0 mL filled into a 1 mL long BD syringe) of belimumab in a final drug product of 200 mg / mL. GMP stability data are currently available for 42 months at the intended storage temperature of 2–8 °C (Table 14). These results indicate that formulation 5 provides sufficient stability for belimumab, and acceptable degradation profiles were observed at the intended storage temperature of 2–8 °C (Table 14).

[0173]

[0174]

[0175]

[0176] Example 2: Anti-IL13 is a high-concentration, high-dose mAb

[0177] Anti-IL13 is a glycosylated humanized mAb (IgG1) against human interleukin-13 (IL13). To achieve very high subcutaneous clinical doses (10 mg / kg), based on a PK / PD model, the drug and pharmaceutical product need to be developed in vials at concentrations of 200 mg / mL. As a direct consequence of achieving higher mAb concentrations in vials, various formulation challenges for anti-IL13 emerge, including: (i) identifying unique formulations to support the stability, manufacturability, analytical, and delivery challenges of high-concentration monoclonal antibodies intended for subcutaneous delivery at high clinical doses; (ii) preventing analytical and stability challenges arising from gelation of high-concentration monoclonal antibodies; and (iii) preventing various associated challenges, particularly during delivery of monoclonal antibodies in injection volumes below 1.5 mL. As a direct finding revealed during formulation development, the monoclonal antibody was identified as having a tendency to form an irreversible gel-like matrix in certain buffer systems at elevated temperatures, thus posing a significant risk of protein instability. Viscosity increases exponentially with concentration and can complicate filtration processes. Higher concentrations of monoclonal antibodies become more prone to polymerization due to increased risk of particle formation and reversible self-association. Therefore, high-throughput formulation (HTF) development studies are conducted to identify optimal buffers and pH for new formulations, supporting the delivery of high-concentration monoclonal antibodies at high clinical doses. Combined with other laboratory studies, these studies identify optimal formulations that prevent gelation at elevated temperatures. Other formulation development studies identify various excipients to be added to selected buffer systems. These are subjected to shaking, freeze-thaw, and elevated temperature studies to assess physical stability after short- and long-term stability studies, thereby evaluating the chemical stability of anti-IL13 mAb. Various other studies are also conducted to ensure compliance with clinical delivery considerations.

[0178] Example 3: Screening of HTF pH-buffers: (Identification of target pH and buffer)

[0179] Previous testing of an acetate-based 50 mg / mL anti-IL13 monoclonal antibody formulation has demonstrated that the stated pH is not optimal. A suboptimal formulation buffer pH can increase the instability of higher concentrations of anti-IL13 monoclonal antibody solutions by altering the charge on the protein and affecting electrostatic interactions. The development of high-concentration formulations begins with identifying the optimal pH and determining the best type of buffer.

[0180] This study was conducted at a mAb concentration of 13 mg / mL. Buffer screening was performed using the HTF method in 96-well plates. The study comprised a wide range of buffer types and pH levels. Each plate consisted of 48 samples, in duplicate, in randomized order. Samples were incubated at 50°C / ambient RH for 3 days. The tests included general appearance (GA), concentration at A280 and A260 nm, pH, size exclusion chromatography (SEC), capillary isoelectric focusing electrophoresis (cIEF), and dynamic light scattering (DLS). The factors tested are shown in Table 15.

[0181] Table 15: Screening Study of HTF Buffers

[0182]

[0183] Results of general appearance tests on the plates showed high levels of precipitation in some samples, particularly in acetate, citrate, and phosphate buffers. Succinate buffer was the only type of buffer that did not show precipitation.

[0184] The deamidation and aggregation properties of all samples were obtained using cIEF and SEC, respectively. Figure 14 This is a graph showing both cIEF and SEC data. The graph shows that formulations with the highest monomer percentage via SEC also have a lower main peak percentage via cIEF, indicating that anti-IL13 aggregates at low pH and deamidates at high pH. It can be observed that the monomer percentage is higher at pH 6–7 than at pH 4–5.5, regardless of buffer type.

