Biopharmaceutical compositions

Abstract

The present invention provides compositions and methods for treating diseases mediated by interleukin-5 (IL-5). [Solution] A composition is provided comprising an antibody having a heavy chain amino acid sequence and a light chain amino acid sequence having a specific sequence, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% acidic antibody variants.

Description

[Technical Field]

[0001] This disclosure relates to compositions and related methods for treating interleukin-5 (IL-5) mediated diseases. [Background technology]

[0002] IL-5 plays a role in several different diseases, including asthma, severe eosinophilic asthma, severe asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis. These serious diseases affect hundreds of millions of people worldwide.

[0003] Mepolizumab is a monoclonal antibody that binds to soluble IL-5 and blocks its binding to its receptor. The structure of IL-5 indicates it is a secreted protein, and there is no evidence of membrane-bound IL-5 in any cell type. Therefore, Fc effector function is not part of mepolizumab's mechanism of action. Based on the mechanism of action and pharmacokinetic properties of mepolizumab, there are two functional domains involved in the biological activity of this monoclonal antibody: a) the complementarity-determining region (CDR) which binds to IL-5 to provide the mechanism of action; and b) the Fc region which binds to the neonatal Fc receptor (FcRn) to determine its half-life. Through detailed characterization studies conducted during product development, deamidation, oxidation, and aggregation were determined to be important quality characteristics of mepolizumab. Importantly, it was found that specific levels of these variants must be maintained to ensure appropriate biological function.

[0004] Therefore, there is a need for compositions to maintain the biological function of mepolizumab and to treat IL-5-mediated diseases. Such compositions and related methods are provided in this disclosure. [Overview of the project]

[0005] One aspect of the present disclosure is a composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% of acidic antibody variants.

[0006] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% acidic antibody variants and ≤20% agglutinating antibody variants.

[0007] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0008] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence; ≤55% of antibody variants oxidized at M64 of the heavy chain amino acid sequence; ≤3% of variants oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of agglutinated antibody variants.

[0009] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0010] Another aspect of the present disclosure is a composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence; ≤35% of antibody variants deamidated at N386 of the heavy chain amino acid sequence; ≤55% of antibody variants oxidized at M64, M254, and M430 of the heavy chain amino acid sequence; ≤3% of antibody variants oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of agglutinated antibody variants.

[0011] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, wherein the composition has a pH of 6.8 to 7.2, the buffer is histidine, phosphate, citrate, citrate or a salt thereof, the purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody is produced by Chinese hamster ovary cells.

[0012] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, wherein the composition has a pH of 6.8 to 7.2, the buffer is a phosphate or a salt thereof, the purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody is produced by Chinese hamster ovary cells.

[0013] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) the main form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0014] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the main form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the acidic form of the antibody, which accounts for about 20% to about 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0015] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) the major form of the antibody that occupies more than 50% of the protein in the composition when measured using capillary isoelectric focusing electrophoresis of the composition; and c) a basic form of the antibody that occupies from about 1% to about 15% of the protein in the composition when measured using capillary isoelectric focusing electrophoresis of the composition.

[0016] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) the major form of the antibody that occupies more than 50% of the protein in the composition when measured using capillary isoelectric focusing electrophoresis of the composition; c) an acidic form of the antibody that occupies from about 20% to about 45% of the protein in the composition when measured using capillary isoelectric focusing electrophoresis of the composition; and d) a basic form of the antibody that occupies from about 1% to about 15% of the protein in the composition when measured using capillary isoelectric focusing electrophoresis of the composition.

[0017] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a deamidated form of the antibody comprising at least one selected from deamidated heavy chain amino acid residues at asparagine 299, deamidated heavy chain amino acid residues at asparagine 317, deamidated heavy chain amino acid residues at asparagine 386, and deamidated light chain amino acid residues at asparagine 31.

[0018] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) an oxidized form of said antibody comprising at least one selected from a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4, and a light chain amino acid residue oxidized at cysteine 2

[0019] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: ;; b) a deamidated form of said antibody comprising at least one selected from a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine ;; a heavy chain amino acid residue deamidated at asparagine 386, and a light chain amino acid residue deamidated at asparagine 31; and c) an oxidized form of said antibody comprising at least one selected from a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4, and a light chain amino acid residue oxidized at cysteine 220

[0020] Another aspect of the present disclosure is an anti-IL-5 antibody comprising: a) a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31.

[0021] Another aspect of the present disclosure is an anti-IL-5 antibody comprising: a) a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) an oxidized form of the antibody comprising heavy chain amino acid residues oxidized with methionine 64.

[0022] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; b) an oxidized form of the antibody comprising heavy chain amino acid residues oxidized with methionine 64; and c) a deamidated form of the antibody comprising light chain amino acid residues deamidated with asparagine 31.

[0023] Another aspect of the present disclosure is an anti-IL-5 antibody comprising a) a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and a composition comprising a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31.

[0024] Another aspect of the present disclosure is an anti-IL-5 antibody comprising a) a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and a composition comprising b) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4.

[0025] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31; and c) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4.

[0026] Another aspect of this disclosure is a) the amino-terminal pyroglutamic acid residue of amino acid residue 1, the carboxy-terminal glycine amino acid residue of amino acid residue 448, the deamidated asparagine residue 299, the deamidated asparagine residue 317, the deamidated asparagine residue 386, the oxidized tryptophan residue 52, the oxidized methionine residue 64, the oxidized methionine residue 82, the oxidized methionine residue 85, the oxidized cysteine ​​residue 222, the oxidized methionine residue 254, the oxidized methionine residue 360 ​​and 4 The composition comprises a group of anti-IL-5 antibodies having: a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of oxidized methionine residue number 30; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, oxidized methionine residue number 4, and oxidized cysteine ​​residue number 220.

[0027] Another aspect of the present disclosure is a composition comprising a population of anti-IL-5 antibodies having a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of deamidated asparagine residue 299, deamidated asparagine residue 317, deamidated asparagine residue 386, oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 82, oxidized methionine residue 85, oxidized cysteine ​​residue 222, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of deamidated asparagine residue 31, oxidized methionine residue 4, and oxidized cysteine ​​residue 220.

[0028] Another aspect of the present disclosure is a composition comprising a population of anti-IL-5 antibodies having: a) a modified form of the antibody heavy chain amino acid sequence represented by SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of deamidated asparagine residue 299, deamidated asparagine residue 317, and deamidated asparagine residue 386; and b) a modified form of the antibody light chain amino acid sequence represented by SEQ ID NO: 2, comprising a deamidated asparagine residue at amino acid residue 31.

[0029] Another aspect of the present disclosure is a composition comprising a group of anti-IL-5 antibodies having: a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 82, oxidized methionine residue 85, oxidized cysteine ​​residue 222, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one selected from oxidized methionine residue 4 and oxidized cysteine ​​residue 220.

[0030] Another aspect of the present disclosure is a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) a composition comprising the main form of the antibody, which accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0031] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the primary form of the antibody, which accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the acidic form of the antibody, which accounts for up to approximately 80% of the protein in the composition when measured by capillary isoelectric focusing of the composition. [Brief explanation of the drawing]

[0032] [Figure 1] A representative capillary isoelectric focusing (cIEF) electrophoresis diagram of a reference standard (RS) composition containing mepolizumab. [Figure 2] Representative cIEF electrophoresis maps of different batches of a reference standard composition (control) containing mepolizumab and a composition containing mepolizumab that underwent forced degradation at pH 9.0 for 3 days. [Figure 3] A representative full-size exclusion chromatography (SEC) chromatogram of an RS composition containing mepolizumab. [Figure 4] A representative enlarged SEC chromatogram of an RS composition containing mepolizumab. [Figure 5] A typical SEC multi-angle light scattering (MALS) chromatogram of an RS composition containing mepolizumab. [Figure 6] A representative SEC-MALS chromatogram of a batch of a composition containing mepolizumab. [Figure 7] Representative SEC chromatograms of different batches of an RS composition containing mepolizumab and a pH 3.5 stress composition containing mepolizumab on day 7. [Modes for carrying out the invention]

[0033] This disclosure provides compositions for treating interleukin-5 (IL-5)-mediated diseases, and related subject matter.

[0034] As used herein, the term “asthma” means an inflammatory disorder of the airways characterized by reversible airway obstruction and bronchospasm. Common symptoms include wheezing, cough, chest tightness, and shortness of breath.

[0035] In the method of this disclosure, “asthma” may mean “severe eosinophilic asthma.” Subjects with severe eosinophilic asthma may have asthma and a blood eosinophil count of 300 or more per μL of blood in the past 12 months. Subjects with severe eosinophilic asthma may meet one or more of the criteria listed in Table 1.

[0036] [Table 1]

[0037] Importantly, subjects with severe eosinophilic asthma that meet these criteria may have fewer than 150 eosinophils per μL of blood at the start of treatment. Mepolizumab is a monoclonal antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2. Mepolizumab, and antigen-binding proteins comprising the heavy chain CDR and light chain CDR of mepolizumab, in particular the antibody molecule, can be used to treat severe eosinophilic asthma according to the methods of this disclosure. For example, mepolizumab, or related antigen-binding proteins, may be indicated for add-on maintenance therapy in patients with severe eosinophilic asthma identified by blood eosinophil counts of 300 cells / μL or higher in the past 12 months and / or blood eosinophil counts of 150 cells / μL or higher at the start of treatment and / or blood eosinophil counts of less than 150 cells / μL at the start of treatment. Alternatively, mepolizumab, or a related antigen-binding protein, may be prescribed for add-on maintenance therapy in patients with severe eosinophilic asthma, characterized by a serum eosinophil count of 300 cells / μL or higher in the past 12 months and / or a serum eosinophil count of 150 cells / μL or higher at the start of treatment. Mepolizumab, or a related antigen-binding protein, may be prescribed for add-on maintenance therapy in patients with severe eosinophilic asthma, characterized by a serum eosinophil count of 300 cells / μL or higher in the past 12 months and / or a serum eosinophil count of less than 150 cells / μL at the start of treatment. Such patients may be 12 years of age or older. Mepolizumab treatment may reduce asthma exacerbations in patients (e.g., patients with a history of exacerbations). The methods of this disclosure may be used when mepolizumab treatment is prescribed (i.e., such mepolizumab treatment may be used in combination with the methods of this disclosure). Mepolizumab treatment is: a) Reduced exacerbation frequency. Compared to placebo, treatment with mepolizumab, e.g., 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, may reduce the rates of 1) clinically significant exacerbations, 2) exacerbations requiring hospitalization or ED visits, and 3) exacerbations requiring hospitalization. This benefit may potentially lead to a reduction in morbidity and fatal asthma events.

[0038] b) Reduction in daily OCS dose: Treatment with mepolizumab, e.g., 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, may allow subjects to reduce their daily dose of parallel corticosteroids without experiencing a lack of asthma management. Subjects treated with mepolizumab may achieve a median percentage reduction of 50% from baseline in their daily oral corticosteroid (OCS) dose, compared to 0% in subjects treated with placebo. In addition, 54% of subjects treated with mepolizumab may achieve a reduction in their OCS dose to 5.0 mg, compared to 32% in subjects treated with placebo (p=0.025).

[0039] c) Improved lung function: Compared to placebo, mepolizumab treatment, e.g., 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, may demonstrate clinically relevant changes in FEV1 before and after bronchodilator administration. Any improvement in lung function is particularly significant because in this subject population, most patients receive maximal asthma therapy, including high-dose ICS (inhaled corticosteroids) and / or OCS + management medication.

[0040] d) Controls that improve asthma management: Mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, may result in statistically significant and clinically relevant improvements in ACQ-5 compared to placebo, indicating that subjects can achieve asthma management by adding mepolizumab to their existing asthma treatment.

[0041] e) Improved quality of life: Mepolizumab, administered subcutaneously at a dose of 100 mg per subject or intravenously at a dose of 75 mg per subject, may demonstrate statistically significant and clinically relevant changes in SGRQ scores compared to placebo. Subjects may experience significant improvements in asthma management and performance of daily activities.

[0042] f) Providing sustained efficacy and pharmacodynamic effects: During a 32-week and / or 52-week treatment period, sustained reductions in asthma exacerbations and blood eosinophils, as well as improvements in lung function, asthma management, and quality of life, may be observed without the development of resistance.

[0043] Furthermore, g) it leads to a reduction in blood eosinophils. Treatment with a composition comprising mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, can result in a rapid reduction in blood eosinophils (approximately 80% by the first assessment at 4 weeks after the first treatment; e.g., from 250-290 cells / μL to 40-60 cells / μL).

[0044] In the methods of this disclosure, “asthma” may mean “severe asthma.” Subjects with severe asthma meet the definition of severe asthma as described in the European Respiratory Society / American Thoracic Society (ERS / ATS) guidelines for severe asthma. Thus, severe asthma is asthma that has required treatment with a drug suggested by the Global Initiative for Asthma (GINA) guidelines for stage 4-5 asthma (high-dose inhaled corticosteroid [ICS] + long-acting β2-agonist [LABA] or leukotriene modifier / theophylline) in the past year, or systemic corticosteroid (CS) treatment in ≥50% of the past year to maintain control of the subject’s asthma. Treatment with a composition comprising mepolizumab may be used to treat severe asthma according to the methods of this disclosure.

[0045] In the method of this disclosure, "asthma" may refer to "poorly controlled uncontrolled occlusive asthma." Subjects with poorly controlled occlusive asthma meet the criteria set out in Table 2.

[0046] [Table 2]

[0047] Treatment with a composition comprising mepolizumab may be used to treat poorly controlled dysacinophilic asthma according to the methods disclosed herein.

[0048] In the method disclosed herein, "asthma" may refer to "eosinophilic asthma." Subjects with controlled eosinophilic asthma meet the criteria set out in Table 3.

[0049] [Table 3]

[0050] In the method disclosed herein, “asthma” may refer to “subeosinophilic asthma.” Subjects with controlled subeosinophilic asthma meet the criteria set out in Table 4.

[0051] [Table 4]

[0052] Treatment with a composition comprising mepolizumab can be used to treat eosinophilic asthma, and can also be used to treat eosinophilic asthma according to the method of this disclosure.

[0053] The term "bullous pemphigoid" (BP), as used herein, refers to an acute or chronic autoimmune skin disease involving the formation of herpes, more appropriately known as vesicles, in the space between the epidermis and dermis of the skin layers. BP is the most common autoimmune herpetic skin disease. BP typically affects older adults (>70 years) with an annual incidence of 5–35 per million people. The incidence of BP is increasing dramatically, averaging 17% per year. BP often begins with highly pruritic skin lesions resembling eczema or urticaria before vesicles and herpes develop. In 10–30% of patients, BP also involves the oral mucosa. The severity of the disease can be determined by means of the autoimmune bullous skin disorder intensity score (ABSIS), which assesses the affected areas and disease activity. This disease is caused by an autoimmune response to structural components of the junctional adhesion complex, resulting in damage to the dermal-epidermal junction accompanied by subepidermal herpes formation. Specifically, autoreactive B and T cell responses to hemiadhesion plaque antigens BP180 and BP230 have been identified. Serum levels of autoantibodies against BP180 reflect the severity and activity of the disease. T cells are memory CD4+ cells that produce both Th1 and Th2 cytokines, primarily IL-4, IL-5, and IL-13. IL-5 and eotaxin are abundant in herpes fluid. IL-5 production is indeed associated with serological eosinophilia and marked eosinophilic infiltration in the skin of BP patients. Eosinophils appear to be clinically involved in herpes formation by releasing toxic granular proteins (ESP, MBP) and proteolytic enzymes.

[0054] The term "eosinophilic esophagitis" (EoE), as used herein, refers to an allergic inflammatory condition of the esophagus involving eosinophils. Symptoms include dysphagia, food obstruction, and heartburn. EoE is characterized by dense infiltrations containing eosinophilic leukocytes in the esophageal lumen. EoE is considered an allergic reaction to ingested food, given the important role eosinophils play in allergic reactions. An EoE diagnostic panel can be used to diagnose EoE. EoE may also be diagnosed when gastroesophageal reflux does not respond to a 6-week trial of high-dose proton-pump inhibitors (PPIs) twice daily, or when a negative ambulatory pH study does not rule out gastroesophageal reflux disease (GERD). Endoscopically, ridges, grooves, or rings may be observed in the esophageal wall. In some cases, multiple rings may be seen in the esophagus, leading to the terms "corrugated esophagus" or "feline esophagus" due to the resemblance of these rings to those of a cat's esophagus. The presence of white exudate in the esophagus is also a diagnostic indicator. Biopsies taken during endoscopy generally show a large number of eosinophils in the surface epithelium. A minimum of 15 eosinophils per high-magnification field of view is required for diagnosis. Eosinophilic inflammation is not limited to the esophagus but can extend throughout the entire digestive tract. Importantly, degranulogenic eosinophils may also be present, as well as microabscesses and basal folds. Radiologically, the term "ringed esophagus" is used for the appearance of eosinophilic esophagitis on barium swallowing, in contrast to the appearance of transient transverse folds (called "feline esophagus") sometimes seen in esophageal reflux.

[0055] Treatment with a composition comprising mepolizumab may be used to treat COPD according to the methods disclosed herein.

[0056] Subjects with chronic obstructive pulmonary disease (COPD) may meet one or more of the following criteria: a) Previous COPD diagnosis: Subjects with a clinically documented history of COPD for at least one year, according to the definition by the American Thoracic Society / European Respiratory Society; b) Severity of COPD: Subjects may meet the following criteria to confirm a diagnosis of COPD: the ratio of forced expiratory capacity (FEV1 / FVC) measurements before and after salbutamol in one second may be <0.70; the FEV1 measurement after salbutamol may be >20 percent and the predicted normal value may be <=80 percent, calculated using the National Health and Nutrition Examination Survey (NHANES) III reference formula; c) History of exacerbations: A well-documented history of at least two moderate COPD exacerbations within a 12-month period (e.g., review of medical records). Moderate is defined as the use of systemic corticosteroids (IM, intravenous, or oral) and / or treatment with antibiotics, or at least one severe COPD exacerbation. Severe is defined as requiring hospitalization. Note: At least one exacerbation should have occurred if the subject was taking inhaled corticosteroids (ICS) + long-acting β2-agonist (LABA) + long-acting muscarinic antagonist (LAMA). Note: Prior use of antibiotics alone is not eligible as a moderate exacerbation unless its use was specifically for the treatment of a COPD exacerbation; and d) Parallel COPD therapy: Need for well-documented optimized standard of care (SoC) background therapy including ICS + 2 additional COPD doses (i.e., triple therapy) over the past 12 months and meeting the following criteria: at least 3 months of inhaled corticosteroids (dose >= 500 micrograms (mcg) / day equivalent to fluticasone propionate added) or LABA and LAMA use immediately prior to visit to a healthcare provider.

[0057] Treatment with a composition comprising mepolizumab may be used to treat COPD according to the methods disclosed herein.

