Assays for fixed dose combinations

Abstract

This invention provides assays for analyzing the quality and quantity properties of fixed-dose combinations. Specifically, this document describes assays for fixed-dose combinations of two anti-HER2 antibodies, and assays for subcutaneous formulations comprising pertuzumab and trastuzumab.

Description

[Technical Field]

[0001] This invention relates to assays for analyzing the quality and quantity properties of fixed-dose combinations. Specifically, this invention relates to assays for fixed-dose combinations of two anti-HER2 antibodies, and for assays for subcutaneous formulations containing pertuzumab and trastuzumab. [Previous Technology]

[0002] To ensure the safety and efficacy of biopharmaceutical products, continuous monitoring of product quality is essential. Certain specific criteria, including Critical Quality Attributes (CQAs), must be met before any batch of product is released. CQAs are physical, chemical, biological, or microbiological properties or characteristics that must be within appropriate limits, ranges, or distributions to ensure the desired product quality, safety, and efficacy.

[0003] Performance tests and numerous other tests are performed as part of product consistency testing, comparability studies, and stability testing. These tests are used to measure product attributes related to product quality and manufacturing controls, and are performed to ensure the identity, purity, strength (potency), and stability of the product used throughout all phases of clinical studies. Similarly, performance measurements are used throughout all phases of clinical studies and after market approval to confirm that only batches of the product that meet the defined specifications or acceptance criteria are administered.

[0004] Ion exchange chromatography (IEX) is widely used for detailed characterization of therapeutic proteins and is considered a powerful and reliable technique for qualitative and quantitative assessment of charge heterogeneity. IEX is typically a flare method where specific specifications are set for the distribution of each acidic, major, and basic component of a monoclonal antibody (mAb). These charged components can be considered product-related impurities that can affect potency. Furthermore, it is one of the few methods that can characterize proteins in their native conformation because no denaturing agents are added. IEX is also used as an identification method for certain biologics and is a routine test for stability and shelf-life validation.

[0005] Quantity is usually measured as CQA (Concentration Quality Assurance) for protein content. It is crucial for biotechnology and bioproducts and should be determined using appropriate assays, typically physicochemical assays. For most biopharmaceuticals, protein content is measured by UV absorption.

[0006] Fixed-dose combination (FDC) combines two different active ingredients into a single-dose formulation. The combination of two anti-HER2 antibodies, trastuzumab and pertuzumab, with hyaluronidase is the first clinically developed combination of two highly similar monoclonal antibodies. It is believed that the mechanisms of action of pertuzumab and trastuzumab are complementary, as both bind to the HER2 receptor, but to different sites. The combination of pertuzumab and trastuzumab is thought to provide a more comprehensive dual blockade of the HER2 signaling pathway. The standard IV regimen (perjeta-based) of perjeta in combination with the IV herceptin and chemotherapy is approved in over 100 countries for the treatment of early and metastatic HER2-positive breast cancer. In the neoadjuvant early breast cancer (eBC) setting, the pCR rate of the perjeta-based regimen has been shown to be almost twice that of Herceptin and chemotherapy alone. In addition, this combination has been shown to significantly reduce the risk of recurrence, aggressive disease, or death in the adjuvant eBC setting. In the metastatic setting, this combination has demonstrated unprecedented survival benefits in previously untreated (first-line) HER2-positive metastatic breast cancer patients.

[0007] The hyaluronidase in FDC enables and optimizes the subcutaneous delivery of appropriate co-administered therapeutic agents. Recombinant human hyaluronidase PH20 (rHuPH20) is an enzyme that temporarily degrades hyaluronic acid (glycosaminoglycans or natural sugar chains in the body) to help disperse and absorb other injected therapeutic agents.

[0008] Trastuzumab and pertuzumab share more than 93% sequence identity and differ by only 30 Da in total. Both antibodies have a molecular weight of approximately 148 kDa and nearly identical isoelectric points. They bind to the same target (HER2) and exhibit synergistic effects in vivo. Due to their structural and functional similarity, most commonly used analytical methods cannot be applied to this combination formulation. [Summary of the Invention]

[0009] In one embodiment, a binding assay for a fixed-dose combination (FDC) of two anti-HER2 antibodies is provided, comprising: a. contacting the FDC with a capture reagent comprising a modified HER2 ECD subdomain; b. contacting the sample with a detectable antibody; c. quantifying the amount of antibody bound to the capture reagent using a detection method for the detectable antibody.