[0185] Overall, the HTF pH-buffer screening yielded the following conclusions: (i) citrate and acetate buffers produced the highest number of turbid pores (indicating precipitation) as determined by GA; and (ii) phosphate buffers promoted precipitation, accompanied by increased aggregation and deamidation, based on cIEF and SEC results; and (iii) anti-IL13 aggregated at low pH and deamidated at high pH.

[0186] Example 4: HTF pH-buffer screening: (determining the optimal pH)

[0187] A second HTF study was conducted using a 3x3 factorial (3 buffers, 3 pH) DOE design, comprising a total of 90 samples. Buffers selected for evaluation included acetate, histidine, and succinate, with final pH ranges between 5.5 and 6.5. A fixed concentration of 25 mM was chosen for the buffers. The design was performed in six replicates for each formulation, along with six replicates of the acetate formulation serving as a control group for comparison on stress plates.

[0188] Each plate was subjected to stress at 50°C / ambient RH for 3 days. The test included the general appearance (GA) of the selected samples, concentration at A280 and A260 nm, pH, SEC, cIEF, DLS, and DSC (differential scanning calorimetry).

[0189] The research results were analyzed using statistical software known as Design Expert. Interesting trends were revealed when the same software was used to perform analysis of variance (ANOVA) on all test results. The type of buffer and pH were found to be significant factors in the results for concentration, correction concentration, DLS, SEC, and cIEF.

[0190] Figure 15 The interaction between pH and buffer type on SEC monomer percentage is shown. For acetate and histidine buffers, monomer percentage increases with increasing pH. Figure 16 The interaction between pH and buffer concentration on the percentage of the main peak in cIEF is shown. Both pH and buffer concentration were found to be important factors. The figure shows that pH in the range of 5.5–6.5 did not affect the percentage of the main peak in acetate or succinate, but it did affect the percentage of the main peak in histidine.

[0191] In summary, 6.25 was selected as the final optimal pH for the screening of HTF pH buffers.

[0192] Example 5: Determining the optimal thermal stress conditions based on the thermal stability properties determined by DSC.

[0193] Among the various potential factors influencing protein gelation, such as concentration, pH, and salt content, one crucial factor dominates this phenomenon – temperature. Thermostability conditions for anti-IL13, selected by HTF and other developmental research tests, were evaluated using DSC. Typically, medium-sized globulins begin to unfold at approximately 25°C, while for monoclonal antibodies, they begin to unfold at approximately 60°C. In all tested buffers (acetate, histidine, and succinate), the mAb began to unfold at approximately 61°C, indicating it is a thermostable molecule and confirming that the folded anti-IL13 monoclonal antibody allows for accelerated storage at 50°C. Since there was a difference of over 10°C between the onset of unfolding and the accelerated storage conditions at 50°C, 50°C was chosen as the storage temperature for screening.

[0194] Table 16 lists the Tm values ​​determined from the original scans. The Tm1 value spans the range of 71.3–71.6°C, and the Tm2 value spans an even wider range, 83.5–84.1°C. There are no significant changes in Tm values ​​for the same buffer at different pH values. The variations in Tm1 and Tm2 measured for all conditions are less than 1°C, therefore the tested protein solutions exhibit similar thermodynamic stability.

[0195] Table 16: Tm values ​​determined by the original scan

[0196] buffer Tm1,C Tm2,C Acetate 6.0 71.3 84.1 Acetate 6.5 71.4 83.9 Histidine 6.0 69.9 83.6 Histidine 6.5 71.3 84.1 Succinate 6.0 71.4 83.6 Succinate 6.5 71.6 83.5

[0197] Example 6: Feasibility assessment of high-concentration formulations

[0198] The clinical need for subcutaneous (SC) administration of high-dose protein drugs (>100 mg / mL) typically introduces additional technical development challenges regarding manufacturing, analytical testing, stability, and delivery. A common property of high-concentration protein formulations is high viscosity, which is directly due to the reversible self-association of proteins. High viscosity can also pose additional clinical development challenges due to high injection forces (increased pain at the injection site) and can alter the pharmacokinetic properties of the drug. Therefore, a crucial aspect of product development is identifying formulations with low viscosity. The effects of viscosity can be mitigated by altering the pH or adding excipients.