[0058] The term "eosinophilic granulomatosis with polyangiitis" (EGPA), as used herein, refers to an autoimmune condition that causes inflammation (vasculitis) of small and medium-sized blood vessels in individuals with a history of airway allergic hypersensitivity (atopic dermatitis). EGPA may also be called Churg-Strauss syndrome (CSS) or allergic granulomatosis. EGPA typically manifests in three stages. The initial (prodromal) stage is characterized by airway inflammation, with almost all patients experiencing asthma and / or allergic rhinitis. The second stage is characterized by an abnormally large number of eosinophils (hypereosinophilia), which most often leads to tissue damage in the lungs and gastrointestinal tract. The third stage consists of vasculitis, which can eventually lead to cell death and be fatal.

[0059] Subjects with EGPA may meet one or more of the following criteria: a) asthma; b) serum eosinophil levels greater than 10% of white blood cell percentage; c) presence of mononeuropathy or polyneuropathy; d) uncertain pulmonary infiltrates; e) presence of sinus abnormalities; and e) histological evidence of extracapillary eosinophils. For classification purposes, a patient should be considered to have EGPA if at least four of the above six criteria are positive.

[0060] Treatment with a composition comprising mepolizumab may be used to treat EGPA according to the method of the present disclosure. The composition of the present disclosure may be administered to EGPA patients in a dose of 300 mg once every four weeks.

[0061] The term "eosinophilic syndrome" (HES), as used herein, refers to a persistently high total blood eosinophil count (≥1500 eosinophils / mm³) for at least six months, without any known cause, involving the heart, nervous system, or bone marrow. 3 This refers to a disease characterized by the following:

[0062] Subjects with eosinophilic syndrome may meet one or more of the following criteria: a) a recorded history of eosinophilic syndrome; b) a total blood eosinophil count greater than 1500 cells for the past six months; c) signs and symptoms of organ system involvement; and d) no evidence of parasitic, allergy or other cause of eosinophilia after overall assessment.

[0063] Treatment with a composition comprising mepolizumab may be used to treat eosinophilic syndrome according to the method of the present disclosure. The composition of the present disclosure may be administered to patients with eosinophilic syndrome at a dose of 300 mg once every four weeks.

[0064] As used herein, the term "nasal polyp" refers to a condition characterized by the presence of nasal polyps. Such polyps may be located in the upper nasal cavity and / or originate within the ostiomeatal complex.

[0065] A subject with nasal polyps may meet one or more of the following criteria: a) a recorded history of nasal polyps; or b) clear nasal polyps present during examination (e.g., endoscopy).

[0066] Treatment with a composition comprising mepolizumab may be used to treat nasal polyps according to the method of the present disclosure. The composition of the present disclosure may be administered to a patient with nasal polyps in a dose of 750 mg once every four weeks.

[0067] As used herein, the term “antibody” means a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this kind. Monoclonal antibodies can be produced by eukaryotic cell clones that express the antibody. Monoclonal antibodies can also be produced by eukaryotic cell lines that can recombinantly express the heavy and light chains of the antibody because they have nucleic acid sequences encoding them introduced into the cells. Methods for producing antibodies from various eukaryotic cell lines, such as Chinese hamster ovary cells, hybridomas, or immortalized antibody cells derived from animals (e.g., humans), are well known.

[0068] Antibodies may originate from rats, mice, primates (e.g., cynomolgus macaques, Old World monkeys, or great apes), humans, or other sources such as nucleic acids created using molecular biology techniques that encode antibody molecules.

[0069] The antibody may consist of a constant region which may be of any isotype or subclass. The constant region may be of an IgG isotype, e.g., IgG1, IgG2, IgG3, IgG4 or a variant thereof. The antigen-binding protein constant region may be IgG1.

[0070] The antigen-binding protein may comprise one or more modifications selected from the mutant constant domain such that the antibody has enhanced effector function / ADCC and / or complement activation.

[0071] Antibodies may have the ability to bind to target antigens. An example of such a target antigen is human IL-5, which contains the amino acid sequence shown in SEQ ID NO: 11.

[0072] Mepolizumab, comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2, is an example of an antibody. Mepolizumab binds to human IL-5 and antagonizes its activity.

[0073] Mepolizumab is a recombinant humanized monoclonal antibody (IgG1, κ). Mepolizumab has two light chains and two heavy chains.

[0074] The mepolizumab heavy chain is encoded by the nucleic acid sequence shown in Sequence ID No. 13. The mepolizumab heavy chain contains 449 amino acids and has an estimated molecular weight of approximately 49 kDa. The predicted mature heavy chain amino acid sequence of mepolizumab is as follows: [ka]

[0075] In the heavy chain amino acid sequence shown above, the heavy chain frameworks and CDRs according to the Kabat definition are identified, in order from the amino proximal portion to the carboxyl terminal of the shown sequence, as zigzag underlined framework 1, solid underlined CDR 1, zigzag underlined framework 2, solid underlined CDR 2, zigzag underlined framework 3, solid underlined CDR 3, and zigzag framework 4. In the heavy chain amino acid sequence shown above, an asterisk to the right of a single-letter amino acid code indicates that the amino acid residue to its left may be an N-glycosylation site.

[0076] The mepolizumab light chain is encoded by the nucleic acid sequence shown in Sequence ID No. 14. The mepolizumab light chain contains 220 amino acid residues and has an estimated molecular weight of approximately 24 kDa. The mature light chain amino acid sequence is as follows: [ka]

[0077] In the above light chain amino acid sequence, the light chain frameworks and CDRs according to the Kabat definition are identified, in order from the amino proximal portion to the carboxyl terminal portion of the shown sequence, as zigzag underlined framework 1, solid underlined CDR 1, zigzag underlined framework 2, solid underlined CDR 2, zigzag underlined framework 3, solid underlined CDR 3, and zigzag framework 4.

[0078] The mepolizumab heavy and light chains are covalently linked by a single disulfide bond, and the heavy chains are linked to each other by two disulfide bonds, forming a typical IgG molecule. Both heavy chains can be glycosylated with a complex branched oligosaccharide at asparagine 299. The predicted polypeptide molecular weight is approximately 146 kDa, the predicted carbohydrate molecular weight is approximately 3 kDa, and the estimated total molecular weight of mepolizumab is 149.2 kDa. The encoded mepolizumab has 1338 amino acid residues (220 amino acid residues per light chain and 449 amino acid residues per heavy chain). The major pI of mepolizumab is approximately 8.7–9.1. The equilibrium dissociation constant (K) of the molecular interaction between mepolizumab and human IL-5, as measured using a standard surface plasmon resonance assay, is... D ) is 2.29 × 10 -11 It is less than M.

[0079] Mepolizumab can be provided as a lyophilized powder containing an antibody and an excipient, which can be reconstituted with a pharmaceutically acceptable carrier (e.g., sterile water). This reconstituted pharmaceutical composition can then be administered subcutaneously or intravenously (e.g., after further dilution). Mepolizumab can also be provided as a liquid formulation containing an antibody, an excipient, and a pharmaceutically acceptable carrier. This liquid pharmaceutical composition can then be administered subcutaneously or intravenously (e.g., after further dilution).

[0080] The term "antibody variant," as used herein, means an antibody that differs from the parent antibody due to at least one amino acid modification (e.g., having a different amino acid side chain), post-translational modification, or other modification in at least one heavy chain, light chain, or combination thereof, resulting in a structural change (e.g., a different amino acid side chain, a different post-translational modification, or other modification) relative to the parent antibody. Mepolizumab is an example of such a parent antibody. Structural changes can be determined directly by various methods well known in the art, such as LC-MS and direct sequencing, or indirectly by methods such as isoelectric focusing. Such methods are well known to those skilled in the art.

[0081] As used herein, the term "IL-5" means human IL-5 comprising the amino acid sequence shown in Sequence ID No. 11.

[0082] When used herein in relation to antigen-binding proteins, the term "specifically binds" means that the antigen-binding protein binds to the target antigen and discontinuous domains or discontinuous amino acid sequences within the target antigen without binding to or with significant binding to other (e.g., unrelated) proteins. However, this term does not preclude the possibility that antigen-binding proteins may cross-react with closely related molecules (e.g., those with a high degree of sequence identity or those derived from a different genus or species). The antigen-binding proteins described herein may bind to human IL-5 or the human IL-5 receptor with at least 2, 5, 10, 50, 100, or 1000 times higher affinity than they would with closely related molecules.

[0083] Antigen-binding protein-target antigen interaction binding affinity (K D ) may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, K D It can be between 5 and 10 nM; or between 1 and 2 nM. Dcan be between 1 pM and 500 pM; or between 500 pM and 1 nM. The binding affinity of an antigen-binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). The binding affinity can be measured by BIACORE™, for example, by capturing a test antibody on the surface of a protein A-coated sensor and flowing a target antigen over this surface. Alternatively, the binding affinity can be measured by FORTEBIO, for example, by capturing a test antibody receptor on a protein A-coated needle and flowing a target antigen over this surface.

[0084] K d is 1×10 -3 Ms -1 Hereinafter, 1×10 -4 Ms -1 Hereinafter, or 1×10 -5 Ms -1 can be hereinafter. K d is 1×10 -5 Ms -1 ~1×10 -4 Ms -1 between; or 1×10 -4 Ms -1 ~1×10 -3 Ms -1 can be between. Slow K d can result in slow dissociation of the antigen-binding protein-target antigen complex and improved neutralization of the target antigen.

[0085] The term "specific antigen-binding activity" as used herein means the antigen-binding activity measured by surface plasmon resonance (SPR). The IL-5 specific binding activity can be determined by SPR using, for example, a BIACORE™ device operated in the binding mode. It is the quotient of the binding activity divided by the total protein (e.g., mepolizumab) content in the sample.

[0086] The term "FcRn binding activity," as used herein, refers to the neonatal Fc(FcRn) receptor binding activity measured by surface plasmon resonance (SPR). FcRn binding can be determined using a BIACORE® instrument. It is the quotient obtained by dividing the FcRn receptor binding activity by the total protein concentration of the sample.

[0087] For SPR methods involving specific antigen binding and FcRn binding, a mepolizumab reference standard is used. The mepolizumab reference standard can be used in the assay to obtain system suitability and sample comparability data, ensuring that the method is performed correctly. The reference standard allows for the establishment of a calibration curve, from which the sample concentration is extrapolated.

[0088] For example, the reference standard is a composition comprising SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the reference standard is a composition comprising SEQ ID NO: 1 and SEQ ID NO: 2, along with 98% or more HC C-terminal lysine deletion mutants and 95% or more HC N-terminal pyroglutamate mutants. In yet another embodiment, the reference standard is a composition comprising SEQ ID NO: 1 and SEQ ID NO: 2, along with 98% or more HC C-terminal lysine deletion mutants, 95% or more HC N-terminal pyroglutamate mutants, and 6% or less deamidation mutants. In yet another embodiment, the reference standard is a composition comprising SEQ ID NO: 1 and SEQ ID NO: 2, along with 98% or more HC C-terminal lysine deletion mutants, 95% or more HC N-terminal pyroglutamate mutants, 6% or less deamidation mutants, 4% or less methionine or cysteine ​​oxidation mutants, and 0.1% tryptophan oxidation mutants. In further embodiments, the reference standard is a composition comprising SEQ ID NOs: 1 and 2, as well as 98% or more HC C-terminal lysine deletion mutants, 95% or more HC N-terminal pyroglutamate mutants, 6% or less deamidation mutants, 4% or less methionine or cysteine ​​oxidation mutants, 0.1% or less tryptophan oxidation mutants, and 0.4% or less agglutination mutants. In another embodiment, the reference standard is a composition comprising isoforms represented by peaks 65, 78, 88, 92, the major peak, and 112 shown in Figure 1. In one embodiment, the reference standards were SEQ ID NOs: 1 and 2, with approximately 62.9% major peaks, approximately 35.9% acidic peaks, approximately 1.2% basic peaks, approximately 99.6% monomers, approximately 0.4% aggregates, approximately 0% fragments, approximately 0.8% HC deamidated N317, approximately 5.5% HC deamidated N386, approximately 5.2% HC deamidated N31, approximately 0.2% HC deamidated N299, approximately 0.9% HC oxidized M64, approximately 3.5% HC oxidized M254, approximately 0.5% HC oxidized M360, approximately 0.5% HC oxidized M430, approximately 0.3% HC oxidized M82 and M85, approximately 0.2% LC oxidized M4, approximately 0.0% LC oxidized C220, approximately 0.1% HC oxidized W52, and over 98% HC The composition comprises a C-terminal lysine deletion mutant and 95% or more of HC N-terminal pyroglutamic acid mutants.

[0089] In one embodiment, the composition has a specific IL-5 binding activity of ≥0.70 and an FcRn binding activity of ≥70%. For example, compared to a reference standard with a specific IL-5 binding activity of 1.0 and an FcRn binding activity of 100%, the specific antigen binding is in the range of 0.70 to 1.30; and / or the FcRn binding is in the range of 70% to 130%.

[0090] IL-5 Neutralizing ED 50 The ratio is the ED of the reference antibody standard (e.g., a mepolizumab antibody standard comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2). 50 The ED of the antibody sample (for example, a mepolizumab mutant sample or a sample from a production batch of a composition comprising a mepolizumab antibody containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2) 50 This is the quotient when divided by [a certain factor].

[0091] "Isolated" means that a molecule, such as an antigen-binding protein or nucleic acid, is intended to be removed from the environment in which it would normally be found. For example, this molecule may be purified from a substance in which it would normally be found. For instance, the mass of this molecule in the sample may be 95% of the total mass.

[0092] The term “V H " and "V L In this specification, " and " refer to the heavy chain variable region and the light chain variable region, respectively, of the antigen-binding protein.

[0093] "CDR" is defined as the complementarity-determining region amino acid sequence of an antigen-binding protein. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable region of an immunoglobulin contains three CDR heavy chains and three light chain CDRs (or CDR regions). Thus, as used herein, "CDR" means all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least one CDR, where at least one CDR is CDRH3. Framework regions follow each of these CDR regions. Acceptable heavy chain variable regions and light chain variable regions Framework 1, Framework 2, and Framework 3 regions are readily recognizable to those skilled in the art. Acceptable heavy chain constant regions (including hinge regions) and light chain constant regions are also readily recognizable to those skilled in the art. Acceptable antibody isotypes are also readily recognizable to those skilled in the art.

[0094] Throughout this specification, amino acid residues in variable domain sequences and full-length antibody sequences are numbered according to Kabat numbering rules. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, and “CDRH3” used herein also conform to Kabat numbering rules.

[0095] It will be obvious to those skilled in the art that there are other numbering rules for the amino acid residues in variable domain sequences and full-length antibody sequences. Furthermore, some CDR sequences also follow other numbering rules, such as the Chothia numbering rule. The structure of antibodies and protein folding may imply that other residues are also part of the CDR sequence, and this will be understood by those skilled in the art.

[0096] Other numbering rules for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. To identify the "minimum binding unit," the minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM, and contact methods. The minimum binding unit may be a portion of the CDR.

[0097] Table 5 below shows one definition for each CDR or binding unit using each numbering rule. In Table 5, the Kabat numbering scheme is used to number the variable domain amino acid sequences. Note that CDR definitions may vary depending on the individual publication used.

[0098] [Table 5]

[0099] The "identity percentage" between the query nucleic acid sequence and the subject nucleic acid sequence is an "identity" value expressed as a percentage, calculated by the BLASTN algorithm when the subject nucleic acid sequence has 100% query coverage with the query nucleic acid sequence after pairwise BLASTN alignment has been performed. Such pairwise BLASTN alignment between the query nucleic acid sequence and the subject nucleic acid sequence is performed with the low complexity region filter removed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute website. Importantly, the query sequence may be described by a nucleic acid sequence identified in one or more claims of this specification.

[0100] Nucleic acid sequences useful in and potentially included in the compositions and related methods of this disclosure may have identity with the nucleic acid sequences identified in this disclosure (e.g., nucleic acids encoding antibody heavy chains or antibody light chains) in the ranges of about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100%. In this disclosure, the identity percentages between the nucleic acid sequences described may also include any sub-range of discontinuities within the identity percentage ranges listed above (e.g., any range of integer values ​​within a particular range or any sub-range of discontinuities within a particular range).

[0101] The "identity percentage" between the query amino acid sequence and the subject amino acid sequence is an "identity" value expressed as a percentage, calculated by the BLASTP algorithm when the subject amino acid sequence has 100% query coverage with the query amino acid sequence after pairwise BLASTP alignment has been performed. Such pairwise BLASTP alignment between the query amino acid sequence and the subject amino acid sequence is performed with the low complexity region filter removed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute website. Importantly, the query sequence may be described by the amino acid sequence specified in one or more claims herein.

[0102] Amino acid sequences that are useful in and may be included in the compositions and related methods of this disclosure may have identity between approximately 85% and approximately 100%, approximately 90% and approximately 100%, approximately 95% and approximately 100%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, and approximately 100% of the amino acid sequences identified in this disclosure. In this disclosure, the percentage of identity between the amino acid sequences described may also include any sub-range of discontinuities within the identity percentage ranges listed above (e.g., any range of integer values ​​within a particular range or any sub-range of discontinuities within a particular range).

[0103] The terms "peptide," "polypeptide," "protein," and "peptide chain" each refer to molecules consisting of two or more amino acid residues. Peptides can be monomers or polymers.

[0104] It is well recognized in the art that certain amino acid substitutions are considered “conservative.” Amino acids are grouped based on common side-chain properties, and substitutions within a group that maintain all or substantially all of the binding affinity of an antigen-binding protein are considered conservative substitutions. See Table 6. Antigen-binding proteins disclosed herein may include such “conservative” amino acid substitutions.

[0105] [Table 6]

[0106] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to a patient.

[0107] The pharmaceutical compositions described herein may comprise the purified antibody preparations described herein.

[0108] For example, a pharmaceutical preparation may comprise a purified antibody preparation described herein in combination with a pharmaceutically acceptable carrier.

[0109] Generally, such pharmaceutical compositions comprise a pharmaceutically acceptable carrier required by known and acceptable pharmaceutical practices. Examples of such carriers include sterile carriers buffered to a pH in the range of 5 to 8 with a optionally preferred buffer, such as physiological saline, Ringer's solution, or dextrose solution.

[0110] The pharmaceutical composition may be administered by injection or infusion (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, or portal vein). Such compositions preferably do not contain visible granular material. The pharmaceutical composition may contain antigen-binding protein between 1 mg and 10 g, for example, between 5 mg and 1 g. Alternatively, the composition may contain antigen-binding protein between 5 mg and 500 mg, for example, between 5 mg and 50 mg.