[0010] In one embodiment, the fixed-dose combination comprises an antibody that binds to HER2 extracellular subdomain II and an antibody that binds to HER2 extracellular subdomain IV.

[0011] In one embodiment, a binding assay is provided for a fixed-dose combination (FDC) of two anti-HER2 antibodies, wherein the binding of the antibody to the extracellular subdomain II of HER2 is quantitatively measured.

[0012] In one embodiment, the capture reagent comprises recombinant HER2 extracellular domain II. In one embodiment, the capture reagent comprises SEQ ID NO: 2 or SEQ ID NO: 23. In one embodiment, the capture reagent comprises recombinant HER2 extracellular domains I, II, and III. In one embodiment, the capture reagent comprises SEQ ID NO: 24. In one embodiment, the capture reagent does not comprise HER2 subdomain IV.

[0013] In one embodiment, a binding assay is provided for a fixed-dose combination (FDC) of two anti-HER2 antibodies, wherein the binding of the antibody to HER2 subdomain II is quantified. In one embodiment, the capture reagent comprises recombinant HER2 extracellular domain IV.

[0014] In one embodiment, the capture reagent comprises SEQ ID NO: 4 or SEQ ID NO: 28. In one embodiment, the capture reagent does not contain HER2 subdomain II. In one embodiment, the capture reagent comprises recombinant HER2 extracellular domains I, III, IV and EGFR domain II. In one embodiment, the capture reagent comprises SEQ ID NO: 29.

[0015] In one embodiment, a binding assay is provided for a fixed-dose combination (FDC) of two anti-HER2 antibodies, wherein the binding assay is used to analyze the bioactivity of one of the anti-HER2 antibodies. In one embodiment, bioactivity is quantified by correlating the amount of antibody bound to the capture reagent with the bioactivity of the isolated antibody measured in a cell-based assay.

[0016] In one embodiment, a capture reagent is coated onto a microplate. In one embodiment, the F(ab')2 portion of an antibody targeting an anti-HER2 antibody can be detected.

[0017] In one embodiment, the fixed-dose combination to be analyzed in the binding assay further comprises hyaluronidase.

[0018] In one embodiment, an isolated protein comprising SEQ ID NO: 24 is provided. In one embodiment, an isolated protein comprising SEQ ID NO: 29 is provided.

[0019] Further provided is a kit for binding antibodies to HER2 extracellular subdomain II in a specific quantitative fixed-dose combination (FDC), the fixed-dose combination (FDC) having a first antibody and a second anti-HER2 antibody binding to HER2 extracellular subdomain II, the kit comprising: a. a container containing a protein as a capture agent, the protein comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 34; b. a description of the binding of the antibody to HER2 extracellular subdomain II.

[0020] Further, a kit is provided for binding an antibody to HER2 extracellular subdomain IV in a specific quantitative fixed-dose combination (FDC), the fixed-dose combination (FDC) having an antibody binding to HER2 extracellular subdomain IV and a secondary anti-HER2 antibody, the kit comprising: a. a container containing a protein as a capture agent, the protein comprising SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 4; b. a description of the binding of the antibody to HER2 extracellular subdomain IV.

[0021] In another aspect of the present invention, a method for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab is provided, the method comprising: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0022] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0023] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0024] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0025] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab to be analyzed further comprises hyaluronidase.

[0026] In one embodiment, a method for preparing a composition is provided, comprising: (1) producing a fixed-dose composition comprising pertuzumab, trastuzumab and one or more variants thereof, and (2) subjecting the composition thus produced to an analytical assay to assess the amount of the variant, wherein the variant comprises: (i) pertuzumab deacetylated with HC-Asn-391, a pertuzumab FC sialic acid variant, and a pertuzumab lysine glycation variant, (ii) a natural pertuzumab antibody, (iii) a natural trastuzumab antibody, and (vi) a trastuzumab having HC-Asp-102 monoisomeric to isaspartic acid at one heavy chain.

[0027] In one embodiment, a method for preparing a composition is provided, wherein the analytical assay of step (2) comprises: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0028] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0029] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0030] In one embodiment, the analytical assay in step (2) further includes the following steps: c. Selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0031] In one embodiment, the method is performed at a temperature of 32-40°C.

[0032] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab in step (1) further includes hyaluronidase.

[0033] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab in step (1) comprises 40-60 mg / mL trastuzumab and 60-80 mg / mL pertuzumab.