[0199] Because the viscosity was expected to increase exponentially with mAb concentrations up to 200 mg / mL, an initial feasibility study was conducted to investigate the viscosity and injectability of high-concentration anti-IL13 solutions. In a previously established acetate-based formulation, a solution of ~150 mg / mL was concentrated to ~210 mg / mL. Viscosities were measured using a cone-plate rheometer at the following mAb concentrations: 50 mg / mL, 150 mg / mL, and 200 mg / mL.

[0200] Figure 17 The graphs show different viscosity levels plotted against the aforementioned concentrations, where it was observed that the viscosity increases exponentially with increasing concentration. The viscosity of the concentrated solution at 207.7 mg / mL was 28.6 cP.

[0201] The maximum injection force was determined by placing 207.7 mg / mL of anti-IL13 into a 1 mL glass syringe fitted with a 27-gauge needle, with the injection speed set at 3 mm / min, and measured using an Instron electromechanical testing system. Table 17 shows the numerical results for both viscosity and injectability obtained at the maximum concentration of 207.7 mg / mL.

[0202] The viscosity (28.6 centipoise) determined at 207.7 mg / mL and the maximum injection force of 30.3 Newtons necessitate efforts to develop additional formulations to allow and enable the feasibility of manufacturing and administering high concentrations of anti-IL13.

[0203] Table 17: Summary of Feasibility Study Results for High Concentration Levels

[0204] concentration 207.7 mg / mL Viscosity 28.6 centipoise Maximum injection force 30.3 Newtons

[0205] Example 7: Evaluating Physical Properties Through Oscillation Studies

[0206] Histidine and succinate buffers at pH 6.25 were identified as the optimal buffering system. Further formulation development studies were then required to identify suitable formulations for high-concentration mAb solutions that readily exhibit high viscosity and gelatinization due to their high concentration (as a function of temperature).

[0207] The formulations used in the oscillation study were derived from the HTF screening study. The HTF study identified two buffer systems (histidine and succinate, pH 6.25) that provided good stability. A third buffer system (50 mM acetate, pH 5.5) was also included as a control. Samples of the histidine and succinate buffer system at pH 6.25 were prepared using small-scale buffer exchange and concentration techniques. The following excipients were then added: 0.02% polysorbate 80 (PS80) to protect the protein from shear stress; and 150 mM sodium chloride as a potential viscosity reducer.

[0208] The sample was filled into 3 mL glass vials at a volume of 1.2 mL (or 1 mL) and shaken at 250 rpm on a horizontal shaker in the dark for 72 hours at 2–8 °C. The sample was then tested using various analytical techniques. Table 18 below lists the formulations used for the shear stress / oscillation studies. Lower concentration formulations are included because 50 mg / mL was not attempted in previous oscillation studies. Control formulations without excipients (such as PS80) are also included. For acetate samples, a NaCl control is included.

[0209] Table 18. Oscillation Study Design and Samples

[0210]

[0211] No significant changes were observed in general appearance, SEC, DLS, and MFI in all samples prepared using NaCl and polysorbate 80. Based on SEC-HPLC results, the high-concentration protein formulation was unstable in the acetate (control) formulation.