[0111] Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. The pharmaceutical composition may contain, optionally, an antigen-binding protein in a unit dose of 1 mg to 10 g, along with instructions for use. The pharmaceutical composition may be freeze-dried for reconstitution before administration according to methods well known or obvious to those skilled in the art. If the antibody has the IgG1 isotype, a copper chelator such as citrate (e.g., sodium citrate) or EDTA or histidine may be added to the pharmaceutical composition to reduce the degree of degradation of this isotype of antibody mediated by copper. The pharmaceutical composition may also contain a solubilizer such as arginine, a surfactant / anti-aggregating agent such as polysorbate 80, and an inert gas such as nitrogen to replace oxygen in the headspace of the vial.

[0112] The term "therapeutically effective amount," as used herein, means the amount of a drug (such as an antibody or pharmaceutical composition) that provides a therapeutic benefit in the treatment or management of one or more symptoms of the condition being treated (e.g., asthma, severe eosinophilic asthma, controlled poor eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic syndrome, and nasal polyps). Examples of such treatment or management for one or more symptoms of severe eosinophilic asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, or subeosinophilic asthma include: 1) reduction in the frequency of asthma exacerbations; 2) reduction in time to a clinically significant first exacerbation requiring oral or systemic corticosteroids, hospitalization, and / or emergency department (ED) visit; 3) reduction in the frequency of exacerbations requiring hospitalization (including intubation and admission to the intensive care unit) or ED visit; 4) reduction in time to a first exacerbation requiring hospitalization or ED visit; 5) change from baseline in FEV1 before clinical bronchodilator administration; 6) change from baseline in FEV1 after clinical bronchodilator administration; 7) change from baseline in Asthma Control Questionnaire (ACQ) score; 8) vital capacity measurement (e.g., vital capacity (VC), forced vital capacity (CVC)). Improvements in lung function, including: forced expiratory volume (FEV), 25-75% forced expiratory flow (FEF25-75), and maximal voluntary ventilation (MVV) at time intervals of 0.5, 1.0 (FEV1), 2.0, and 3.0 seconds; actual lung volume including total lung volume, idal volume, residual volume, expiratory reserve, inspiratory reserve, inspiratory volume, inspiratory vital capacity, vital capacity, functional residual volume, residual volume expressed as a percentage of total lung volume, alveolar gas volume, volume of the inductive airways, and forced vital capacity; and reduction of asthma exacerbations requiring management with steroids (e.g., oral steroids or steroid-like prednisone, prednisolone, etc. - administered via any route).Such a reduction in asthma exacerbations requiring steroid management can amount to approximately a 50% reduction in exacerbations requiring steroids (e.g., oral steroids).

[0113] The therapeutically effective dosage and treatment plan are generally determined empirically and may vary depending on factors such as the patient's age, weight, and health status, as well as the disease or disorder being treated. Such factors are within the discretion of the attending physician.

[0114] The dosage of antigen-binding protein administered to a subject is generally between 1 μg / kg and 150 mg / kg, between 0.1 mg / kg and 100 mg / kg, between 0.5 mg / kg and 50 mg / kg, between 1 and 25 mg / kg, between approximately 0.3 mg / kg and approximately 3 mg / kg, or between 1 and 10 mg / kg of subject's body weight. For example, the dosage may be 10 mg / kg, 30 mg / kg, or 60 mg / kg. The dosage may also be between 10 mg / kg and 110 mg / kg, 15 mg / kg and 25 mg / kg, or 15 mg / kg and 100 mg / kg. Antigen-binding protein can be administered, for example, parenterally, subcutaneously, intravenously, or intramuscularly. Doses may also be administered on a per-subject basis, such as approximately 20 mg to approximately 750 mg per subject, approximately 75 mg to approximately 750 mg per subject, or approximately 20 mg to approximately 200 mg per subject. Doses may be any discontinuous sub-range within these dose ranges. For example, the dose (e.g., the dose of mepolizumab or a pharmaceutical composition comprising mepolizumab) may also be administered subcutaneously on a per-subject basis, such as approximately 100 mg per subject (e.g., once every four weeks), or 300 mg per subject (or other doses may be administered subcutaneously, as long as bioavailability approximately the same as or comparable to intravenous administration is achieved—e.g., three doses of 100 mg per subject to achieve a total subcutaneous dose of 300 mg per subject).

[0115] Any range described herein includes all values ​​within a given range and values ​​around the endpoints of that range.

[0116] Depending on the circumstances, the effective daily dose of the therapeutic composition may be administered in units of 2, 3, 4, 5, or 6 or more doses, each administered separately at appropriate intervals throughout the day.

[0117] The dose may be administered by slow intravenous infusion over a period of 2 to 24 hours, for example, 2 to 12 hours or 2 to 6 hours. Such administration may reduce side effects.

[0118] The dosage may be repeated, if necessary, one or more times, for example, three times a day, once daily, once every two days, once a week, once every 14 days, once a month, once every three months, once every four months, once every six months, or once every twelve months. This antigen-binding protein may also be administered as maintenance therapy once a week for a period of six months or more. This antigen-binding protein may also be administered as intermittent therapy in a cycle, for example, for a period of three to six months, followed by three to six months without administration, and then again for three to six months.

[0119] For example, the dose may be administered subcutaneously in multiple doses on each administration day, once every 14 or 28 days. In one embodiment, the dose of the composition is 100 mg once every 4 weeks (28 days).

[0120] This antigen-binding protein can be administered to a subject in a way that targets the therapy to a specific site.

[0121] The antigen-binding protein of the method disclosed herein may be used in combination with one or more other therapeutically active agents, such as antibodies or small molecule inhibitors.

[0122] The term “to treat” and its grammatical variations, as used herein, mean therapeutic treatment. With respect to a particular medical condition, to treat means (1) improving one or more of the biological signs of that condition; (2) a) interfering with one or more points in the biological cascade leading to or contributing to that condition; or b) interfering with one or more of the biological signs of that condition; (3) alleviating one or more symptoms, effects, or side effects associated with the condition or its treatment; (4) slowing the progression of one or more of the condition or its biological signs; or (5) preventing the onset of one or more of the biological signs of that condition. Prophylactic treatment is also intended by this. Those skilled in the art will recognize that “prevention” is not an absolute term. In medicine, “prevention” is understood to mean the prophylactic administration of a drug to substantially reduce the likelihood or severity of a medical condition or its biological signs, or to delay the onset of such a condition or its biological signs.

[0123] The terms “individual,” “subject,” and “patient” are used interchangeably herein. The subject is generally human. The subject may also be a mammal such as a mouse, rat, or primate (e.g., marmoset or monkey). The subject may also be a non-human animal. The antigen-binding proteins, compositions, and methods of this disclosure also have veterinary uses. The subject treated may be an agricultural animal, such as a cow or uncastrated bull, sheep, pig, castrated bull, goat, or horse, or a domestic animal such as a dog or cat. The animal may be of any age or may be a mature adult.

[0124] The treatment may be therapeutic, preventative, or prophylactic. The subject is one who needs it. Those who need treatment may include individuals who are at risk of developing the disease in the future, as well as individuals who already suffer from a particular medical condition.

[0125] Therefore, the methods, antigen-binding proteins, and compositions disclosed herein can also be used for preventive or prophylactic measures, if explicitly stated. In this case, the methods, antigen-binding proteins, and compositions disclosed herein can be used to prevent or delay the onset of one or more aspects or symptoms of a disease. The subject may be asymptomatic. The subject may have a genetic composition for the disease. A preventively effective amount of antigen-binding protein is administered to such an individual. A preventively effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

[0126] The methods, antigen-binding proteins, and compositions of this disclosure do not need to act to achieve a complete cure or to eradicate all symptoms or signs of the disease in order to make a viable therapeutic treatment. As is recognized in the art, a drug used as a therapeutic agent in a method of treatment may reduce the severity of a given condition, but does not need to eliminate all signs of the disease in order to be recognized as a useful therapeutic agent. Similarly, a treatment administered prophylactically does not need to be completely effective in preventing the onset of the disease in order to make a viable prophylactic agent. It is sufficient to merely reduce the effects of the disease (e.g., by reducing the number or severity of its symptoms, or by enhancing the effectiveness of another treatment, or by producing another beneficial effect), or by reducing the likelihood that the disease occurs (e.g., by delaying the onset of the disease) or worsens in the subject.

[0127] One aspect of the present disclosure is a composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% of acidic antibody variants.

[0128] In one embodiment, the composition has a) ≥ 0.70% IL-5 specific antigen binding; and / or b) ≥ 70% FcRn binding. IL-5 specific antigen binding, for example, to human IL-5 comprising the amino acid sequence of SEQ ID NO: 11, can be measured using standard assays such as surface plasmon resonance (e.g., BIACORE®), which are well known in the art. Similarly, FcRn binding can also be measured using standard assays such as surface plasmon resonance (e.g., BIACORE®), which are well known in the art.

[0129] In another embodiment, a) specific antigen binding is in the range of 0.70–1.30; and / or b) FcRn binding is in the range of 70%–130%. In some embodiments, specific antigen binding may be in the range of about 0.9–1.1, 0.75–about 1, about 0.7–about 0.8, about 0.7, about 0.91–about 0.95, about 0.994–about 0.997, or about 0.7–about 0.9. In some embodiments, FcRn bonding may range from about 70% to about 100%, about 100% to about 130%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 80% to about 90%, about 80% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 80% to about 120%, and about 90% to about 110%.

[0130] In one embodiment, the composition comprises ≤80% acid antibody variants. For example, the composition may comprise ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, or ≤45% acid antibody variants.

[0131] In another embodiment, the composition comprises ≤35% of the deamidation antibody variant.

[0132] In another embodiment, the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence. For example, the composition may comprise ≤22.5%, ≤20%, ≤17.5%, ≤15%, ≤12.5%, ≤10%, or ≤7.5% of antibody variants deamidated at N31 of the light chain amino acid sequence.

[0133] In another embodiment, the composition comprises ≤35% of antibody variants deamidated at N386 of the heavy chain amino acid sequence. For example, the composition may comprise ≤32.5%, ≤30%, ≤25.5%, or ≤20%, ≤17.5%, ≤15%, ≤12.5%, ≤10%, or ≤7.5% of antibody variants deamidated at N386 of the heavy chain amino acid sequence.

[0134] In another embodiment, the composition comprises ≤55% of oxidative antibody variants.

[0135] In another embodiment, the composition comprises ≤55% of an oxidized antibody variant with any one or a combination of a) heavy chain amino acid sequence M64; b) heavy chain amino acid sequence M254; and / or c) heavy chain amino acid sequence M430. For example, the composition may comprise ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, or ≤25%, ≤20%, ≤15%, ≤10%, or ≤5% of antibody variants oxidized with heavy chain amino acid sequences M64, M254, and / or M430.

[0136] In another embodiment, the composition comprises ≤3% of antibody variants oxidized at W52 of the heavy chain amino acid sequence. For example, the composition may comprise ≤2.5%, ≤2%, ≤1.5%, ≤1%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.25%, ≤0.2%, ≤0.15%, or ≤0.1% of antibody variants oxidized at W52 of the heavy chain amino acid sequence.

[0137] In another embodiment, the amount of deamidation antibody variants and / or oxidative variants is determined by peptide mapping LC-MS / MS.

[0138] In another embodiment, the composition comprises ≤20% of agglutinating antibody variants. For example, the composition may comprise ≤17.5%, ≤15%, ≤12.5%, ≤10%, ≤7.5%, ≤5%, or ≤4% of agglutinating variants. The composition may comprise 3%, 2%, 1%, or 0.5% or less of agglutinating antibody. The composition may comprise 98% or more of monomeric antibody.

[0139] In another embodiment, the agglutinating antibody variant comprises a dimer. Such an agglutinating antibody may comprise two antibody molecules (e.g., two IgG1 antibody molecules).

[0140] In another embodiment, the amount of aggregated antibody variant is determined by size exclusion chromatography (SEC). Methods for performing size exclusion chromatography and determining the size of protein molecules are well known in the art.

[0141] In another embodiment, the composition comprises ≥50% of a heavy chain amino acid sequence C-terminal lysine K449 deletion antibody variant. For example, the composition may comprise ≥60%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95% of a heavy chain amino acid sequence C-terminal lysine K449 deletion antibody variant.

[0142] In another embodiment, the composition comprises ≥50% of the heavy chain amino acid sequence pyroglutamic acid N-terminal antibody variant. For example, the composition may comprise ≥60%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95% of the heavy chain amino acid sequence pyroglutamic acid N-terminal antibody variant.

[0143] In another embodiment, the composition comprises host cell proteins (HCPs). The HCPs may be derived from CHO cells. HCPs are process-related impurities compared to mepolizumab product-related substances (i.e., mepolizumab + mepolizumab variants). Industry standard tolerance limits for HCPs may be up to 100 ppm (equivalent to 100 ng / mg). The HCP content in the compositions described herein may be ≤50 ng / mg, ≤40 ng / mg, ≤30 ng / mg, or ≤20 ng / mg. For example, the HCP content of the composition may be ≤10 ng / mg. In certain embodiments, the HCP content of the composition may be ≤5 ng / mg or ≤2 ng / mg.

[0144] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% acidic antibody variants and ≤20% agglutinating antibody variants.

[0145] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0146] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence; ≤5% of antibody variants oxidized at M64 of the heavy chain amino acid sequence; ≤3% of variants oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of agglutinated antibody variants.

[0147] Another aspect of the present disclosure is an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or a composition comprising an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0148] Another aspect of the present disclosure is a composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence; ≤35% of antibody variants deamidated at N386 of the heavy chain amino acid sequence; ≤55% of antibody variants oxidized at M64, M254, and M430 of the heavy chain amino acid sequence; ≤3% of antibody variants oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of agglutinated antibody variants.

[0149] The compositions of this disclosure may further comprise a buffer selected from the group consisting of disodium heptahydrate, phosphates, citric acid, citrates, sodium phosphate, potassium phosphate, sodium citrate, and histidine, providing a pH between 6.8 and 7.2 or a pH between 6.2 and 6.6 (a pH of 6.3 is preferred). The buffer in the compositions of this disclosure may be present in a range of about 10 to 30 mM, about 10 to 20 mM, about 20 mM, or about 15.5 mM. For example, the buffer in the compositions of this disclosure may be present in about 20 mM or about 15.5 mM of disodium heptahydrate.

[0150] The compositions of this disclosure may comprise a disodium heptahydrate and a citrate buffer providing a pH of 6.2 to 6.6 (a pH of 6.3 is preferred). The disodium heptahydrate buffer may be present in a range of about 15 to 16.4 mM, and the citrate buffer may be present in a range of about 3.8 to 4.9 mM. For example, the compositions of this disclosure may comprise about 15.5 mM of disodium heptahydrate and about 4.5 mM of citrate monohydrate.

[0151] The compositions of the present disclosure may further contain sugars. The compositions of the present disclosure may further contain sucrose. Sucrose may be present in the compositions of the present disclosure in amounts ranging from about 5 to 20%; about 10 to 15%, about 11 to 13%, or about 12% by weight / volume.

[0152] The compositions of this disclosure may further comprise polysorbate 80. Polysorbate 80 may be present in an amount ranging from about 0.01% to 0.1% by weight / volume. For example, polysorbate 80 may be present in the compositions of this disclosure in an amount of about 0.02% by weight / volume or about 0.05% by weight / volume.

[0153] The compositions of this disclosure may further comprise EDTA, which may be present in a concentration of about 0.01 to 0.1 mM. For example, EDTA may be present in a concentration of about 0.05 mM.

[0154] In one embodiment, the composition of the present disclosure further comprises 20 mM disodium phosphate heptahydrate, 12% by weight of sucrose by volume, and 0.05% by weight of polysorbate 80 by volume.

[0155] In another embodiment, the composition of the present disclosure further comprises 15.5 mM disodium phosphate, 3.9 mM citric acid monohydrate, 12% by weight of sucrose by volume, 0.02% by weight of polysorbate 80 by volume, and 0.05 mM EDTA.

[0156] The compositions of this disclosure may comprise an aqueous liquid formulation with a pH of 6.2 containing 16.1 mM disodium phosphate heptahydrate, 3.9 mM citric acid monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0157] The compositions of this disclosure may comprise an aqueous liquid formulation with a pH of 6.2 containing 15.2 mM disodium phosphate heptahydrate, 4.8 mM citric acid monohydrate, 12% by weight of sucrose by volume, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA by volume.

[0158] The compositions of this disclosure may comprise an aqueous liquid formulation with a pH of 6.4 containing 15.8 mM disodium phosphate heptahydrate, 4.2 mM citric acid monohydrate, 12% by weight of sucrose by volume, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA by volume.

[0159] The compositions of this disclosure may comprise an aqueous liquid formulation with a pH of 6.6 containing 16.3 mM disodium phosphate heptahydrate, 3.7 mM citric acid monohydrate, 12% by weight of sucrose by volume, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA by volume.

[0160] The compositions of the present disclosure may comprise an aqueous liquid formulation at pH 6.3 containing 15.5 mM disodium phosphate heptahydrate, 4.5 mM citric acid monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA by volume. Importantly, the tangential filtration and ultrafiltration exchange steps of Example 1 below may be prepared to produce the compositions of the present disclosure, for example, the compositions of the present disclosure comprising 15.5 mM disodium phosphate heptahydrate, 4.5 mM citric acid monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA at pH 6.3, or other such liquid formulations.

[0161] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, wherein the composition has a pH of 6.8 to 7.2, the buffer is histidine, phosphate, citrate, citrate or a salt thereof, the purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody is produced by Chinese hamster ovary cells. In the present composition, the heavy chain may comprise an amino acid sequence having at least 95%, 96%, 96.88%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1. In this composition, the light chain may comprise an amino acid sequence having at least 98%, 98.63%, or 99% identity with the amino acid sequence of Sequence ID No. 2.

[0162] In one embodiment, the buffer is at least one selected from disodium phosphate heptahydrate, phosphate, citric acid, and citrate.

[0163] In another embodiment, the buffer is sodium phosphate, potassium phosphate, or sodium citrate.

[0164] In another embodiment, the composition further comprises sugars, carbohydrates, and / or salts.

[0165] In another embodiment, the composition comprises sucrose.

[0166] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, wherein the composition has a pH of 6.8 to 7.2, the buffer is a phosphate or a salt thereof, the purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody is produced by Chinese hamster ovary cells.

[0167] In another embodiment, the buffer is at least one selected from disodium phosphate heptahydrate, phosphate, citric acid, and citrate.

[0168] In another embodiment, the composition further comprises sugar.

[0169] In another embodiment, the sugar is sucrose.

[0170] In another embodiment, the composition comprises polysorbate 80.

[0171] In another embodiment, the composition comprises one selected from: a first formulation of 20 mM disodium phosphate heptahydrate, 12% by weight of sucrose and 0.05% by weight of polysorbate 80 by volume; a second formulation of 15.5 mM disodium phosphate heptahydrate, 3.9 mM citric acid monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA by volume; and a third formulation of 26 mM disodium phosphate heptahydrate, 15% by weight of sucrose and 0.065% by weight of polysorbate 80 by volume. The composition may have a pH between about 6.8 and about 7.2, about 6.1 and about 6.5, or about 6 and about 6.6.