[0034] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 28% of pertuzumab natural antibody, at least 16% of trastuzumab natural antibody and less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0035] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 38% of pertuzumab natural antibody, at least 16% of trastuzumab natural antibody and less than 9% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0036] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 21% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 28% of pertuzumab natural antibody, at least 23% of trastuzumab natural antibody and less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0037] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of the total peak area of ​​peaks 1 to 3, at least 28% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 16% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 12% of the peak area of ​​peak 8, as determined by a method comprising the following steps: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. extracting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0038] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0039] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0040] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0041] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0042] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0043] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of the total peak area of ​​peaks 1 to 3, at least 38% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 16% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 9% of the peak area of ​​peak 8, as determined in a method comprising the following steps: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0044] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0045] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0046] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0047] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0048] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0049] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 21% of the total peak area of ​​peaks 1 to 3, at least 28% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 23% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 12% of the peak area of ​​peak 8, as determined in a method comprising the following steps: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0050] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0051] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0052] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0053] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0054] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0055] In another aspect of the invention, the composition provided herein can be obtained by a method comprising the following steps: a. adding a predetermined amount of pertuzumab to a mixing vessel; b. adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or in a 1:2 ratio of trastuzumab to pertuzumab; c. adding rHuPH20.

[0056] In another aspect, a method is provided for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, comprising: a. providing an RP-HPLC phenyl column; b. loading the fixed-dose combination (FDC) of two anti-HER2 antibodies onto the RP-HPLC column; c. separating the two anti-HER2 antibodies at a flow rate of 0.2-0.4 mL / min, wherein the column temperature is 64°C to 76°C.

[0057] In one embodiment, the fixed-dose combination comprises pertuzumab and trastuzumab. In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase.

[0058] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, a water-2-propanol / acetonitrile gradient is used to achieve the separation in step c).

[0059] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the flow rate in step c) is about 0.3 mL / min.

[0060] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the antibody system is separated within 10 to 20 minutes. In another embodiment, the antibody system is separated within 15 minutes. In one embodiment, the antibodies are separated over 15 minutes at a flow rate of 0.3 mL / min.

[0061] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the column temperature is 70°C ± 2°C.

[0062] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the phenyl column is selected from the group consisting of: Agilent Zorbax RRHD 300-Diphenyl, Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, and Agilent AdvanceBio RP mAb Diphenyl.

Implementation Method

[0064] I. Definition

[0065] The term "about" as used in this patent specification is intended to indicate that a specific value provided may vary to a certain extent, for example, meaning a variation within a range of +10% in a given value. In one embodiment, the variation within a range of + / -5% in a given value is included.

[0066] "HER receptor" refers to the tyrosine kinase receptor protein belonging to the HER receptor family and includes EGFR, HER2, HER3, and HER4 receptors. HER receptors typically include: an extracellular domain that binds to HER ligands and / or dimers with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain containing several phosphorylated tyrosine residues. HER receptors can be the "natural sequence" HER receptor or its "amino acid sequence variants." Preferably, the HER receptor system is the natural sequence human HER receptor.

[0067] The terms "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (GenBank Accession Number: X03363). The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to the gene encoding rat p185neu. The preferred HER2 is the natural sequence human HER2.

[0068] In this document, "HER2 extracellular domain" or "HER2 ECD" refers to the HER2 domain anchored to the cell membrane or circulating outside the cell, including its fragments. The amino acid sequence of HER2 is shown in Figure 1. In one embodiment, the extracellular domain of HER2 may include the following four subdomains: "Subdomain I" (amino acid residues 1-195; SEQ ID NO:1), "Subdomain II" (amino acid residues 196-319; SEQ ID NO:2), "Subdomain III" (amino acid residues 320-488; SEQ ID NO:3), and "Subdomain IV" (amino acid residues 489-630; SEQ ID NO:4) (the residue numbers do not include signal peptides). See Garrett et al., Mol. Cell. 11: 495-505 (2003); Cho et al., Nature 421: 756-760 (2003); Franklin et al., Cancer Cell 5: 317-328 (2004); and Plowman et al., Proc. Natl. Acad. Sci. 90: 1746-1750 (1993); and Figure 1 of this paper. "Recombinant HER2 extracellular subdomain" or "recombinant HER2 ECD subdomain" comprises individual native HER2 ECD subdomains in full-length or truncated form. To make the conformation of the modified HER2 ECD as close as possible to that of the native HER2 ECD, the recombinant HER2 ECD subdomain can preferably be truncated by up to 6 amino acids at its C-terminus.