[0212] The target concentration of anti-IL13 mAb for all tests was 200 mg / mL; however, due to limitations in processing yield and the inherent variability of viscosity measurements, the nominal concentrations of the 200 mg / mL formulations in histidine and succinate were within ±10%. Viscosity measurements of the samples used in the oscillation studies showed that a viscosity reduction of 1 / 6 was achievable in a formulation with a nominal concentration of 200 mg / mL containing 150 mM sodium chloride, independent of the buffer system. Figure 18In formulations containing only buffers, histidine exhibited a significantly higher viscosity compared to sodium acetate and sodium succinate. This indicates that the sodium ions in the aforementioned sodium acetate and sodium succinate buffers contribute to viscosity reduction, thus explaining the association between sodium ions in sodium chloride and viscosity-reducing effects. However, when histidine was combined with 150 mM sodium chloride, the degree of viscosity reduction was the same as with other buffer systems. This also suggests a synergistic effect between histidine buffer and NaCl, which effectively reduces viscosity.

[0213] The study also included lower concentration formulations 1, 2, and 3 at 50 mg / mL; however, no differences were observed in these formulations, highlighting the importance of stability data generated at the target concentration high concentration formulations (data not shown).

[0214] Figure 18 The study summarized the dependence of viscosity on protein concentration and sodium content of samples. Viscosity readings for samples at 50 mg / mL (lower concentrations) were all less than 2 cps. The viscosity of the acetate sample with a nominal concentration of 200 mg / mL and containing 150 mM NaCl was approximately 3 cps lower than that of the corresponding histidine and succinate samples, mainly due to poor processing yields resulting in concentrations lower than the nominal 200 mg / mL. Figure 18 As shown, the total concentration is less than 200 mg / mL due to losses during the preparation of the concentrated sample.

[0215] The results of this study clearly demonstrate that the presence of 150 mM NaCl significantly reduces the viscosity of all tested buffer systems. A unique synergistic relationship between the histidine buffer and NaCl was also observed.

[0216] Note: The target concentration of anti-IL13 mAb for all tests is 200 mg / mL; however, due to limitations in processing yield and the inherent variability of viscosity measurements, the nominal concentration of the 200 mg / mL formulation in histidine and succinate is within ±10%.

[0217] Example 8: Evaluation of the physical properties of increased temperature

[0218] The following study was designed to evaluate the physical properties of high concentrations of anti-IL13 at a nominal concentration of 200 mg / mL when exposed to heat stress at appropriately determined elevated temperatures.

[0219] Incubate the samples at an elevated temperature of 50°C / 60% RH for 7 or 10 days.

[0220] Analytical tests included general appearance (GA), viscosity, concentration obtained at A280 nm, SEC-HPLC, MFI, and DLS. Table 19 summarizes the sample formulations used in this elevated temperature study.

[0221] Table 19: Formulations used in temperature elevation studies

[0222]

[0223] As shown in Table 20 and Figure 19 As shown in the results, general appearance results indicate that Formulation 1 (acetate) gelled after 7 days. Formulation 2 (succinate) showed semi-gelling after 10 days of storage, while Formulation 3 (histidine) showed no gelling after 10 days of storage. No particles were observed in any of the non-gelled samples. The succinate and histidine samples at 7 and 10 days were classified as “milky iridescent.” Iridescence is equivalent to decreased transparency, indicating that particles and aggregates are visible under a microscope at high concentrations. MFI results (not shown) indicate that Formulation 1 (acetate) at t=0 had approximately 50% more particles compared to the other formulations.

[0224] Table 20: GA Results of the Planned 2-Week Temperature Elevation Study at 50℃ / 60%RH

[0225]

[0226] The results clearly demonstrate that the combination of 150 mM NaCl and a histidine buffer at pH 6.25 prevents gelation. The results also show that the type of buffer has a significant impact on physical stability (histidine > succinate > acetate). These results suggest a synergistic relationship between histidine buffers and NaCl that enhances the physical stability of high-concentration mAb formulations.

[0227] Example 9: Freezing-thawing study at 200 mg / mL

[0228] The aim of this study was to evaluate the effect of three freeze-thaw cycles between 2–8 °C and -70 °C on the physical and chemical stability of an anti-IL13 mAb at a nominal concentration of 200 mg / mL. The freeze-thaw study examined the stability in a 15 mM histidine buffer (containing 100 mM or 150 mM NaCl, 0.05 mM EDTA, and 0.02% polysorbate 80) at pH 6.25. An acetate-based formulation was included as a control (pH 5.5). Samples were filled into 3 mL glass vials at 1.2 mL filling volumes and subjected to three freeze-thaw cycles between 2–8 °C and -70 °C, protected from light. Table 21 shows the samples used in this study.