[0172] In another embodiment, the antibody is approximately 3.5 × 10⁶ against human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. -11 It has a dissociation constant less than or equal to M.

[0173] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) a major form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition. The major form of the antibody may also account for 57.9%, 59.4%, and 60% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0174] In another embodiment, the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0175] Another aspect of this disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the primary form of the antibody accounting for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the acidic form of the antibody accounting for approximately 20% to approximately 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition. The acidic form of the antibody may also account for 37.6%, 37.8%, 38.4%, and 39.8% of the protein in the composition when measured by capillary isoelectric focusing of the composition. The total acidic peak area determined by cIEF can be as high as 72%, while retaining 0.74 IL-5 specific binding and 80% FcRn binding.

[0176] In another embodiment, the acidic form of the antibody comprises at least one selected from the acidic form at peak 65, the acidic form at peak 78, the acidic form at peak 88, and the acidic form at peak 92.

[0177] In another embodiment, the acidic form of the antibody comprises at least one deamidated amino acid residue selected from the group consisting of a heavy-chain amino acid residue deamidated with asparagine 299, a heavy-chain amino acid residue deamidated with asparagine 317, a heavy-chain amino acid residue deamidated with asparagine 386, and a light-chain amino acid residue deamidated with asparagine 31. The acceptable level of deamidation on LC N31 is 17% or greater, or 17.4% or greater, as measured by peptide mapping LC MS / MS. The acceptable level of deamidation on HC 386 is 30% or greater, as measured by peptide mapping LC MS / MS. The upper acceptable level may be the level of a specific variant that causes the antibody molecule in the composition to retain an antigen-binding activity of about 0.70 to about 1.30 as measured by SPR and an FcRn-binding activity of about 70% to about 130% as measured by SPR, or other antigen-binding activity values ​​or FcRn-binding activity values, or ranges disclosed herein.

[0178] In another embodiment, the acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. Acceptable levels of oxidation on the heavy chain residues of the antibody, as measured by peptide mapping LC-MS / MS, may be about 50% for HC M64, M254, and M430, and about 3% for HC W52. The acceptable upper limit may be the level of a specific variant that causes the antibody molecule in the composition to retain an antigen-binding activity of about 0.70 to about 1.30 as measured by SPR and an FcRn-binding activity of about 70% to about 130% as measured by SPR, or other antigen-binding activity values ​​or FcRn-binding activity values ​​or ranges disclosed herein.

[0179] In another embodiment, the main form of the antibody includes at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. And so; the acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0180] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the major form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the basic form of the antibody, which accounts for about 1% to about 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition. The basic form of the antibody may also account for 2.2% and 2.3% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0181] In another embodiment, the basic form of the antibody includes the basic form of peak 112.

[0182] In another embodiment, the basic form of the antibody comprises a heavy chain having a carboxyl-terminal residue that is glycine 448.

[0183] In another embodiment, the basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0184] In another embodiment, the main form of the antibody includes at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​222.

[0185] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the main form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; c) the acidic form of the antibody, which accounts for about 20% to about 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition; and d) the basic form of the antibody, which accounts for about 1% to about 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0186] In another embodiment, the acidic form of the antibody contains at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​222.

[0187] In another embodiment, the primary form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; the acidic form of the antibody comprises a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, and a light chain amino acid residue oxidized with methionine 254 The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of heavy chain amino acid residues oxidized with methionine 360, heavy chain amino acid residues oxidized with methionine 430, light chain amino acid residues oxidized with methionine 4, and light chain amino acid residues oxidized with cysteine ​​220; the basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of heavy chain amino acid residues oxidized with tryptophan 52, heavy chain amino acid residues oxidized with methionine 64, heavy chain amino acid residues oxidized with methionine 82, heavy chain amino acid residues oxidized with methionine 85, heavy chain amino acid residues oxidized with cysteine ​​222, heavy chain amino acid residues oxidized with methionine 254, heavy chain amino acid residues oxidized with methionine 360, heavy chain amino acid residues oxidized with methionine 430, light chain amino acid residues oxidized with methionine 4, and light chain amino acid residues oxidized with cysteine ​​220.

[0188] Another aspect of the present disclosure is an anti-IL-5 antibody comprising a) a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and a composition comprising b) a deamidated form of the antibody comprising at least one selected from a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386, and a light chain amino acid residue deamidated with asparagine 31.

[0189] Another aspect of the present disclosure is an anti-IL-5 antibody comprising a) a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and a composition comprising b) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0190] Another aspect of the present disclosure is a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) a deamidated form of the antibody comprising at least one selected from a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386 and a light chain amino acid residue deamidated with asparagine 31; and c) tryptophan 5 The composition comprises an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with 2, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0191] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31. In this composition, CDRH2 may comprise an amino acid sequence having at least 85% or 87.5% identity with the amino acid sequence of SEQ ID NO: 6. In this composition, CDRL1 may comprise an amino acid sequence having 93%, 94%, or 94.11% identity with the amino acid sequence of SEQ ID NO: 8.

[0192] Another aspect of the present disclosure is an anti-IL-5 antibody comprising: a) a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) an oxidized form of the antibody comprising heavy chain amino acid residues oxidized with methionine 64.

[0193] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; b) an oxidized form of the antibody comprising heavy chain amino acid residues oxidized with methionine 64; and c) a deamidated form of the antibody comprising light chain amino acid residues deamidated with asparagine 31.

[0194] Another aspect of the present disclosure is an anti-IL-5 antibody comprising: a) a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31. In the present composition, the heavy chain variable region may comprise an amino acid sequence having at least 90%, 95%, or 95.57% identity with the amino acid sequence of SEQ ID NO: 3. In the present composition, the light chain variable region may comprise an amino acid sequence having at least 90%, 98%, or 98.31% identity with the amino acid sequence of SEQ ID NO: 3.

[0195] Another aspect of the present disclosure is an anti-IL-5 antibody comprising a) a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and a composition comprising b) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4.

[0196] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) a deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31; and c) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4.

[0197] In another embodiment, the total protein concentration is approximately 75 mg / mL. The total protein concentration may also be around any pair or single value in the range of approximately 75 mg / mL to approximately 150 mg / mL, for example, approximately 75 mg / mL to approximately 100 mg / mL, approximately 67.3 to approximately 87.5 mg / mL, approximately 76 g protein / L to approximately 82 g protein / L, approximately 46 g protein / L to approximately 66 g protein / L, or approximately 100 mg / mL. In this composition, the purity of the anti-human-IL-5 antibody in the sample is 97.0%, 96%, 95%, or 80%, 85% or higher.

[0198] In another embodiment, the composition further comprises a) the main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0199] In another embodiment, the composition further comprises: a) the primary form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and b) the acidic form of the antibody, which accounts for about 20% to about 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0200] In another embodiment, the composition further comprises: a) the major form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and b) the basic form of the antibody, which accounts for about 1% to about 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0201] In another embodiment, the composition further comprises: a) the primary form of the antibody, which accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; b) the acidic form of the antibody, which accounts for about 20% to about 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the basic form of the antibody, which accounts for about 1% to about 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0202] Another aspect of this disclosure is a) the amino-terminal pyroglutamic acid residue of amino acid residue 1, the carboxy-terminal glycine amino acid residue of amino acid residue 448, the deamidated asparagine residue 299, the deamidated asparagine residue 317, the deamidated asparagine residue 386, the oxidized tryptophan residue 52, the oxidized methionine residue 64, the oxidized methionine residue 82, the oxidized methionine residue 85, the oxidized cysteine ​​residue 222, the oxidized methionine residue 254, and the oxidized methionine residue 360. The composition comprises a group of anti-IL-5 antibodies having: a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of nin and oxidized methionine residue 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, oxidized methionine residue 4, and oxidized cysteine ​​residue 220.

[0203] In another embodiment, the composition comprises: a) a population comprising an amino-terminal pyroglutamic acid residue at amino acid residue 1 of the antibody heavy chain; b) a population comprising a carboxy-terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain; c) a population comprising 6.0% or less comprising a deamidated asparagine residue at amino acid residue 386 of the antibody heavy chain; d) a population comprising 1.5% or less comprising an oxidized methionine residue at amino acid residue 64 of the antibody heavy chain; e) a population comprising 4.5% or less comprising oxidized methionine at amino acid residue 254 of the antibody heavy chain; f) a population comprising 0.8% or less comprising an oxidized methionine residue at amino acid residue 430 of the antibody heavy chain; and g) a population comprising 6.6% or less comprising a deamidated asparagine residue at amino acid residue 31 of the antibody light chain.

[0204] In another embodiment, the composition comprises: a) approximately 92% to 99% of the population containing an amino-terminal pyroglutamic acid residue at amino acid residue 1 of the antibody heavy chain; b) approximately 95% to 99.5% of the population containing a carboxy-terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain; c) approximately 0.3% to 1.5% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; d) approximately 1.5% to 4.5% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; and e) approximately 0.5% to 1.5% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain. Furthermore, f) approximately 0.2% to 1.5% of the population contains an oxidized methionine residue at position 82 of the antibody heavy chain, or an oxidized methionine residue at position 85 of the antibody heavy chain; g) approximately 2.5% to 3.5% of the population contains oxidized methionine at position 254 of the antibody heavy chain; h) approximately 0.4% to 0.8% of the population contains an oxidized methionine residue at position 430 of the antibody heavy chain; i) approximately 3.3% to 6.6% of the population contains a deamidated asparagine residue at amino acid residue 31 of the antibody light chain; and j) approximately 0.1% to 1% of the population contains an oxidized methionine residue at position 4 of the antibody light chain.

[0205] In another embodiment, the composition comprises: a) approximately 93.7% to 98.6% of the population containing an amino-terminal pyroglutamic acid residue at amino acid residue 1 of the antibody heavy chain; b) approximately 97.6% to 99.2% of the population containing a carboxy-terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain; c) approximately 0.4% to 1.2% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; d) approximately 1.6% to 4.2% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; and e) approximately 0.7% to 0.9% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain. The population comprises the following: f) approximately 0.3% to 1.1% of the population contains an oxidized methionine residue at position 82 of the antibody heavy chain, or an oxidized methionine residue at position 85 of the antibody heavy chain; g) approximately 2.6% to 3.3% of the population contains oxidized methionine at position 254 of the antibody heavy chain; h) approximately 0.5% to 0.7% of the population contains an oxidized methionine residue at position 430 of the antibody heavy chain; i) approximately 3.4% to 6.5% of the population contains a deamidated asparagine residue at amino acid residue 31 of the antibody light chain; and j) approximately 0.2% to 0.8% of the population contains an oxidized methionine residue at position 4 of the antibody light chain.

[0206] Another aspect of the present disclosure is a composition comprising a population of anti-IL-5 antibodies having a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine ​​residue at position 222, an oxidized methionine residue at position 254, an oxidized methionine residue at position 360, and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4, and an oxidized cysteine ​​residue at position 222.

[0207] In another embodiment, the composition comprises: a) about 0.3% to about 1.5% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; b) about 1.5% to about 4.5% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.5% to about 1.5% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain; and d) about 0.2% to about 1.5% of the population containing an oxidized methionine residue at position 82 of the antibody heavy chain, or an antibody The heavy chain contains an oxidized methionine residue at position 85; e) approximately 2.5% to 3.5% of the population contains oxidized methionine at position 254 of the antibody heavy chain; f) approximately 0.4% to 0.8% of the population contains an oxidized methionine residue at position 430 of the antibody heavy chain; g) approximately 3.3% to 6.6% of the population contains a deamidated asparagine residue at amino acid residue 31 of the antibody light chain; and h) approximately 0.1% to 1% of the population contains an oxidized methionine residue at position 4 of the antibody light chain.

[0208] In another embodiment, the composition comprises: a) about 0.4% to about 1.2% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; b) about 1.6% to about 4.2% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.7% to about 0.9% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain; and d) about 0.3% to about 1.1% of the population containing an oxidized methionine residue at position 82 of the antibody heavy chain, or the antibody heavy The antibody chain contains an oxidized methionine residue at position 85, and e) approximately 2.6% to 3.3% of the population contains oxidized methionine at position 254 of the antibody heavy chain, f) approximately 0.5% to 0.7% of the population contains an oxidized methionine residue at position 430 of the antibody heavy chain, g) approximately 3.4% to 6.5% of the population contains a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and h) approximately 0.2% to 0.8% of the population contains an oxidized methionine residue at position 4 of the antibody light chain.

[0209] Another aspect of the present disclosure is a composition comprising a group of anti-IL-5 antibodies having: a) a modified form of the antibody heavy chain amino acid sequence represented by SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, and a deamidated asparagine residue at position 386; and b) a modified form of the antibody light chain amino acid sequence represented by SEQ ID NO: 2, comprising a deamidated asparagine residue at amino acid residue 31.

[0210] In another embodiment, the composition comprises a) about 0.3% to about 1.5% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; b) about 1.5% to about 4.5% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.3% to about 6.6% of the population containing a deamidated asparagine residue at amino acid residue 31 of the antibody light chain.

[0211] In another embodiment, the composition comprises a) about 0.4% to about 1.2% of the population containing a deamidated asparagine residue at position 317 of the antibody heavy chain; b) about 1.6% to about 4.2% of the population containing a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.4% to about 6.5% of the population containing a deamidated asparagine residue at amino acid residue 31 of the antibody light chain.

[0212] Another aspect of the present disclosure is a composition comprising a group of anti-IL-5 antibodies having a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 82, oxidized methionine residue 85, oxidized cysteine ​​residue 222, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of oxidized methionine residue 4 and oxidized cysteine ​​residue 220.

[0213] In another embodiment, the composition comprises a) about 0.5% to about 1.5% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain, b) about 0.2% to about 1.5% of the population containing an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain, c) about 2.5% to about 3.5% of the population containing oxidized methionine at position 254 of the antibody heavy chain, d) about 0.4% to about 0.8% of the population containing an oxidized methionine residue at position 430 of the antibody heavy chain, and e) about 0.1% to about 1% of the population containing an oxidized methionine residue at position 4 of the antibody light chain.

[0214] In another embodiment, the composition comprises a) approximately 0.7% to 0.9% of the population containing an oxidized methionine residue at position 64 of the antibody heavy chain, b) approximately 0.3% to 1.1% of the population containing an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain, c) approximately 2.6% to 3.3% of the population containing oxidized methionine at position 254 of the antibody heavy chain, d) approximately 0.5% to 0.7% of the population containing an oxidized methionine residue at position 430 of the antibody heavy chain, and e) approximately 0.2% to 0.8% of the population containing an oxidized methionine residue at position 4 of the antibody light chain.

[0215] Another aspect of the present disclosure is a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) a composition comprising the main form of the antibody, which accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0216] Another aspect of the present disclosure is a composition comprising: a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) the primary form of the antibody, which accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) the acidic form of the antibody, which accounts for up to approximately 80% of the protein in the composition when measured by capillary isoelectric focusing of the composition.

[0217] In another embodiment, the composition is for the treatment of diseases selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, controlled poor eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis.

[0218] Another embodiment is a method for treating a disease in a subject, comprising: a) identifying a subject having a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, controlled poor eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis; and b) administering a therapeutically effective amount of the composition of the present disclosure to the subject; thereby treating the disease in the subject.

[0219] Another embodiment is a method for producing the composition of the present disclosure, comprising: a) expressing in host cells an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence; b) growing the cells at a pH of about 6.75 to about 7.00; c) collecting the cell culture supernatant; d) contacting the cell culture supernatant with a protein A resin or a protein G resin to bind antibody molecules; e) eluting the antibody molecules from the resin to obtain a first eluate; f) treating the first eluate at a pH of about 3.3 to about 3.7 for about 15 to about 240 minutes to obtain a treated first eluate; g) The method comprises the steps of: h) contacting the eluate with an ion exchange resin at a load pH of approximately 8.3 to approximately 8.7; i) recovering a second eluate (flow-through eluate) from the ion exchange resin and holding it for no more than approximately 96 hours; j) treating the second eluate with guanidine and ammonium sulfate to obtain a solution; k) contacting the solution with a hydrophobic interaction chromatography resin bed at a load ratio of approximately 12 g protein / L resin to approximately 27 g protein / L resin; k) eluting a third eluate containing antibody molecules from the hydrophobic interaction chromatography resin at an elution gradient volume of approximately 9 resin bed volume to approximately 11 resin bed volume and an elution peak cut-off stop of approximately 17% to approximately 23% of the maximum peak height; and l) formulating the third eluate; thereby producing the composition of the present disclosure. The method of this disclosure provides any nucleic acid sequence suitable for expressing an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence. For example, the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 can be used to express an antibody in eukaryotic cells.Alternatively, other nucleic acid sequences having different sequences encoding the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 or the antibody light chain amino acid sequence shown in SEQ ID NO: 2 (e.g., for the use of selective codons) may also be used. In the method of this disclosure, deamidation can be controlled by growing cells at a pH of about 6.75 to about 7.00. In the method of this disclosure, deamidation can be controlled by growing cells for about 12 to about 18 days at an in vitro cell age of 166 days or less. In the method of this disclosure, deamidation can be controlled by contacting the treated first eluate with an ion exchange resin at a load pH of about 8.3 to about 8.7, recovering the second eluate from the ion exchange resin, and holding it for up to about 96 hours. In the method of the present disclosure, aggregation can be controlled by contacting a hydrophobic interaction chromatography resin bed with a phenylSEPHAROSE® fast-flow chromatography at a loading ratio of approximately 12 g protein / L resin to approximately 27 g protein / L resin; and eluting a third eluate containing antibody molecules from the hydrophobic interaction chromatography resin at an elution gradient volume of approximately 9 resin bed volume to approximately 11 resin bed volume and an elution peak cut-off stop of approximately 17% to approximately 23% of the maximum peak height. Aggregation can also be limited to approximately 6 hours after the final filtration, filling, and freezing of the pharmaceutical composition of the present disclosure. Importantly, any of the steps of the disclosed method may be omitted or combined to produce the composition of the present disclosure.

[0220] Post-translational modifications can occur during antibody production. These may include cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns, deamidation (e.g., at asparagine or glutamine residues), oxidation (e.g., at methionine, tryptophan, or free cysteine ​​residues), disulfide bond scrambling, isomerization (e.g., at aspartic acid residues), C-terminal resync-clipping (e.g., from one or both heavy chains), and N-terminal glutamine cyclization (e.g., in the heavy and / or light chains).

[0221] The antibody composition may comprise (i) an antibody (i.e., an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2); and (ii) an antibody variant comprising one or more or a combination of charge variants (e.g., acidic and basic variants), amino acid sequence variants, and antibody structure variants (e.g., agglutination and fragmentation variants).