[0069] "Anti-HER2 antibody" or "HER2 antibody" refers to an antibody that binds to the HER2 receptor. Depending on the situation, HER2 antibodies may further interfere with the activation or function of HER2. The anti-HER2 antibodies of interest in this article are pertuzumab and trastuzumab.

[0070] The antibody "binding to extracellular subdomain II of HER2" binds to residues in domain II (SEQ ID NO: 2) of HER2 and, where appropriate, residues in other subdomains (e.g., subdomains I and III (SEQ ID NO: 1 and 3, respectively)). Preferably, the antibody binding to extracellular subdomain II binds to the junction between extracellular subdomains I, II, and III of HER2. In one embodiment, the antibody binding to extracellular subdomain II is pertuzumab or a variant thereof.

[0071] For purposes herein, "pertuzumab" and "rhuMAb 2C4" (which are used interchangeably) refer to antibodies comprising the variable light chain amino acid sequence and the variable heavy chain amino acid sequence of SEQ ID NO: 7 and 8, respectively. In the case of a complete pertuzumab antibody, it preferably comprises an IgG1 antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 11 or 15 and the heavy chain amino acid sequence of SEQ ID NO: 12 or 16. The antibody is generated by recombinant Chinese hamster ovary (CHO) cells, as appropriate. The terms "pertuzumab" and "rhuMAb 2C4" herein cover biosimilar forms having a United States Adopted Name (USAN) or International Nonproprietary Name (INN): pertuzumab.

[0072] The antibody “binding to the extracellular subdomain IV of HER2” binds to residues in domain IV (SEQ ID NO: 4) of HER2 and, where applicable, residues in other subdomains. In one embodiment, the antibody binding to the extracellular subdomain IV is trastuzumab or a variant thereof.

[0073] For purposes herein, “trastuzumab” and “rhuMAb4D5” (which are used interchangeably) refer to antibodies comprising the variable light chain amino acid sequence and the variable heavy chain amino acid sequence of SEQ ID No: 13 and 14, respectively. In the case of a complete trastuzumab antibody, it preferably comprises an IgG1 antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 13 and the heavy chain amino acid sequence of SEQ ID NO: 14. The antibody is generated using Chinese hamster ovary (CHO) cells, as appropriate. The terms “trastuzumab” and “rhuMAb4D5” herein cover biosimilar forms having the US Adopted Name (USAN) or International Nonproprietary Name (INN): trastuzumab.

[0074] The term “combination formulation” is used herein to refer to a single standby pharmaceutical formulation containing two or more active ingredients, including, for example, a single standby pharmaceutical formulation containing pertuzumab and trastuzumab formulated together for subcutaneous (SC) administration.

[0075] "Fixed-dose combination" or "FDC" as used herein refers to a single standby pharmaceutical preparation containing two or more active ingredients, including, for example, a single standby pharmaceutical preparation containing pertuzumab and trastuzumab formulated together for subcutaneous (SC) administration. "Pertuzumab-trastuzumab FDC" includes pertuzumab, trastuzumab, and, as appropriate, hyaluronidase.