[0229] Table 21: Sample formulations used for freeze-thaw study testing

[0230]

[0231] The following analytical techniques were used to analyze the samples: GA, pH, viscosity, concentration obtained at A280 nm, potency obtained by SPR, SDS-PAGE, SEC-HPLC, cIEF, and MFI.

[0232] Freezing / thawing (F / T). Based on general appearance, pH, potency obtained via SPR, concentration, SEC, SDS-PAGE, or viscosity, there were no significant differences between the F / T and control results for any formulation. For histidine formulations, there was no decrease in the percentage of the main cIEF relative to the initial value. Table 22 lists all general appearance, pH, potency, concentration, and viscosity data. Table 23 lists all SDS-PAGE data. Table 24 lists all SEC and cIEF data.

[0233] The overall results of this study clearly demonstrate that the combination of 150 mM NaCl with a histidine buffer at pH 6.25 is the optimal formulation and remains stable after exposure to freeze-thaw stress.

[0234] Table 22: Analytical test results of GA, pH, potency, concentration and viscosity after freezing and thawing.

[0235]

[0236]

[0237] Table 23: Results of the freeze-thaw study (SDS-PAGE)

[0238]

[0239] Table 24: Results of freeze-thaw studies (SEC, cIEF)

[0240]

[0241] Example 10: Short-term chemical stability study and biophysical properties of mAb-based drug products at a nominal concentration of 200 mg / mL

[0242] Developmental stability studies were conducted to evaluate the short-term chemical stability of the 200 mg / mL mAb formulation containing NaCl, PS80, and EDTA. The short-term chemical stability studies examined the stability in a 15 mM histidine buffer (containing 100 mM or 150 mM NaCl, 0.05 mM EDTA, and 0.02% polysorbate 80) at pH 6.25. Acetate-based formulations were included as controls. Samples were filled into 3 mL glass vials at 1.2 mL filling volumes and stored at 5°C, 25°C, and 40°C. Table 25 below shows the samples used in this study.

[0243] Table 25: Short-term chemical stability of samples and initial osmotic pressure results

[0244]

[0245] The samples were analyzed using the following analytical techniques: GA, pH, viscosity, concentration obtained at A280 nm, potency obtained by SPR, SDS-PAGE, SEC-HPLC, and cIEF. The study lasted a total of 3 months, and various biophysical properties were also tested at the initial and final time points using DSC, DLS, CD, and MFI.

[0246] The results of analytical (concentration, potency, SEC, SDS-PAGE, cIEF) and biophysical (DSC, fluorescence, CD) tests showed that formulation B (200 mg / mL anti-IL13, dissolved in 15 mM histidine, 150 mM NaCl, 0.05 mM EDTA, and 0.02% polysorbate 80 at pH 6.25) had the best short-term chemical stability among the three formulations.

[0247] Summary and discussion of the test results:

[0248] Table 26 summarizes the 5°C data over a 3-month period using all analytical techniques, and Table 27 summarizes the stress and acceleration data at 25°C and 40°C over a 3-month period.