[0222] Acidic or basic antibody variants can be characterized and identified from antibodies based on their overall acidic or basic charge. For example, the charge distribution of an antibody composition can be detected using capillary isoelectric focusing (cIEF) or ion-exchange chromatography. Acidic variants may include deamidated antibody variants, glycated antibody variants, sialylated antibody variants, and oxidized antibody variants. Cysteine ​​and tryptophan oxidation in antibody variants results in a pI shift (i.e., a difference in charge) which is detected in other acidic antibody variants. Methionine oxidation in antibody variants can be monitored by changes in antigen binding or by peptide mapping, for example, by LC-MS / MS.

[0223] Deamidation is an enzymatic reaction that primarily converts asparagine (N) to isoaspartic acid (isoaspartaginate) (iso-D) and aspartic acid (aspartaginate) (D) in a ratio of approximately 3:1. Therefore, this deamidation reaction is related to the isomerization of aspartaginate (D) to isoaspartaginate. Both the deamidation of asparagine and the isomerization of aspartaginate involve the intermediate succinimide. Deamidation can also occur at glutamine residues, albeit to a much lesser degree. Deamidation can occur within the CDR, Fab (non-CDR region), or Fc region.

[0224] Deamidation alters the charge of the antibody, resulting in a deamidated antibody variant being more acidic than the original antibody. An antibody composition may contain ≤35% deamidated antibody variants. For example, N31 of the light chain may be deamidated to iso-D, D, or succinimide. An antibody composition may also contain ≤25% of antibody variants deamidated at position 31 of the light chain. This can result in, for example, a single amino acid change in the sequence of the antibody's light chain in ≤25% of the antibody composition.

[0225] For example, N386 of the heavy chain may be deamidated to iso-D, D, or succinimide. The antibody composition may contain ≤35% of antibody variants deamidated at position 386 of the heavy chain. This may result in, for example, ≤35% of the antibody composition having one amino acid change in the sequence of the antibody's heavy chain. The composition may contain a mixture of antibody variants. Since deamidation events can accumulate, two or more asparagine residues may be deamidated. Therefore, the antibody composition may contain at least one amino acid change in the sequence of the antibody's heavy chain and / or at least one amino acid change in the sequence of the antibody's heavy chain. For example, the antibody composition may contain antibody variants deamidated at position 31 of the light chain and antibody variants deamidated at position 386 of the heavy chain.

[0226] Oxidation can occur during production and storage (i.e., in the presence of oxidative conditions) and results in covalent modifications of proteins, induced directly by reactive oxygen species or indirectly by reactions with secondary byproducts of oxidative stress. Oxidation primarily occurs at methionine residues, but can also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in CDR, Fab (non-CDR), or Fc regions.

[0227] Oxidation can cause a change in the charge of an antibody, resulting in oxidized antibody variants being more acidic than the antibody. Some oxidized antibody variants have the same charge as the antibody. An antibody composition may contain ≤55% oxidized antibody variants. For example, one or a combination of M64, M254, and / or M430 in the heavy chain may be oxidized. An antibody composition may contain ≤55% antibody variants oxidized at one or a combination of M64, M254, and / or M430 in the heavy chain. For example, W52 in the heavy chain may be oxidized. An antibody composition may contain ≤3% antibody variants oxidized at W52 in the heavy chain.

[0228] This composition may comprise a mixture of antibody variants. Accordingly, the antibody composition may comprise at least one amino acid change in the sequence of the antibody heavy chain and / or at least one amino acid change in the sequence of the antibody heavy chain. For example, the antibody composition may comprise an antibody variant deamidated at position 31 of the light chain; and / or an antibody variant deamidated at position 386 of the heavy chain; and / or oxidation of any one or a combination of M64, M254, and / or M430 and / or W52 of the heavy chain.

[0229] Scrambling of disulfide bonds can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds may break or fail to form properly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryl (-SH) can promote shuffling.

[0230] The N-terminal glutamine (Q) and glutamate (glutamic acid) (E) of the heavy and / or light chains are thought to form pyroglutamates (pGlu) via cyclization. While most pGlu formation is thought to occur within the production bireactor, it may also be formed non-enzymatically depending on the pH and temperature of the processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in natural human antibodies. The antibody compositions described herein may contain pGlu at the N-terminus of the antibody in ≥50% of cases. pGlu may be present in the heavy chain. This may result in a single amino acid change in the sequence of the heavy or light chain of the antibody in, for example, ≥50% of the antibody compositions.

[0231] This composition may comprise a mixture of antibody variants. Since sequence changes can accumulate, this composition may comprise two or more sequence changes in the heavy chain and / or light chain. Accordingly, this antibody composition may comprise at least one amino acid change in the sequence of the antibody heavy chain and / or at least one amino acid change in the sequence of the antibody heavy chain. For example, the antibody composition may comprise an antibody variant deamidated at position 31 of the light chain; and / or an antibody variant deamidated at position 386 of the heavy chain; and / or oxidation of any one or a combination of M64, M254, and / or M430 and / or W52 of the heavy chain; and / or pGlu at the N-terminus of the heavy chain and / or light chain.

[0232] C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and native human antibodies. Variations of this process include the removal of lysine from one or both heavy chains by cellular enzymes from recombinant host cells. Administration to human subjects / patients may result in the removal of any remaining C-terminal lysine. The antibody compositions described herein may contain ≥50% C-terminal lysine deletion at the C-terminus of the antibody. K449 may be deleted in one or both heavy chains of the antibody. Thus, two antibody variants exist: single lysine deletion in the heavy chain and double lysine deletion in the heavy chain. This antibody (i.e., an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2) has both lysines intact / present. This means, for example, that ≥50% of the antibody compositions may result in a single amino acid change in the sequence of the antibody's heavy chain.

[0233] The composition may comprise a mixture of antibody variants. For example, the antibody composition may comprise an antibody variant deamidated at position 31 of the light chain; and / or an antibody variant deamidated at position 386 of the heavy chain; and / or oxidation of any one or a combination of M64, M254, and / or M430 and / or W52 of the heavy chain; and / or pGlu at the N-terminus of the heavy chain and / or light chain; and / or a C-terminal lysine deletion at the C-terminus.

[0234] Aggregated or fragmented antibody variants can be characterized and identified from the antibody based on their size. For example, the size distribution of an antibody composition can be detected using size exclusion chromatography (SEC).

[0235] The antibody composition may comprise ≤20% of agglutinating antibody variants. The agglutinating antibody variants may comprise dimers. The composition may comprise a mixture of antibody variants. For example, the antibody composition may comprise an antibody variant deamidated at position 31 of the light chain; and / or an antibody variant deamidated at position 386 of the heavy chain; and / or oxidation of any one or a combination of M64, M254, and / or M430 and / or W52 of the heavy chain; and / or pGlu at the N-terminus of the heavy chain and / or light chain; and / or C-terminal lysine deletion at the C-terminus; and / or agglutinating antibody variants.

[0236] The compositions described may have undergone or are subject to one or more post-translational modifications. These modifications may occur in the CDR, variable framework region, or constant region. These modifications may result in changes to the molecular charge. The post-translational modifications and changes in the primary amino acid sequence described above do not result in significant changes to the antigen-binding affinity, bioactivity, PK / PD, aggregation, immunogenicity, or Fc receptor binding of the compositions. The compositions are substantially free of impurities.

[0237] Antibody compositions comprising the above-mentioned antibodies and antibody variants retain specific antigen binding and / or FcRn binding. For example, an antibody composition comprising the above-mentioned antibodies and antibody variants has ≥0.70% IL-5 specific antigen binding; and / or ≥70% FcRn binding. Therefore, these levels (%) of variants can be tolerated in antibody compositions without affecting function.

[0238] The compositions described herein may be produced by any of several prior arts. For example, the compositions may be expressed in a recombinant expression system and purified therefrom. In one embodiment, the compositions are produced by culturing host cells under conditions suitable for the expression of a polypeptide comprising SEQ ID NO: 1 and SEQ ID NO: 2, thereby expressing the compositions, optionally purifying them, and optionally incorporating them into a pharmaceutical composition.

[0239] Several different expression systems and purification schemes are available for producing this composition. Generally, host cells are transformed with a recombinant expression vector encoding an antibody. A wide range of host cells are available, including mammalian eukaryotic cell lines (e.g., CHO, Perc6, HEK293, HeLa, NS0). Suitable host cells include mammalian cells such as CHO (e.g., CHOK1 and CHO-DG44).

[0240] The host cell can be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., a plant or animal). The host cell may be a non-human host cell.

[0241] Suitable cloning and expression vectors and cloning methods for use with eukaryotic or mammalian cell hosts are known in the art.

[0242] The cells can be cultured under conditions that promote the expression of the antibody. For example, a production bioreactor is used to culture the cells. The production bioreactor capacity may be (i) about 20,000 liters, about 10,000 liters; about 5,000 liters; about 2,000 liters; about 1,000 liters; or about 500 liters; or (ii) between 500 and 20,000 liters; between 500 and 10,000 liters; between 500 and 5,000 liters; between 1,000 and 10,000 liters; or between 2,000 and 10,000 liters. For example, the cells can be cultured in the production bioreactor at a pH of about 6.75 to 7.00. Alternatively, the cells can be cultured in the production bioreactor for about 12 to about 18 days. Alternatively, the cells can be cultured in a production bioreactor at a pH of approximately 6.75 to 7.00 for approximately 12 to 18 days. This culture process can help control the level of deamidation antibody variants, for example, to reduce the level of deamidation antibody variants.

[0243] This composition can be recovered and purified by conventional protein purification procedures. For example, the composition may be collected directly from the culture medium. Collection of the cell culture medium may be done, for example, by centrifugation and / or deep filtration for clarification. After the recovery of the composition, purification is performed to ensure sufficient purity.

[0244] For purification, one or more chromatography steps, for example, one or more chromatography resins; and / or one or more filtration steps may be used. To purify the composition, affinity chromatography using a resin such as protein A, G, or L may be used. Alternatively, or in addition thereto, an ion exchange resin such as a cation exchange resin may be used to purify the composition. Alternatively, or in addition thereto, a hydrophobic interaction chromatography resin may be used to purify the composition. Or, the purification step may consist of an affinity chromatography resin step, followed by a cation exchange resin step, followed by a hydrophobic interaction chromatography resin step.

[0245] For example, the sample is brought into contact with a protein A resin. A solution containing this composition is eluted from the protein A resin and treated at pH 3.3 to 3.7 for 15 to 240 minutes. This protein A resin step can help control the level of agglutinating antibody variants, for example, to reduce the level of agglutinating antibody variants.

[0246] Next, the solution containing the composition may be further clarified by deep filtration and / or double-layer filtration.

[0247] Alternatively, or in addition to the above, an anion exchange resin may be used. A solution containing this composition can be brought into contact with an anion exchange resin (e.g., Q-SEPHAROSE® fast-flow anion exchange chromatography) at a load pH of 8.3 to 8.7. The solution containing this composition can be eluted from the anion exchange resin and retained for up to 96 hours. This anion exchange resin step can help control the level of deamidation antibody variants, for example, to reduce the level of deamidation antibody variants.

[0248] Optionally, guanidine and / or ammonium sulfate may be added to the solution containing the composition and held for 15 to 240 minutes.

[0249] Alternatively, or in addition to the above, a hydrophobic interaction chromatography resin may be used. A solution containing the composition may be contacted with a hydrophobic interaction chromatography resin (e.g., Phenyl SEPHAROSE® Fast Flow Chromatography) at a load ratio of 12 to 27 g protein / L resin. For example, a solution containing the composition may be eluted using an elution gradient volume (bed volume; BV) of about 9 to about 11. An elution peak cut stop (%) of about 17 to about 23 may be used during elution from the hydrophobic interaction chromatography resin. This hydrophobic interaction chromatography resin step can help control the level of agglutinated antibody variants, for example, to reduce the level of agglutinated antibody variants.

[0250] Next, the solution containing the composition may be filtered to remove the virus. The solution containing the composition may then be formulated at antibody concentrations of approximately 76 g protein / L to approximately 82 g protein / L, or approximately 100 g protein / L. The solution containing the composition may be filled into containers and frozen. Aliquots of the solution containing the composition may be freeze-dried. The freeze-dried product may be reconstituted by adding water to prepare a composition with a pH of approximately 6.8 to approximately 7.2, comprising 75 mg / L of protein, monoclonal anti-IL-5 mepolizumab antibody, 20 mM disodium phosphate heptahydrate, 12% by weight of sucrose and 0.05% by weight of polysorbate 80 by volume.

[0251] In another embodiment, the compositions of the present disclosure are produced using this method for producing the compositions of the present disclosure.

[0252] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, the composition having a pH of 6.2 to 6.6, the buffer being histidine, phosphate, citric acid, citric acid monohydrate, citrate or a salt thereof, the purified preparation comprising isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprising a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody being produced by Chinese hamster ovary cells.

[0253] Another aspect of the present disclosure is a composition comprising a purified monoclonal antibody preparation and a buffer, wherein the composition has a pH of 6.2 to 6.6, the buffer is a phosphate or a salt thereof, the purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, a major peak and 112 as shown in Figure 1, the antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and the antibody is produced by Chinese hamster ovary cells.

[0254] In another embodiment of the composition of the present disclosure, the buffer is at least one selected from disodium phosphate heptahydrate, phosphates, and citric acid.

[0255] Another embodiment of the composition of the present disclosure includes a first formulation comprising 16.1 mM disodium phosphate heptahydrate, 3.9 mM citric acid, 12% by weight sucrose by volume, 0.02% by weight polysorbate 80 and 0.05 mM EDTA by volume; a second formulation comprising 15.2 mM disodium phosphate heptahydrate, 4.8 mM citric acid, 12% by weight sucrose by volume, 0.02% by weight polysorbate 80 and 0.05 mM EDTA by volume; and 15.8 mM disodium phosphate heptahydrate, 4.2 mM citric acid, and 12% by weight sucrose by volume. A third formulation comprising 0.02% by weight of polysorbate 80 and 0.05 mM of EDTA relative to the volume; a fourth formulation comprising 16.3 mM of disodium phosphate heptahydrate, 3.7 mM of citric acid, 12% by weight of sucrose relative to the volume, 0.02% by weight of polysorbate 80 and 0.05 mM of EDTA relative to the volume; and a fifth formulation comprising 15.5 mM of disodium phosphate heptahydrate, 4.5 mM of citric acid, 12% by weight of sucrose relative to the volume, 0.02% by weight of polysorbate 80 and 0.05 mM of EDTA relative to the volume.

[0256] In summary, this disclosure includes the following: 1. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% of acidic antibody variants.

[0257] 2. a) IL-5 specific antigen binding ≥ 0.70; and / or b) FcRn binding ≥ 70% The composition according to claim 1, having the following characteristics.

[0258] 3. The composition according to 2, wherein a) the specific antigen binding is in the range of 0.70 to 1.30; and / or b) the FcRn binding is in the range of 70% to 130%.

[0259] 4. A composition according to any one of 1 to 3, comprising ≤ 35% of a deamidation antibody mutant.

[0260] 5. A composition according to any one of 1 to 4, comprising ≤ 25% of an antibody variant deamidated at N31 of the light chain amino acid sequence.

[0261] 6. A composition according to any one of 1 to 5, comprising ≤ 35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence.

[0262] 7. a) M64 of the heavy chain amino acid sequence; b) M254 of the heavy chain amino acid sequence; and / or c) M430 of the heavy chain amino acid sequence A composition according to any one of 1 to 6, comprising any one or a combination of the following oxidative antibody variants in an amount of ≤55%.

[0263] 8. A composition according to any one of 1 to 7, comprising ≤ 3% of an antibody variant oxidized at W52 of the heavy chain amino acid sequence.

[0264] 9. The composition according to any one of 4 to 8, wherein the amount of deamidated antibody variant and / or oxidized antibody variant is determined by peptide mapping LC-MS / MS.

[0265] 10. The composition according to any one of 1 to 9, comprising ≦20% of aggregated antibody variants.

[0266] 11. The composition according to 10, wherein the aggregated antibody variant comprises a dimer.

[0267] 12. The composition according to 10 or 11, wherein the amount of the aggregated antibody variant is determined by SEC.

[0268] 13. The composition according to any one of 1 to 12, comprising ≧50% of a heavy chain amino acid sequence C-terminal lysine K449 deletion antibody variant.

[0269] 14. The composition according to any one of 1 to 13, comprising ≧50% of a heavy chain amino acid sequence pyroglutamic acid N-terminal antibody variant.

[0270] 15. A composition comprising an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, the composition comprising ≦80% of acidic antibody variants and ≦20% of aggregated antibody variants.

[0271] 16. A composition comprising an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, the composition comprising ≦25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; and ≦20% of aggregated antibody variants.

[0272] 17. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤55% of an antibody variant oxidized at M64 of the heavy chain amino acid sequence; ≤3% of an antibody variant oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0273] 18. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0274] 19. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence; ≤55% of an antibody variant oxidized at M64, M254, or M430 of the heavy chain amino acid sequence; ≤3% of an antibody variant oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

[0275] 20. Includes a purified preparation of monoclonal antibodies and a buffer, The pH is 6.8-7.2. The buffering agent is histidine, phosphate, citric acid, citrate, or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112 shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and The aforementioned antibodies are produced by Chinese hamster ovary cells. composition.

[0276] 21. 20 compositions wherein the buffering agent is at least one selected from disodium phosphate heptahydrate, phosphate, citric acid, and citrate.

[0277] 22. The composition according to 22, wherein the buffering agent is sodium phosphate, potassium phosphate, or sodium citrate.

[0278] 23. The composition of 22, further comprising sugars, carbohydrates and / or salts.

[0279] 24. 23 compositions comprising sucrose.

[0280] 25. A purified preparation of monoclonal antibody and a buffer, The pH is 6.8-7.2. The buffering agent is a phosphate or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112, as shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and The composition wherein the antibody is produced by Chinese hamster ovary cells. Composition.

[0281] 26. The composition according to 25, wherein the buffer is at least one selected from disodium phosphate heptahydrate, phosphate, citrate and citrate.

[0282] 27. The composition according to 26, further comprising sugar.

[0283] 28. The composition according to 27, wherein the sugar is sucrose.

[0284] 29. The composition according to 28, comprising polysorbate 80.

[0285] 30. The composition according to 29, comprising one selected from the first formulation of 20 mM disodium phosphate heptahydrate, 12% by weight of sucrose based on volume and 0.05% by weight of polysorbate 80 based on volume; the second formulation of 15.5 mM disodium phosphate heptahydrate, 3.9 mM citric acid monohydrate, 12% by weight of sucrose based on volume, 0.02% by weight of polysorbate 80 based on volume and 0.05 mM EDTA; and the third formulation of 26 mM disodium phosphate heptahydrate, 15% by weight of sucrose based on volume and 0.065% by weight of polysorbate 80 based on volume.