[0249] Compared to the acetate control, the histidine formulations (A and B) exhibited higher peak percentages (%) at 5°C and 25°C after 3 months, as determined by cIEF. Purity determined by SDS-PAGE indicated that the samples in both histidine formulations (A and B) had higher heavy and light chain percentages (%) at 25°C after 3 months, relative to the acetate control. At 40°C / 75% RH storage conditions, all three formulations showed lower H+L percentages (H+L%) relative to the initial values ​​after 1 month; however, formulations A and B were superior to formulation C (90.6% and 91.8%, respectively, compared to 88.2%). Significant reductions in peak percentages were observed for all samples stored at 40°C for 1 month, but the reductions in histidine formulations were smaller (8.8% or 7.5%) compared to the acetate formulation (9.9%), with formulation B showing the smallest reduction. The highest potency was observed in formulation B, containing 150 mM NaCl, at 3 months (25°C / 60% RH) and 1 month (40°C / 75% RH), compared to the lowest potency observed in the previous and control formulation C, which did not provide adequate stability for high concentrations of mAb. The initial viscosity results for formulations A and B (18.0 and 16.3 cps, respectively) were lower than those for formulation C (18.7 cps), precisely due to the higher NaCl concentrations in formulations A and B (100 mM and 150 mM NaCl, respectively). Formulation B exhibited the lowest viscosity results across all conditions and time points.

[0250] Table 26: Short-term chemical stability data at 5°C

[0251]

[0252] Table 27: Stress and Acceleration Data on Short-Term Chemical Stability

[0253]

[0254] Summary and discussion of biophysical characteristics results:

[0255] Table 28 shows the initial DLS data and the fluorescence data at the initial and 3-month stages.

[0256] MFI results showed that no visible particles were observed in any sample within 3 months. DLS (which measures the hydrodynamic radius of the mAb) revealed no significant differences among the three samples. Fluorescence and circular dichroism data indicated no fluctuations in the secondary and tertiary structures of the histidine formulation after 3 months of storage. Figure 20The CD comparison plots of the spectra are shown. The lower signal in formulation C indicates a change in tertiary structure. The difference observed in the quenching percentage for formulation C also indicates a change in tertiary structure for this formulation. Table 29 shows the DSC data for the initial and 3-month samples. The trend of higher Tm and total kcal / mol results for both histidine / NaCl formulations suggests that the other salts achieve greater thermodynamic stability compared to the acetate formulation.

[0257] Table 28: DLS and fluorescence data for short-term chemical stability

[0258]

[0259] Table 29: DSC data on short-term chemical stability

[0260]

[0261]

[0262] Example 11: Long-term chemical stability study and biophysical properties of a mAb-based drug product with a nominal concentration of 200 mg / mL

[0263] As listed in Table 26, the stability of all samples was tested at 16-month time points. The results at these time points were used to assess the long-term chemical stability of mAb drug products formulated with His, NaCl, PS80, and EDTA at a nominal concentration of 200 mg / mL. Limited assays were performed to confirm long-term stability.

[0264] The samples were analyzed using the following analytical techniques: GA, pH, viscosity, concentration obtained at A280 nm, SDS-PAGE, SEC-HPLC, and cIEF. Biophysical properties were also tested, including fluorescence, DSC, and CD.

[0265] Table 26 includes 5°C data at 16-month time points obtained through all analytical techniques.

[0266] Summary and discussion of the analytical test results: Samples of histidine preparations (A and B) showed higher monomer content (%) after 16 months at 5°C. Preparation C had a 0.9% higher content than the two histidine preparations.

[0267] Summary and discussion of biophysical properties: Overall, the biophysical properties were comparable between the 3-month and 16-month time points. Fluorescence and circular dichroism data indicated no fluctuations in the secondary and tertiary structures of the histidine formulation after 16 months of storage. No significant changes were observed in formulations A and B at 5°C and 16 months. A significant increase in intensity was observed in both formulations C, indicating molecule unfolding and exposure of more fluorescent groups. Analysis of the MFI results for formulations A and B showed no increase in visible particles at the 16-month time point. DSC data for the samples at the 16-month time point are also provided. The trend towards higher Tm and total kcal / mol values ​​in both histidine / NaCl formulations suggests that the other salts achieve greater thermodynamic stability compared to the acetate formulation.

[0268] Table 28 shows the fluorescence data at 16 months. Table 29 shows the DSC results at 16 months.