[0286] 31. The composition according to 29, wherein the antibody has a dissociation constant of about 3.5×10 -11 M or less against human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11.

[0287] 32. The composition according to 31, wherein the monoclonal antibody concentration is about 75 mg / mL or about 100 mg / mL.

[0288] 33. The composition according to 30, wherein the antibody has a dissociation constant of about 3.5×10 -11 M or less against human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11.

[0289] 34. The composition of 33, wherein the monoclonal antibody concentration is approximately 75 mg / mL or approximately 100 mg / mL.

[0290] 35. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0291] 36. A composition 35 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0292] 37. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The acidic form of the antibody, which accounts for approximately 20% to 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0293] 38. A composition 37 wherein the acidic form of the antibody comprises at least one selected from the acidic form with peak 65, the acidic form with peak 78, the acidic form with peak 88, and the acidic form with peak 92.

[0294] 39. A composition 38 wherein the acidic form of the antibody comprises at least one deamidated amino acid residue selected from the group consisting of a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386, and a light chain amino acid residue deamidated with asparagine 31.

[0295] 40. 39 compositions wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0296] 41. 39 compositions wherein the acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0297] 42. The main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; and, The acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 39 compositions.

[0298] 43. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The basic form of the antibody, which accounts for approximately 1% to 15% of the protein in the composition, as measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0299] 44. The composition of 43, wherein the basic form of the antibody includes the basic form with peak 112.

[0300] 45. The composition of 44, comprising a heavy chain having a carboxyl-terminal residue in which the basic form of the antibody is glycine 448.

[0301] 46. ​​The composition of 45, wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0302] 47. The composition of 45 wherein the basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​220.

[0303] 48. The main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​220; and, The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 45 compositions.

[0304] 49. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; c) The acidic form of the antibody that accounts for approximately 20% to approximately 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition; and d) The basic form of the antibody, which accounts for approximately 1% to 15% of the protein in the composition, as measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0305] 50. 49 compositions wherein the acidic form of the antibody comprises at least one selected from the acidic form with peak 65, the acidic form with peak 78, the acidic form with peak 88, and the acidic form with peak 92.

[0306] 51. A composition 50 wherein the acidic form of the antibody comprises at least one deamidated amino acid residue selected from the group consisting of a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386, and a light chain amino acid residue deamidated with asparagine 31.

[0307] 52. 49 compositions comprising the basic form of the antibody having peak 112.

[0308] 53. The composition of 52, comprising a heavy chain having a carboxyl-terminal residue in which the basic form of the antibody is glycine 448.

[0309] 54. 49 compositions wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0310] 55. 49 compositions wherein the acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​220.

[0311] 56. 49 compositions wherein the basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220.

[0312] 57. The main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a heavy chain amino acid residue oxidized with cysteine ​​222; and, The acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 49 compositions.

[0313] 58. The acidic form of the antibody contains at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; and, The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 49 compositions.

[0314] 59. The main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; and, The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 49 compositions.

[0315] 60. The main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; The acidic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​220, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220; and, The basic form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. 49 compositions.

[0316] 61. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) Deamidation form of the antibody comprising at least one selected from a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386, and a light chain amino acid residue deamidated with asparagine 31. A composition comprising the following.

[0317] 62. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) Oxidation form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. A composition comprising the following.

[0318] 63. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) A deamidated form of the antibody comprising at least one selected from a heavy chain amino acid residue deamidated with asparagine 299, a heavy chain amino acid residue deamidated with asparagine 317, a heavy chain amino acid residue deamidated with asparagine 386, and a light chain amino acid residue deamidated with asparagine 31; and c) Oxidation form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, a heavy chain amino acid residue oxidized with cysteine ​​222, a heavy chain amino acid residue oxidized with methionine 254, a heavy chain amino acid residue oxidized with methionine 360, a heavy chain amino acid residue oxidized with methionine 430, a light chain amino acid residue oxidized with methionine 4, and a light chain amino acid residue oxidized with cysteine ​​220. A composition comprising the following.

[0319] 64. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) Deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31. A composition comprising the following.

[0320] 65. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) Oxidation form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52 and a heavy chain amino acid residue oxidized with methionine 64. A composition comprising the following.

[0321] 66. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; b) an oxidized form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52 and a heavy chain amino acid residue oxidized with methionine 64; and c) Deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31. A composition comprising the following.

[0322] 67. a) An anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) A deamidated form of the antibody comprising light chain amino acid residues deamidated with asparagine 31. A composition comprising the following.

[0323] 68. a) An anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) An oxidation form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4. A composition comprising the following.

[0324] 69. a) An anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) A deamidated form of the antibody comprising a light chain amino acid residue deamidated with asparagine 31; and c) The oxidation form of the antibody comprising at least one selected from a heavy chain amino acid residue oxidized with tryptophan 52, a heavy chain amino acid residue oxidized with methionine 64, a heavy chain amino acid residue oxidized with methionine 82, a heavy chain amino acid residue oxidized with methionine 85, and a light chain amino acid residue oxidized with methionine 4. A composition comprising the following.

[0325] 70. a) A modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification, selected from the group consisting of the amino-terminal pyroglutamic acid residue of amino acid residue 1, the carboxy-terminal glycine amino acid residue of amino acid residue 448, the deamidated asparagine residue at position 299, the deamidated asparagine residue at position 317, the deamidated asparagine residue at position 386, the oxidized tryptophan residue at position 52, the oxidized methionine residue at position 64, the oxidized methionine residue at position 82, the oxidized methionine residue at position 85, the oxidized cysteine ​​residue at position 222, the oxidized methionine residue at position 254, the oxidized methionine residue at position 360, and the oxidized methionine residue at position 430; and b) A modification of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification, selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4, and an oxidized cysteine ​​residue at position 220. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

[0326] 71. a) Approximately 92% or more of the population have an amino-terminal pyroglutamic acid residue in amino acid residue 1 of the antibody heavy chain, b) Approximately 90% or more of the population have a carboxy-terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) Less than 6.0% of the population has a deamidated asparagine residue at position 386 of the antibody heavy chain. d) Approximately 1.5% or less of the population has an oxidized methionine residue at position 64 of the antibody heavy chain. e) Approximately 4.5% or less of the population has oxidized methionine at position 254 of the antibody heavy chain. f) Approximately 0.8% or less of the population has an oxidized methionine residue at position 430 of the antibody heavy chain, and g) Approximately 6.6% or less of the population has an antibody light chain containing a deamidated asparagine residue at amino acid residue 31. 70 compositions.

[0327] 72. a) Approximately 93.7% to 98.6% of the population have an amino-terminal pyroglutamic acid residue in amino acid residue 1 of the antibody heavy chain. b) Approximately 97.6% to 99.2% of the population have a carboxy-terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain. c) Approximately 0.4% to 1.2% of the population have a deamidated asparagine residue at position 317 of the antibody heavy chain. d) Approximately 1.6% to 4.2% of the population have a deamidated asparagine residue at position 386 of the antibody heavy chain. e) Approximately 0.7% to 0.9% of the population have an oxidized methionine residue at position 64 of the antibody heavy chain. f) Approximately 0.3% to 1.1% of the population have an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain. g) Approximately 2.6% to 3.3% of the population have oxidized methionine at position 254 of the antibody heavy chain. h) Approximately 0.5% to 0.7% of the population have an oxidized methionine residue at position 430 of the antibody heavy chain. i) Approximately 3.4% to 6.5% of the population have a deamidated asparagine residue in amino acid residue 31 of the antibody light chain, and j) Approximately 0.2% to 0.8% of the population have an oxidized methionine residue at position 4 of the antibody light chain. 71 compositions.

[0328] 73. a) Modified forms of the antibody heavy chain amino acid sequence represented by Sequence ID No. 1, comprising at least one amino acid residue modification selected from the group consisting of the deamidated asparagine residue at position 299, the deamidated asparagine residue at position 317, the deamidated asparagine residue at position 386, the oxidized tryptophan residue at position 52, the oxidized methionine residue at position 64, the oxidized methionine residue at position 82, the oxidized methionine residue at position 85, the oxidized cysteine ​​residue at position 222, the oxidized methionine residue at position 254360, and the oxidized methionine residue at position 430; and b) Modification of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4, and an oxidized cysteine ​​residue at position 220. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

[0329] 74. a) Approximately 0.3% to 1.5% of the population have a deamidated asparagine residue at position 317 of the antibody heavy chain. b) Approximately 1.5% to 4.5% of the population have a deamidated asparagine residue at position 386 of the antibody heavy chain. c) Approximately 0.5% to 1.5% of the population have an oxidized methionine residue at position 64 of the antibody heavy chain. d) Approximately 0.2% to 1.5% of the population consists of an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain. e) Approximately 2.5% to 3.5% of the population have oxidized methionine at position 254 of the antibody heavy chain. f) Approximately 0.4% to 0.8% of the population have an oxidized methionine residue at position 430 of the antibody heavy chain. g) Approximately 3.3% to 6.6% of the population have a deamidated asparagine residue in the antibody light chain amino acid residue 31, and h) 73 compositions, wherein approximately 0.1% to 1% of the population contains an oxidized methionine residue at position 4 of the antibody light chain.

[0330] 75. a) Approximately 0.4% to 1.2% of the population have an amidated asparagine residue at position 317 of the antibody heavy chain. b) Approximately 1.6% to 4.2% of the population have a deamidated asparagine residue at position 386 of the antibody heavy chain. c) Approximately 0.7% to 0.9% of the population have an oxidized methionine residue at position 64 of the antibody heavy chain. d) Approximately 0.3% to 1.1% of the population have an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain. e) Approximately 2.6% to 3.3% of the population have oxidized methionine at position 254 of the antibody heavy chain. f) Approximately 0.5% to 0.7% of the population have an oxidized methionine residue at position 430 of the antibody heavy chain. g) Approximately 3.4% to 6.5% of the population have a deamidated asparagine residue in amino acid residue 31 of the antibody light chain, and h) Approximately 0.2% to 0.8% of the population have an oxidized methionine residue at position 4 of the antibody light chain. 74 compositions.

[0331] 76. a) Modified form of the antibody heavy chain amino acid sequence represented by SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of the deamidated asparagine residue at position 299, the deamidated asparagine residue at position 317, and the deamidated asparagine residue at position 386; and b) Modified form of the antibody light chain amino acid sequence shown in Sequence ID No. 2, wherein amino acid residue 31 contains a deamidated asparagine residue. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

[0332] 77. a) Approximately 0.3% to 1.5% of the population have a deamidated asparagine residue at position 317 of the antibody heavy chain. b) Approximately 1.5% to 4.5% of the population have a deamidated asparagine residue at position 386 of the antibody heavy chain, c) Approximately 3.3% to 6.6% of the population have an antibody light chain containing a deamidated asparagine residue at amino acid residue 31. 76 compositions.

[0333] 78. a) Approximately 0.4% to 1.2% of the population have a deamidated asparagine residue at position 317 of the antibody heavy chain. b) Approximately 1.6% to 4.2% of the population have a deamidated asparagine residue at position 386 of the antibody heavy chain, c) Approximately 3.4% to 6.5% of the population have an antibody light chain containing a deamidated asparagine residue at amino acid residue 31. 77 components.

[0334] 79. a) Modified forms of the antibody heavy chain amino acid sequence represented by SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 82, oxidized methionine residue 85, oxidized cysteine ​​residue 222, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430; and b) Modification of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, comprising at least one amino acid residue modification selected from the group consisting of oxidized methionine residue 4 and oxidized cysteine ​​residue 220. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

[0335] 80. c) Approximately 0.5% to 1.5% of the population have an oxidized methionine residue at position 64 of the antibody heavy chain. d) Approximately 0.2% to 1.5% of the population consists of an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain. e) Approximately 2.5% to 3.5% of the population have oxidized methionine at position 254 of the antibody heavy chain. f) Approximately 0.4% to 0.8% of the population have an oxidized methionine residue at position 430 of the antibody heavy chain, and g) Approximately 0.1% to 1% of the population consists of 79 compositions in which the antibody light chain has an oxidized methionine residue at position 4.

[0336] 81. a) Approximately 0.7% to 0.9% of the population have an oxidized methionine residue at position 64 of the antibody heavy chain. b) Approximately 0.3% to 1.1% of the population have an oxidized methionine residue at position 82 of the antibody heavy chain or at position 85 of the antibody heavy chain. c) Approximately 2.6% to 3.3% of the population have oxidized methionine at position 254 of the antibody heavy chain. d) Approximately 0.5% to 0.7% of the population contains an oxidized methionine residue at position 430 of the antibody heavy chain, and e) Approximately 0.2% to 0.8% of the population have an oxidized methionine residue at position 4 of the antibody light chain. 80 compositions.

[0337] 82. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) The main form of the antibody that accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0338] 83. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The acidic form of the antibody, which accounts for up to approximately 80% of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

[0339] 84. A composition according to any one of 1 to 83 for the treatment of a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, controlled poor eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis.

[0340] 85. A method for treating a disease in a subject, a) Identifying subjects having a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis; and b) A method comprising the step of administering to a subject a therapeutically effective amount of any of the compositions described in 1 to 84, thereby treating the subject's disease.

[0341] 86. a) IL-5 specific antigen binding ≥ 0.70; and / or b) FcRn binding ≥ 70% A composition comprising 20 to 83.

[0342] 87. Compositions 20 to 83 in which (a) specific antigen binding is in the range of 0.70 to 1.30; and / or (b) FcRn binding is in the range of 70% to 130%.

[0343] 88. Compositions 20 to 83 comprising ≤ 20% agglutinated antibody variants.

[0344] 89. Compositions 20 to 83 comprising the agglutinating antibody variant in a dimer.

[0345] A method for producing compositions 90.1 to 83, a) A step of expressing in host cells an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence; b) A step of growing the cells in vitro for about 12 to 18 days at a pH of about 6.75 to about 7.00, with an in vitro cell age of 166 days or less; c) Step of collecting cell culture supernatant; d) A step of contacting the cell culture supernatant with a protein A resin or a protein G resin to bind antibody molecules; e) A step of eluting the antibody molecule from the resin to obtain a first eluate; f) A step of treating the first eluate with a pH of approximately 3.3 to approximately 3.7 for approximately 15 to approximately 240 minutes to obtain a treated first eluate; g) A step of bringing the treated first eluate into contact with an anion exchange resin at a load pH of approximately 8.3 to approximately 8.7; h) A step of recovering a second eluate from the anion exchange resin and holding it for approximately 96 hours; i) A step of treating the second eluate with guanidine and ammonium sulfate to obtain a solution; j) A step of bringing the solution into contact with a hydrophobic interaction chromatography resin bed at a loading ratio of approximately 12 g protein / L resin to approximately 27 g protein / L resin; k) A step of eluting a third eluate containing the antibody molecule from the hydrophobic interaction chromatography resin with an elution gradient volume of about 9 resin bed volume to about 11 resin bed volume and an elution peak cut-stop of about 17% to about 23% of the maximum peak height; and l) The process of formulating the third eluate. A method comprising, thereby producing 1 to 83 compositions.

[0346] Compositions 1 to 83 produced by the method of 91.90.

[0347] 92. Consists of a purified monoclonal antibody preparation and a buffer, The pH is 6.2 to 6.6. The buffering agent is histidine, phosphate, citric acid, citric acid monohydrate, citrate, or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112 shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and The aforementioned antibodies are produced by Chinese hamster ovary cells. composition.

[0348] 93. The composition of 92, wherein the buffering agent is at least one selected from disodium phosphate heptahydrate, phosphate, citric acid, and citric acid monohydrate.

[0349] 94. The composition of 92, wherein the buffering agent is sodium phosphate, potassium phosphate, citric acid, citric acid monohydrate, or sodium citrate.

[0350] 95. The composition of 92, further comprising sugars, carbohydrates and / or salts.

[0351] 96. A composition of 95 comprising sucrose.

[0352] 97. A purified preparation of a monoclonal antibody and a buffer, The pH is 6.2 to 6.6. The buffering agent is a phosphate or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112 shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and The aforementioned antibodies are produced by Chinese hamster ovary cells. composition.

[0353] 98. The composition of 97, wherein the buffering agent is at least one selected from disodium phosphate heptahydrate, phosphate, citric acid, citric acid monohydrate, and citrate.

[0354] 99. The composition of 98, further comprising sugar.

[0355] 100. The composition of 99 wherein the sugar is sucrose.

[0356] 101. A composition comprising polysorbate 80.

[0357] First formulation: 102.16.1 mM disodium phosphate heptahydrate, 3.9 mM citrate monohydrate, 12% by weight sucrose, 0.02% by weight polysorbate 80 and 0.05 mM EDTA; Second formulation: 15.2 mM disodium phosphate heptahydrate, 4.8 mM citrate monohydrate, 12% by weight sucrose, 0.02% by weight polysorbate 80 and 0.05 mM EDTA; 15.8 mM disodium phosphate heptahydrate, 4.2 mM citrate monohydrate, 12% by weight sucrose, 101 compositions comprising one selected from a third formulation of 0.02 wt% polysorbate 80 and 0.05 mM EDTA; a fourth formulation of 16.3 mM disodium phosphate heptahydrate, 3.7 mM citric acid monohydrate, 12 wt% sucrose by volume, 0.02 wt% polysorbate 80 and 0.05 mM EDTA by volume; and a fifth formulation of 15.5 mM disodium phosphate heptahydrate, 4.5 mM citric acid monohydrate, 12 wt% sucrose by volume, 0.02 wt% polysorbate 80 and 0.05 mM EDTA by volume.

[0358] 103. Approximately 3.5 × 10⁶ of the antibody against human interleukin-5 containing the amino acid sequence shown in SEQ ID NO: 11. -11 A composition of 101 having a dissociation constant of M or less.

[0359] 104. The composition of 101, wherein the monoclonal antibody concentration is approximately 75 mg / mL or approximately 100 mg / mL.

[0360] 105. Approximately 3.5 × 10⁶ of the antibody against human interleukin-5 containing the amino acid sequence shown in SEQ ID NO: 11. -11A composition of 102 having a dissociation constant of M or less.

[0361] 106. The composition according to any one of 1 to 105, wherein the antibody is concentrated in a concentration between approximately 75 mg / ml and approximately 100 mg / ml.

[0362] 107. a) A buffer selected from the group consisting of disodium phosphate heptahydrate, phosphates, citrates, sodium phosphate, potassium phosphate, sodium citrate, and histidine, which provides a pH between 6.8 and 7.2; and / or b) Sugars; and / or c) Polysorbate 80; and / or d) EDTA A composition according to any one of 1 to 106, further comprising one or a combination of the above.

[0363] 108. a) A buffer selected from the group consisting of disodium phosphate heptahydrate, phosphates, citrates, citric acid monohydrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, which provides a pH between 6.2 and 6.6; and / or b) Sugars; and / or c) Polysorbate 80; and / or d) EDTA The composition according to any one of 1 to 107, further comprising one or a combination of the following.

[0364] 33. A composition according to any one of 1 to 108 for use in therapy.

[0365] 35. A composition according to any one of 1 to 108 for use in the treatment of asthma, severe eosinophilic asthma, severe asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis. [Examples]

[0366] Example 1 Preparation of composition Multiple batches of a composition containing the monoclonal anti-IL-5 antibody mepolizumab were prepared.

[0367] Inoculations of Chinese hamster ovary cells, stably transfected with an expression vector construct containing the nucleic acid sequences shown in SEQ ID NOs: 13 and 14, were cultured in a 5000 L bioreactor containing liquid cell culture medium. The mature antibody encoded by these nucleic acids is mepolizumab, comprising the heavy-chain amino acid sequence shown in SEQ ID NO: 1 and the light-chain amino acid sequence shown in SEQ ID NO: 2.

[0368] The bioreactor was operated at a temperature of approximately 34.5°C to 35.5°C. Air and oxygen were sprayed into the culture medium, and the pH was maintained at approximately 6.75 to 7.00. The culture period was approximately 12 to 18 days. The in vitro cell age (number of culture days from the initial thawing of the master cell bank to harvesting) was within 166 days. After this, the clear cell culture supernatant was collected by centrifugation and deep filtration of the cell culture medium. Next, this clear supernatant was subjected to protein A chromatography, and impurities were washed from the chromatography column. Then, the binding proteins containing antibody molecules were eluted from the protein A column and treated at a pH of approximately 3.3 to 3.7 for approximately 15 to 240 minutes. Subsequently, this treated preparation was adjusted to a pH of approximately 4.3 to 4.7 and held for approximately 20 to 1110 minutes. Next, this treated preparation was clarified using a depth filter and a filtration train of 0.5 / 0.2 μm bilayer filters. Subsequently, the filtered preparation was subjected to Q-SEPHAROSE® fast-flow anion exchange chromatography at a load pH of approximately 8.3–8.7, and eluted from the chromatography column. The eluate was then held for approximately 96 hours. Guanidine and ammonium sulfate were then added. Guanidine was added to a concentration of approximately 1.8 M–2.2 M and held for approximately 15–240 minutes. This solution was then subjected to phenyl-SEPHAROSE® fast-flow chromatography at a load ratio of approximately 12 g protein / L resin to approximately 27 g / L resin, an elution gradient volume (bed volume; BV) of approximately 9–11, and an elution peak cut-off stop (percentage of maximum peak height) of approximately 17–23. Viral filtration was then performed using a Planova 20N virus removal filter. Next, the filtrate was adjusted to a target concentration of approximately 46 g protein / L to approximately 66 g protein / L, and bulk drug substances (BDS) were formulated by tangential filtration and ultrafiltration exchange using a solution containing approximately 20 mM disodium phosphate heptahydrate and 12% by weight of sucrose by volume. Subsequently, this solution was adjusted to a target concentration of 76 g protein / L to approximately 82 g protein / L, and approximately 0.05% by weight of polysorbate 80 by volume was added. Then, this solution was filtered through a 0.5 / 0.2 μm PES filter, the solution was filled into containers, and frozen.The drug product was prepared using a sterile manufacturing method that includes thawing and bulk container combination according to manufacturing methods well known in the art, followed by filtration of the bulk into vials, lyophilization, sealing, and crimping. The final drug product preparation is a lyophilized drug product in a single-use vial.

[0369] Lyophilized products from each produced batch were reconstituted by adding water to prepare a composition containing 100 mg / mL of protein, monoclonal anti-IL-5 mepolizumab antibody, 26 mM disodium phosphate heptahydrate, 15% by weight of sucrose and 0.065% by weight of polysorbate 80, at a pH of approximately 6.8 to approximately 7.2.

[0370] Example 2 Characterization of composition Samples from a batch of compositions containing the monoclonal anti-IL-5 antibody prepared as described above were characterized.

[0371] Capillary isoelectric focusing (cIEF) consistently demonstrated the presence of six antibody isoforms in this composition (e.g., composition reference standard (RS) 101245722). See Figure 1. These isoforms are the isoforms of peaks 65, 78, 88, 92, the major peak, and 112 shown in Figure 1. Samples of this composition were subjected to cIEF using the standard method. Samples analyzed by cIEF included pI7.9 and pI9.46 standards. The cIEF electrophoresis diagrams shown in Figure 1 are representative of the composition from multiple batches.

[0372] The electrophoretic graph shows that the composition comprises the primary form, acidic form, and basic form of the antibody. The primary form can be seen in Figure 1 and may also be identified as peak 100. The acidic form of the antibody corresponds to the forms at peaks 65, 78, 88, and 92 in Figure 1. The basic form of the antibody corresponds to the form at peak 112 in Figure 1.

[0373] The formula used for peak allocation is as follows: Peak name = int{(pI peak - 8) / (pI main - 8) * In formula 100, int = integer and pI peak = pI of the named peak. Table 7 shows the peak naming conventions for cIEF electrophoresis of compositions comprising monoclonal anti-IL-5 antibody. The names of peaks determined as described herein should be confirmed based on the observed electrophoretic / chromatographic pattern. Peaks not within the range described herein should be treated according to the above formula.

[0374] [Table 7]

[0375] Integral analysis was performed on the electrophoretic peaks. See Table 8.

[0376] [Table 8]

[0377] This indicates that the dominant form accounted for more than 50.0% of the total peak area of ​​the sample (values ​​between approximately 58.1% and 62.3% were similarly observed). It also indicates that the acidic form accounted for less than 45.0% of the total peak area of ​​the sample (values ​​between approximately 20% and 45%, e.g., between 32.2% and 40.7% were similarly observed). The basic form accounted for approximately 1% to 15% of the total peak area of ​​the sample (values ​​between approximately 1.2% and 4.9% were similarly observed).

[0378] Next, the peak fractions of the major, acidic, and basic forms obtained by cIEF were further analyzed by weak cation exchange (WCX), trypsin peptide mapping, and liquid chromatography-mass spectrometry (LC-MS / MS). Standard methods were used for these analyses.

[0379] These WCX, trypsin peptide mapping, and LC-MS / MS results showed that the major morphological peak fraction contained two IgG1 mAb modifications. Thus, in the antibody heavy chain amino acid sequence of Sequence ID No. 9, 99.6% of the N-terminal glutamine (Gln, Q) was cyclized to pyroglutamic acid (pGlu), and 99.9% of the C-terminal lysine (Lys, K) 449 of the heavy chain (HC) was cleaved. In general, the pGlu levels in the tested batches were >95.0%, and the levels of HC lacking C-terminal K449 were >98.0%.

[0380] These WCX, trypsin peptide mapping, and LC-MS / MS results also showed a 1-Dalton mass shift characteristic of deamidation with respect to the acidic form peak. Peptide mapping LC-MS / MS indicated that the acidic form peak contained a mixture of deamidating antibody species. Deamidation was primarily observed at HC N386 of the amino acid sequence shown in SEQ ID NO: 1 and LC N31 of the amino acid sequence shown in SEQ ID NO: 2. Lower levels of deamidation were also observed at HC N317 of the amino acid sequence shown in SEQ ID NO: 1.

[0381] Overall, these experimental data showed that the asparagine residues HC N317, HC N386, and HC N299 in the amino acid sequence shown in SEQ ID NO: 1, and LC N31 in the amino acid sequence shown in SEQ ID NO: 2, were sensitive to deamidation.

[0382] These WCX, trypsin peptide mappings, and LC-MS / MS analyses indicated that, with respect to the basic morphology peak, the antibody morphology of this peak possessed at least one intact heavy-chain carboxy-terminal lysine amino acid residue. Antibody species with intact lysine migrate to the basic region due to the extra positive charge derived from these residues compared to other morphologies where they are absent. Therefore, basic morphologies such as peak 112 correspond to antibody morphologies in which one or both heavy-chain amino acid sequences possess the intact carboxy-terminal lysine amino acid sequence shown in SEQ ID NO: 1.

[0383] The primary sequencing of compositions containing monoclonal anti-IL-5 antibodies was also performed using standard LC-MS / MS techniques. These analyses examined the primary structure and amino acid sequence of the antibody molecules in the compositions. In particular, these analyses revealed which amino acid residues were deamidated, oxidized, cyclized, or absent in the anti-IL-5 antibodies, and the percentages of these in the population of anti-IL-5 antibodies present in the compositions (e.g., those expressed from the nucleic acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14). See Table 9.

[0384] [Table 9]

[0385] Antibody variants Mepolizumab binds to soluble IL-5 and blocks its binding to its receptor. The structure of IL-5 indicates that it is a secreted protein, and there is no evidence of membrane-bound IL-5 in any cell type. Therefore, the Fc effector function is not part of the mechanism of action (MOA) of mepolizumab. Based on the MOA and PK properties of mepolizumab, there are two functions involved in the biological activity of this antibody: binding to IL-5 via the CDR and binding to the FcRn receptor via the Fc region.

[0386] Through the detailed characterization studies conducted above and shown below, it was determined that at least deamidation, oxidation, and aggregation can lead to antibody variants in the mepolizumab composition, and that these variants can affect the function of mepolizumab. Certain levels of these variants must be maintained to ensure appropriate biological function. In this specification, function is described within acceptable ranges of 0.70–1.30 specific antigen-binding activity (IL5-binding) and 70%–130% FcRn binding. Therefore, the steps for identifying antibody variants that affect function include (i) determining whether an antibody variant is morphologically formed, (ii) whether the variant affects function, and (iii) what degree of variant can result in a functional composition.

[0387] function IL-5 binding: Statistical analysis was performed to calculate the acceptable antigen-binding activity range using all previously prepared drug substance (DS) and drug product (DP) release and stability data. The calculated statistical range was compared with clinical trials and evaluated based on the known impact of product-related variants on efficacy. Based on this analysis, the acceptable antigen-binding activity range at release and end of shelf life is specific antigen binding of 0.70 to 1.30.

[0388] IL-5 specific binding was determined by surface plasmon resonance (SPR) using a BIACORE® instrument in binding mode. This SPR assay can detect decreased antigen binding resulting from changes in mepolizumab and mepolizumab variants related to efficacy.

[0389] SPR is used to determine the specific antigen-binding activity of mepolizumab. First, a mepolizumab reference standard is injected into the surface of a CM5 sensor chip containing immobilized protein A. Then, a fixed-concentration diluted IL-5 protein is injected to allow IL-5 to bind to the captured mepolizumab sample. The concentration of mepolizumab bound to IL-5, reported as the functional binding of mepolizumab to IL-5, is determined from the corresponding mepolizumab reference standard calibration curve. SPR results were reported as the quotient obtained by dividing the functional binding concentration of mepolizumab to IL-5 by the total protein concentration.

[0390] FcRn binding: The neonatal Fc(FcRn) receptor binding activity of mepolizumab was also measured by surface plasmon resonance (SPR) using a BIACORE® instrument. Based on results obtained during mepolizumab product development, known assay variables, and results obtained for similar mAb products, the acceptable range of FcRn binding activity was determined to be 70–130%.

[0391] The Fc region of mepolizumab binds to FcRn, and this interaction reflects the long serum half-life of mepolizumab (mean terminal half-life = 20 days). In the SPR assay, fixed concentrations of mepolizumab were captured using a nitrilotriacetate (NTA) sensor chip containing immobilized FcRn receptors. First, Ni 2+ Inject at a fixed concentration, and Ni via NTA 2+ The chelated nitrate was then captured on the NTA sensor chip. Next, FcRn receptors were injected at a fixed concentration, and the 6× histidine tag at the C-terminus of the α-chain of the FcRn receptor was captured by the previously captured Ni. 2+ Next, mepolizumab, diluted within the standard curve concentration range, was injected onto the surface of an NTA sensor chip containing the captured FcRn receptor. The concentration of mepolizumab bound to the FcRn receptor was extrapolated from the corresponding mepolizumab reference standard calibration curve. This SPR result was reported as the quotient obtained by dividing the functional binding concentration of mepolizumab to the FcRn receptor by the total protein concentration.

[0392] This SPR method for specific antigen binding and FcRn binding uses a mepolizumab reference standard. The mepolizumab reference standard is used in the assay simply to obtain system suitability and sample comparison data to ensure the method is performed correctly. The reference standard allows for the establishment of a calibration curve, from which the sample concentration is extrapolated.

[0393] Acidic mutant Next, forced degradation tests were performed to determine the effect of increased levels of acidic variants, such as deamidation, on antibody function / efficacy, i.e., antigen-binding and FcRn-binding activity.

[0394] In the forced degradation test at pH 9.0, the composition was adjusted to pH 9.0 with 6N sodium hydroxide and incubated at 40°C for 30 days. Samples were taken on days 0, 3, 7, 14, and 30 and compared to an unstressed composition used as a control. Next, the pH 9.0 stressed samples were analyzed by cIEF. These results are shown in Table 10 and Figure 1 (days 0 and 3). Since the pH 9.0 stressed composition degraded beyond the capacity of the cIEF assay on day 14, only the results up to day 7 are shown in Table 10. Under a 3-day stress condition at pH 9.0, the total acidic region was found to be 74.4% and 71.9% for two different batches of the composition.

[0395] [Table 10]

[0396] Next, pH 9.0 forced degradation test samples of the composition from different batches were tested for specific antigen-binding activity (Table 11) and FcRn-binding activity (Table 12) using standard surface plasmon resonance (SPR) spectroscopy.

[0397] [Table 11]

[0398] [Table 12]

[0399] On day 3 (i.e., approximately 72-74% acidic mutant), the IL5-specific binding activity was 0.74 in both batches of the composition subjected to pH 9.0 forced filtration. FcRn binding activity was 82% and 80%, respectively, in both batches of the composition subjected to pH 9.0 forced filtration. These values ​​were within the acceptance criteria for each assay. The acceptance criteria for specific antigen-binding activity is 0.70-1.30, and the acceptance criteria for FcRn binding is 70%-130%. Therefore, acidic mutants that maintain the function of the mepolizumab composition can be as high as approximately 74%.

[0400] Deamidation Forced degradation tests can determine which residues suspected to be sensitive to deamidation actually undergo deamidation, whether deamidation variants affect function, and how much deamidation is acceptable in a composition to maintain function. The asparagine residues experimentally determined to be sensitive to deamidation are HC N317, HC N386, HC N299, and LC N31. Forced degradation tests were performed to determine the effect of increased levels of deamidated LC N31 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2 on antigen-binding activity. In these tests, compositions from different batches were adjusted to pH 9.0 with 6N sodium hydroxide and incubated at 40°C for 30 days. Samples were taken on days 0, 3, 7, 14, and 30 and compared to a non-stressed composition used as a control (e.g., reference standard). pH 9.0 stressed samples were tested by peptide mapping LC-MS / MS. These results are shown in Table 13. When mepolizumab was retained at pH 9.0 for 3 days, the levels of deamidated LC N31 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2 were 17.4% and 16.8% in different batches of composition. See Table 13. Antigen and FcRN binding data for mepolizumab retained at pH 9.0 for 3 days are shown in Tables 11 and 12.

[0401] [Table 13]

[0402] Therefore, the specific antigen-binding activity of 0.74 on day 3 (Table 11), and the FcRn-binding activity of 84% and 82% (Table 12), along with deamidation at N31 of up to approximately 17% and deamidation at N386 of up to approximately 28% (Table 13), indicate that the functional composition can be maintained within an acceptable range of specific antigen-binding activity of 0.70–1.30 and FcRn-binding of 70%–130%.

[0403] oxidation Forced degradation tests were performed to experimentally investigate the susceptibility of methionine and other amino acid residues in the heavy and light chains of the composition to oxidation. Forced degradation tests can determine which residues suspected to be susceptible to oxidation actually oxidize, whether their oxidized variants affect function, and how much oxidation is acceptable in the composition to maintain function / effectiveness (antigen binding and / or FcRn binding).

[0404] To induce oxidation, samples of the composition were incubated with 0.1% hydrogen peroxide (H2O2) at room temperature (RT) for 48 hours. Samples were harvested at 0, 6, 12, 24, and 48 hours. These were compared to an unstressed composition used as a control (e.g., a reference standard). From these tests, it was determined that the methionine (M) residues with the greatest oxidation tendency included HC M64, HC M82, HC M85, HC M254, HC M360, HC M430 in the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, and LC M4 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2. The methionine (M) residues with the greatest oxidation tendency included M64 located within HC CDR2; M254 and M430 located within the Fc region and protein A binding pocket; and M360 in the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1. Methionine residues with a low degree of oxidation tendency included HC M4, HC M82, and HC M85 in the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1. In addition, LC C220 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2 was determined to have an oxidation tendency under chemical induction conditions. Importantly, LC C220 and HC C222 form an intrachain disulfide bond linking the heavy and light chains.

[0405] The levels of sulfoxides resulting from methionine oxidation and sulfonic acids resulting from cysteine ​​oxidation were measured using peptide mapping LC-MS / MS and summarized in Table 14. Over 70% of HC M64, M254, M360, and M430 were oxidized after 48 hours of incubation with 0.1% hydrogen peroxide (H2O2).

[0406] [Table 14]

[0407] Specific antigen binding by the antibody in the composition was measured by SPR. This showed a greater decrease in specific antigen-binding activity at 48 hours in different H2O2 stress batches, as summarized in Table 15. This decrease in antigen binding correlates with relatively high levels of M64 oxidation observed by peptide mapping LC-MS / MS. Specific antigen-binding activity decreased by approximately 15% and 32% in test samples from different batches of the composition, respectively. Even when mepolizumab was oxidized by 85.6%, the specific antigen-binding activity was still retained at 0.57. Using the linear relationship between the cumulative level of mepolizumab oxidation and specific antigen-binding activity, it was determined that in the worst case, HC M64 could be oxidized by approximately 50%, but the antibody in the composition would still retain antigen-binding activity in the range of 0.70–1.30.

[0408] [Table 15]

[0409] The FcRn binding activity profiles of H2O2 stress samples from different batches of the composition were remarkably similar, and as shown in Table 16, they exhibited a substantial decrease in FcRn binding activity at 48 hours compared to the control (untreated reference standard). HC M254 and HC M430 are located within the Fc region and have been shown to contribute to a decrease in FcRn binding when oxidized. Based on peptide mapping results created during the forced degradation test, when the composition is chemically oxidized with H2O2 for 48 hours, the levels of oxidized HC M254 and HC M430 observed in different batches are ≥95%. FcRn binding results showed an approximately 80% decrease in antigen FcRn binding in H2O2 stress samples from different batches of the composition. Even when mepolizumab was ≥90% oxidized, FcRn binding activity was still retained at 22%. Using a linear relationship between the cumulative level of mepolizumab oxidation and its FcRn binding activity, it was determined that, in the worst-case scenario, HC M254 and HC M430 may be oxidized by 50%, but the antibody in the composition would still retain FcRn binding activity in the range of 70% to 130%.