[0269] The results of analytical (concentration, potency, SEC, SDS-PAGE, cIEF) and biophysical (DSC, fluorescence, CD) tests indicate that formulation A (200 mg / mL anti-IL13 in 15 mM histidine, 150 mM NaCl, 0.05 mM EDTA, and 0.02% polysorbate 80 at pH 6.25) is the best among the three formulations in terms of long-term chemical stability.

[0270]

[0271] sequence list <110> BLAKE-HASKINS, Angela MARSHALL, Tristan PERKINS, Melissa D. O'BERRY, Kristen CROTTS, George H. PURI, Manasi Dunley, Donna M. <120> antibody preparations <130> PU65703 <140> Not yet allocated <141> 2015-05-15 <150> 61 / 994427 <151> 2014-05-16 <150> 62 / 093734 <151> 2014-12-18 <150> 62 / 095181 <151> 2014-12-22 <160> 16 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 285 <212> PRT <213> Artificial sequence <220> <223> BLyS <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 1 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 285 <210> 2 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 3 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 3 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly 100 105 <210> 4 <211> 142 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 4 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe 35 40 45 Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 <210> 5 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 5 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 <210> 6 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 6 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 7 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 7 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 8 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 8 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Gln Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <210> 9 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 10 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 10 Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu 1 5 10 15 Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe 20 25 30 Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys 35 40 45 Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly 50 55 60 Gln Val Leu Tyr Thr Asp Lys Thr Tyr Tyr Ala Met Gly His Leu Ile Gln 65 70 75 80 Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu 85 90 95 Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys 100 105 110 Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu 115 120 125 Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr 130 135 140 Phe Phe Gly Ala Leu Lys Leu Leu 145 150 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 11 Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn 1 5 10 <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 12 Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln 1 5 10 15 Gly <210> 13 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 13 Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 14 Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 15 Gly Lys Asn Asn Arg Pro Ser 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequences identified using molecular biology techniques. <400> 16 Ser Ser Arg Asp Ser Ser Gly Asn His Trp Val 1 5 10

Claims

1. A pharmaceutical preparation of an antigen-binding protein, comprising: a. 200 mg / mL of antigen-binding protein; b. A 10 mM buffer providing a pH of 6.0, wherein the buffer is histidine; c. A 115 mM tensile agent, wherein the tensile agent is sodium chloride; d. A 25 mM stabilizer, wherein the stabilizer is arginine; and e. 0.01% (w / v) of a nonionic surfactant, wherein the nonionic surfactant is polysorbate 80; wherein the antigen-binding protein comprises the following amino acid sequence: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO:

16.

2. The pharmaceutical formulation of claim 1, wherein the antigen-binding protein is a monoclonal antibody or a fragment thereof.

3. The pharmaceutical formulation of claim 2, wherein the monoclonal antibody or a fragment thereof binds to BLyS: SEQ ID NO: 1 or a heterotrimeric or homotrimeric form of BLyS.

4. The pharmaceutical formulation of claim 2, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences having at least 90% or 100% identity with SEQ ID NO: 2 and 3.

5. The pharmaceutical formulation of claim 3, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences having at least 90% or 100% identity with SEQ ID NO: 2 and 3.

6. The pharmaceutical formulation of claim 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising amino acid sequences having at least 90% or 100% identity with SEQ ID NO: 6 and 7, respectively.

7. The pharmaceutical formulation of claim 3, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain each comprise an amino acid sequence having at least 90% or 100% identity with SEQ ID NO: 6 and 7.

8. The pharmaceutical formulation of claim 4, wherein the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising amino acid sequences having at least 90% or 100% identity with SEQ ID NO: 6 and 7, respectively.

9. The pharmaceutical formulation of claim 5, wherein the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising amino acid sequences having at least 90% or 100% identity with SEQ ID NO: 6 and 7, respectively.

10. The pharmaceutical preparation of any one of claims 1-9, which remains stable during freezing-thawing.

11. The pharmaceutical preparation of any one of claims 1-9, for subcutaneous or intramuscular administration.

12. The pharmaceutical preparation of claim 10, for subcutaneous or intramuscular administration.

Images