[0410] [Table 16]

[0411] Photostress tests were performed to determine the effect of light-induced tryptophan oxidation on the antigen-binding activity of antibodies in different batches of the composition. This demonstrated a tendency for oxidation of tryptophan W52 in the antibody heavy chain. In these tests, photostress was induced by exposing compositions from different batches to 1.8 million lux-hr of visible light at 25°C for approximately 60 hours. Samples collected at 0, 3, 7, 14, and 30 hours were compared to an unstressed reference standard of the composition used as a control. The levels of hydroxydioxide / kynurenine resulting from tryptophan oxidation were remarkably similar across different batches of exposure-stressed compositions, as summarized in Table 17. An increase in HC W52 oxidation was detected after 60 hours of exposure.

[0412] [Table 17]

[0413] The specific antigen-binding activity profiles of antibodies from different batches of exposure-stressed compositions showed a decrease in specific antigen-binding activity over time. This is summarized in Table 18. Even when mepolizumab was oxidized by approximately 7%, its specific antigen-binding activity was still retained at 0.53. Using the linear relationship between the cumulative level of tryptophan oxidation in mepolizumab and its specific antigen-binding activity, it was determined that in the worst case, W52 could be oxidized by 3%, but the antibodies in the composition would still retain antigen-binding activity in the range of 0.70–1.30.

[0414] [Table 18]

[0415] Therefore, in order to maintain its function (IL-5 bond and / or FcRn bond), HC M64 can be oxidized up to 50%, HC M254 and HC M430 can be oxidized up to 50%, and W52 can be oxidized up to 3%.

[0416] agglomeration The antibody size distribution in the composition was monitored using standard undenatured size exclusion chromatography (SEC). As shown in Figures 3 and 4, three peaks were detected in the SEC profile of the RS composition. A major peak at 7.9 min, representing 99.4% of the relative percentage area, was identified as monomers, while a minor peak at approximately 6.7 min, representing 0.5% of the relative percentage area, was identified as aggregates. A second minor peak was observed in several batches, eluting after the major peak and indicating the presence of fragments. Generally, this peak is below SEC assay QL 0.1.

[0417] The aggregate peaks were further characterized using SEC in conjunction with multi-angle light scattering (MALS) detection and analytical ultracentrifugation (AUC). The results, as shown in Figure 5 for the RS composition and in Figure 6 for different batches of the composition, show that the SEC-MALS profile contains peaks that elute earlier (dimers) and peaks that elute later (monomers). The lines crossing each peak represent the molar mass of the detected species and the position of the dimer peak (which is difficult to see in the chromatogram due to the low abundance of dimers in the sample).

[0418] SEC-MALS data were used to calculate the molar masses of antibody monomers and dimers. The obtained molar masses of monomers in the RS composition and from different batches of the composition were comparable to the 148,760 kDa hypothesis of mepolizumab monomer, as shown in Table 19. Variations in molecular weight observed for dimers were detected due to the low levels of this type present in the samples.

[0419] [Table 19]

[0420] To confirm the absence of perturbations from self-association equilibrium or exclusion of higher-order aggregates from chromatographic separation, area under the sedimentation velocity curve (AUC) integral analysis was used as a supplementary technique to the fraction-based method including SEC. The results of the AUC analysis showed that the c(s) distribution, as shown in Table 20, contained one major species (major peak) identified as a monomer with a sedimentation coefficient of 2.81S in both the RS batch of the composition and another batch of the composition; as well as one aggregate peak identified as a dimer with a sedimentation coefficient of 4.87S in the RS batch of the composition and 5.10S in the other batch of the composition. The difference in sedimentation coefficient values ​​between PRS and BDS was not considered substantial and was due to the low abundance of dimers in the sample. The only high molecular weight species detected was a dimer, which is consistent with the SEC-UV and SEC-MALS results.

[0421] [Table 20]

[0422] The sedimentation coefficients determined for monomers and dimers were lower than those conventionally observed for IgG1 monoclonal antibodies. The mepolizumab formulation contains 12% (w / v) sucrose, resulting in a highly viscous sample, which produces a lower sedimentation coefficient than those observed for these antibody molecules in the composition. SEC-UV, SEC-MALS, and AUC analyses indicate that the aggregate species in the composition are antibody dimers.

[0423] To investigate the effect of aggregates on antigen-binding activity, low pH tests were performed on compositions from different batches. Composition samples from different batches were adjusted to pH 3.5 with 5N hydrochloric acid. The pH 3.5 adjusted samples were then incubated at 40°C for 30 days to induce chemical modification. Samples were taken at 0, 3, 7, 14, and 30 days and compared to unstressed RS samples of the compositions used as controls. This indicated that aggregation is one of the major degradation pathways when compositions are chemically stressed at low pH 3.5.

[0424] The SEC degradation profiles of pH 3.5 stressed samples of the composition are shown in Figure 7 and summarized in Table 21. Under low pH 3.5 stress conditions, differences in aggregation rates were observed between different batches of the composition. However, by day 30, both batches of the composition reached equilibrium and exhibited similar levels of aggregation. This difference in aggregation rates between the different batches is due to a higher level of covalent dimers compared to non-covalent dimers in these batches. The slower aggregation rate observed in the MDS1 batch of the composition is due to a higher proportion of non-covalent dimers compared to the MDS2 batch, where non-covalent dimers associate and dissociate until equilibrium is achieved, potentially slowing the overall rate of aggregate morphology.

[0425] [Table 21]

[0426] The specific antigen-binding activity profile of the pH 3.5 stress batch samples of the composition showed a decrease in specific antigen-binding activity over time, as summarized in Table 22.

[0427] [Table 22]

[0428] The FcRn binding activity profile of the pH 3.5 stress batch samples of the composition showed a decrease in FcRn binding activity over time, as summarized in Table 23.

[0429] [Table 23]

[0430] In summary, Tables 21-23 show that when aggregates are present in the sample at approximately 40%, antigen binding and FcRn binding decrease by approximately 50%. Even when mepolizumab aggregated at approximately 40%, specific antigen binding activity was still maintained at 0.57 and FcRn binding activity was maintained at 51% (Table 22, MDS2, day 3). Due to the different ratios of covalent and uncovalent dimers, slightly different degradation profiles were observed in aggregate content between MDS1 and MDS2 on day 3 (Table 21).

[0431] The linear relationship between mepolizumab aggregation and specific antigen activity and FcRn binding activity was determined from MDS2, and it was found that in the worst case, mepolizumab may aggregate to 20%, but the antibody in the composition would still retain antigen binding activity in the range of 0.70–1.30 and FcRn binding activity of 70–130%.

[0432] Therefore, although 20% of the antibody in a composition containing mepolizumab may be aggregates, it still maintains IL-5 binding activity in the range of 0.70 to 1.30 and FcRn binding activity in the range of 70% to 130%.

[0433] HCP The level of residual CHO host cell protein in the mepolizumab composition is measured using an enzyme-linked immunosorbent assay (ELISA). This method uses antibodies produced against the native antigen of a CHO cell line grown under conditions that mimic the mepolizumab production process.

[0434] In the mepolizumab composition, the acceptable range for HCP content was determined to be ≤10 ng / mg. This range is derived from release data prepared to date and represents true analytical and process variability. Thirty-seven different batches of the active pharmaceutical ingredient had the following HCP content: 1.1 ng / mg (2 batches), 1.0 ng / mg (5 batches), 0.9 ng / mg (1 batch), 0.8 ng / mg (3 batches), 0.7 ng / mg (1 batch), 0.6 ng / mg (1 batch), 0.5 ng / mg (1 batch), <0.5 ng / mg (4 batches), and <1 ng / mg (19 batches).

[0435] In summary, mepolizumab has two key functions that contribute to its biological activity: binding to IL-5 via the CDR and binding to the FcRn receptor via the Fc domain. Through the detailed characterization studies conducted above, it was determined that certain deamidation antibody variants, particularly oxidative antibody variants and agglutinating antibody variants, can affect the function of the mepolizumab composition. Therefore, specific levels of these variants need to be maintained to ensure appropriate function / efficacy.

[0436] Example 3 Unofficial Sequence List The underlined sequences below identify CDR sequences or nucleic acid sequences encoding these CDR sequences in the variable heavy and variable light chain portions of antibodies, according to the Kabat definition of CDRs. For example, in Sequence ID No. 1, the frameworks and CDRs are represented in order from the amino-proximal portion to the carboxy-terminal portion of the shown sequence as plain text framework 1, underlined CDR 1, plain text framework 2, underlined CDR 2, plain text framework 3, underlined CDR 3, and plain text framework 4. An asterisk to the right of a single-letter amino acid code indicates that the amino acid residue to its left is an N-glycosylation site. This scheme is used, for example, in Sequence IDs No. 1-4, 11, 12, and 19-22. The amino-terminal methionine residues shown in these sequences can be cleaved. Therefore, if these sequences show an amino-terminal methionine residue here, it should be considered that they also disclose cleavage forms of these proteins lacking such an amino-terminal methionine residue. Nucleic acid sequences should be considered as being represented as DNA nucleic acid sequences, containing "t" nucleic acid residues, and the corresponding RNA sequences should be disclosed in such a way that the "t" nucleic acid residues can also be considered to represent "u" nucleic acid residues. In addition, the 5' proximal "atg" start codon and the 3' proximal "taa," "tag," and "tga" stop codons are omitted from the following cDNA nucleic acid sequences, for example, in sequence numbers 31-34.

[0437] Mepolizumab full-length heavy chain Sequence ID 1 [ka]

[0438] Mepolizumab full-length light chain Sequence ID 2 [ka]

[0439] Mepolizumab VH Sequence ID 3 [ka]

[0440] Mepolizumab VL Sequence ID 4 [ka]

[0441] Mepolizumab CDRH1 Sequence ID 5 [ka]

[0442] Mepolizumab CDRH2 Sequence ID 6 [ka]

[0443] Mepolizumab CDRH3 Sequence ID 7 [ka]

[0444] Mepolizumab CDRL1 Sequence ID 8 [ka]

[0445] Mepolizumab CDRL2 Sequence ID 9 [ka]

[0446] Mepolizumab CDRL3 Sequence ID 10 [ka]

[0447] Human IL-5 (mature protein) Sequence ID 11 [ka]

[0448] Human IL-5 receptor subunit α isoform 1 (mature protein) Sequence ID 12 [ka]

[0449] Mepolizumab full-length heavy chain coding DNA Sequence ID 13 [ka]

[0450] Mepolizumab full-length light chain coding DNA Sequence ID 14 [ka]

[0451] The present invention is fully described herein, and it will be obvious to those skilled in the art that many modifications and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% of acidic antibody variants.

2. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤80% acidic antibody variants and ≤20% agglutinating antibody variants.

3. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence and ≤20% of agglutinated antibody variants.

4. A composition comprising a heavy chain amino acid sequence shown in Sequence ID No. 1 and a light chain amino acid sequence shown in Sequence ID No. 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤55% of an antibody variant oxidized at M64 of the heavy chain amino acid sequence; ≤3% of an antibody variant oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

5. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of an antibody variant deamidated at N31 of the light chain amino acid sequence; ≤35% of an antibody variant deamidated at N386 of the heavy chain amino acid sequence; and ≤20% of an agglutinated antibody variant.

6. A composition comprising an antibody having a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence and / or a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence, wherein the composition comprises ≤25% of antibody variants deamidated at N31 of the light chain amino acid sequence; ≤35% of antibody variants deamidated at N386 of the heavy chain amino acid sequence; ≤55% of antibody variants oxidized at M64, M254, and M430 of the heavy chain amino acid sequence; ≤3% of antibody variants oxidized at W52 of the heavy chain amino acid sequence; and ≤20% of agglutinated antibody variants.

7. A composition comprising a purified monoclonal antibody preparation and a buffer, The pH is between 6.8 and 7.

2. The buffering agent is histidine, phosphoric acid, or citric acid, or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112 shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and A composition wherein the aforementioned antibody is produced by Chinese hamster ovary cells.

8. A composition comprising a purified monoclonal antibody preparation and a buffer, The pH is between 6.8 and 7.

2. The buffering agent is phosphoric acid or a salt thereof. The purified preparation comprises isoforms represented by peaks 65, 78, 88, 92, the main peak, and 112 shown in Figure 1. The antibody comprises a heavy chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and A composition wherein the aforementioned antibody is produced by Chinese hamster ovary cells.

9. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

10. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The acidic form of the antibody that accounts for approximately 20% to 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

11. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in Anti-SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The basic form of the antibody, which accounts for approximately 1% to approximately 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

12. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 50% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; c) The acidic form of the antibody that accounts for approximately 20% to approximately 45% of the protein in the composition when measured by capillary isoelectric focusing of the composition; and d) The basic form of the antibody, which accounts for approximately 1% to 15% of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

13. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) Deamidation form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues deamidated with 35% or less asparagine 386 and light chain amino acid residues deamidated with 25% or less asparagine 31 A composition comprising the following.

14. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) The oxidation form of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized with 3% or less tryptophan 52, a heavy chain amino acid residue oxidized with 55% or less methionine 64, a heavy chain amino acid residue oxidized with 50% or less methionine 254, a heavy chain amino acid residue oxidized with 50% or less methionine 360, and a heavy chain amino acid residue oxidized with 50% or less methionine 430. A composition comprising the following.

15. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) A deamidated form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues deamidated with 35% or less of asparagine 386 and light chain amino acid residues deamidated with 25% or less of asparagine 31; and c) The oxidation form of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized with 3% or less tryptophan 52, a heavy chain amino acid residue oxidized with 50% or less methionine 64, a heavy chain amino acid residue oxidized with 50% or less methionine 254, a heavy chain amino acid residue oxidized with 50% or less methionine 360, and a heavy chain amino acid residue oxidized with 50% or less methionine 430. A composition comprising the following.

16. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) Deamidated form of the antibody comprising 25% or less of light chain amino acid residues deamidated with asparagine 31 A composition comprising the following.

17. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) The oxidation form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues oxidized with 3% or less of tryptophan 52 and heavy chain amino acid residues oxidized with 50% or less of methionine 64. A composition comprising the following.

18. a) An anti-IL-5 antibody comprising: a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; b) The oxidation form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues oxidized with 3% or less of tryptophan 52 and heavy chain amino acid residues oxidized with 50% or less of methionine 64; and c) Deamidated form of the antibody comprising 25% or less of light chain amino acid residues deamidated with asparagine 31 A composition comprising the following.

19. a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) a deamidated form of the antibody comprising 25% or less of light chain amino acid residues deamidated with asparagine 31. A composition comprising the following.

20. a) An anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) An oxidation form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues oxidized with 3% or less of tryptophan 52 and heavy chain amino acid residues oxidized with 50% or less of methionine 64. A composition comprising the following.

21. a) An anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) A deamidated form of the antibody comprising 25% or less of light chain amino acid residues deamidated with asparagine 31; and c) The oxidation form of the antibody comprising at least one selected from the group consisting of heavy chain amino acid residues oxidized with 3% or less of tryptophan 52 and heavy chain amino acid residues oxidized with 50% or less of methionine 64. A composition comprising the following.

22. a) An anti-IL-5 antibody comprising a heavy chain sequence having the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having the amino acid sequence shown in SEQ ID NO: 2; b) Modified forms of the antibody heavy chain amino acid sequence represented by Sequence ID No. 1, comprising at least one amino acid residue modification selected from the group consisting of amino acid residue 1 (amino-terminal pyroglutamic acid residue), amino acid residue 448 (carboxy-terminal glycine amino acid residue), deamidated asparagine residue 386, oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430; and c) A modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, wherein amino acid residue 31 contains a deamidated asparagine residue. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

23. a) An anti-IL-5 antibody comprising a heavy chain sequence having the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having the amino acid sequence shown in SEQ ID NO: 2; b) Modified forms of the antibody heavy chain amino acid sequence represented by Sequence ID No. 1, comprising at least one amino acid residue modification selected from the group consisting of the deamidated asparagine residue at position 386, the oxidized tryptophan residue at position 52, the oxidized methionine residue at position 64, the oxidized methionine at position 254, the oxidized methionine at position 254, the oxidized methionine at position 360, and the oxidized methionine residue at position 430; and c) A modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, wherein amino acid residue 31 contains a deamidated asparagine residue. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

24. a) An anti-IL-5 antibody comprising a heavy chain sequence having the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having the amino acid sequence shown in SEQ ID NO: 2; b) A modified form of the antibody heavy chain amino acid sequence represented by SEQ ID NO: 1, comprising a deamidated asparagine residue at position 386; and c) A modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2, wherein amino acid residue 31 contains a deamidated asparagine residue. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

25. a) An anti-IL-5 antibody comprising a heavy chain sequence having the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having the amino acid sequence shown in SEQ ID NO: 2; and b) Modification of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1, comprising at least one amino acid residue modification selected from the group consisting of oxidized tryptophan residue 52, oxidized methionine residue 64, oxidized methionine residue 254, oxidized methionine residue 360, and oxidized methionine residue 430. A composition comprising a group of anti-IL-5 antibodies having [a certain characteristic].

26. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; and b) The main form of the antibody that accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition. A composition comprising the following.

27. a) An anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2; b) The main form of the antibody that accounts for 20% or more of the protein in the composition when measured by capillary isoelectric focusing of the composition; and c) The acidic form of the antibody that accounts for up to approximately 80% of the protein in the composition when measured using capillary isoelectric focusing of the composition. A composition comprising the following.

28. The composition according to any one of claims 1 to 27, wherein the antibody is concentrated in a concentration between approximately 75 mg / ml and approximately 100 mg / ml.

29. (a) A buffer selected from the group consisting of disodium phosphate heptahydrate, phosphates, citrates, sodium phosphate, potassium phosphate, sodium citrate, and histidine, which gives a pH between 6.8 and 7.2; and / or (b) sugars; and / or (c) Polysorbate 80; and / or (d) EDTA The composition according to any one of claims 1 to 28, further comprising one or a combination of the above.

30. The composition according to any one of claims 1 to 29, having at least 0.70 IL-5 specific antigen-binding activity and / or at least 70% FcRn-binding activity compared to a reference standard composition comprising SEQ ID NO: 1 and SEQ ID NO:

2.

31. A method for treating a disease in a subject, a) Identifying subjects having a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis; and b) The step of administering to the subject a therapeutically effective amount of the composition according to any one of claims 1 to 30. A method comprising, wherein a disease in the subject is treated.

32. The method according to claim 31, wherein the composition is administered once every four weeks in a dose of 100 mg.

33. A composition according to any one of claims 1 to 30 for use in therapy.

34. A pharmaceutical composition comprising the composition according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

35. A composition according to any one of claims 1 to 30 for use in the treatment of asthma, severe eosinophilic asthma, severe asthma, poorly controlled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic syndrome, nasal polyps, bullous pemphigoid, and eosinophilic esophagitis.

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