[0076] The term "hyaluronidase" refers to a group of enzymes found throughout the animal kingdom that are generally neutral or acidic in activity. Hyaluronidases vary due to their specificity and mechanism of action (WO 2004 / 078140). There are generally three classes of hyaluronidases: 1. Mammalian hyaluronidase (EC 3.2.1.35), which is an endo-β-N-acetylglucosaminease that produces tetrasaccharides and hexasaccharides as its main end products. It possesses hydrolytic and transglycosidic activities and can degrade hyaluronic acid and chondroitin sulfate (CS), typically C4-S and C6-S. 2. Bacterial hyaluronidase (EC 4.2.99.1), which degrades hyaluronic acid and, to varying degrees, CS and DS. It is an endo-β-N-acetylglucosaminease that functions primarily through β-elimination reactions that produce disaccharide final products. 3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-β-glucuronidases that hydrolyze hyaluronidases via β1-3 linkages to produce tetrasaccharide and hexasaccharide final products. Mammalian hyaluronidases can be further divided into two categories: neutral-active enzymes and acid-active enzymes. Hyaluronidase-like enzymes are also characterized by being typically anchored to the plasma membrane via glycosylphosphatidylinositol (e.g., human HYAL2 and human PH20) [Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA, 2003; 100(8):4580-4585; Phelps et al., Science 1988; 240(4860): 1780-1782] and typically soluble (e.g., human HYAL1) [Frost, IG et al., "Purification, cloning, and expression of human plasma hyaluronidase", Biochem. Biophys. Res. Commun. 1997; 236(1):10-15]. Bovine PH20 is loosely attached to the serous membrane and is not anchored via a phospholipase-sensitive anchor [Lalancette et al., Biol. Reprod., 2001; 65(2):628-36]. This unique characteristic of bovine hyaluronidase allows for the use of soluble bovine testicular hyaluronidase as an extractant for clinical applications (Wydase™, Hyalase™). Other PH20 substances are typically lipid-anchored enzymes that are insoluble without the use of detergents or lipases. For example, human PH20 is anchored to the serous membrane via a GPI anchor. Natural macaque sperm hyaluronidase has been found in both soluble and membrane-bound forms.Although the 64 kDa membrane-bound form is enzymatically active at pH 7.0, the 54 kDa form is active only at pH 4.0 [Cherr et al., Dev. Biol., 1996; 10; 175(1): 142-53]. WO2006 / 091871 describes a soluble hyaluronidase glycoprotein (sHASEGP) that facilitates the delivery of therapeutic drugs to the subepithelial tissue. By rapidly depolymerizing HA in the extracellular space, sHASEGP reduces interstitial viscosity, thereby increasing hydraulic conductivity and allowing for the safe and comfortable delivery of larger volumes to SC tissue. The preferred hyaluronidase is human hyaluronidase, with recombinant human hyaluronidase called rHuPH20 (FuHyaluronidase α) being the most preferred. rHuPH20 is a member of a family of neutral and acidic β-1,4-glycosidic hydrolases. These hydrolases depolymerize hyaluronic acid by hydrolyzing the β-1,4 bond between the C1 position of N-acetylglucosamine and the C4 position of glucuronic acid. Hyaluronidase products approved in the European Union include Hylase® "Dessau" and Hyalase®. Animal-derived hyaluronidase products approved in the United States include Vitrase™, Hydase™, and Amphadase™.

[0077] rHuPH20 is currently the first and only recombinant human hyaluronidase available for therapeutic use. The amino acid sequence of rHuPH20 (HYLENEXTM) is well known and available at CAS Registry No.: 75971-58-7. The approximate molecular weight is 61 kDa. In one embodiment, pertuzumab-trastuzumab FDC contains hyaluronidase at a concentration of 2000 U / mL, depending on the situation.

[0078] The term "loading" dose in this document typically includes the initial dose of the therapeutic agent administered to the patient, followed by one or more maintenance doses. The loading dose (LD) of pertuzumab-trastuzumab FDC comprises 40 mg / mL trastuzumab, 80 mg / mL pertuzumab, and 2000 U / mL rHuPH20.

[0079] The term "maintenance" dose in this document refers to one or more therapeutic doses administered to the patient during the treatment period. Maintenance doses are typically administered at intervals between treatments, such as approximately weekly, approximately every 2 weeks, approximately every 3 weeks, or approximately every 4 weeks, preferably every 3 weeks. The maintenance dose (MD) of the pertuzumab-trastuzumab FDC comprises 60 mg / mL trastuzumab, 60 mg / mL pertuzumab, and 2000 U / mL rHuPH20.

[0080] As used herein, "capture reagent" means any reagent capable of binding to an analyte (e.g., an anti-HER2 antibody). Preferably, "capture reagent" means any reagent specifically bound to an anti-HER2 antibody in a fixed-dose combination of two anti-HER2 antibodies. For specific analysis of the binding of one of the two anti-HER2 antibodies in the fixed-dose combination, the capture reagent must be specific to that antibody; for example, the binding affinity and / or specificity of the analyte to the capture reagent should be higher than that of the second anti-HER2 antibody in the FDC. In one embodiment, the capture reagent provided in the assay is a modified HER2 ECD.

[0081] "Modified HER2 ECD" refers to a genetically engineered protein or peptide comprising one or more recombinant HER2 ECD subdomains. The HER2 ECD is modified so that one of the anti-HER2 antibodies to be evaluated in the FDC can bind to it, while a second anti-HER2 antibody in the FDC does not bind to it. This is achieved by deleting the HER2 ECD subdomain that the second anti-HER2 antibody binds to, or by replacing that subdomain with a structurally compact subdomain that does not bind to either anti-HER2 antibody. Preferably, the modified HER2 ECD is constructed to mimic the native HER2 ECD as closely as possible. The subdomain can be full-length or shortened by a few amino acids at the N-terminus or C-terminus. The inventors of this invention have discovered that the integrity of the three-dimensional structure of the HER2 ECD can be preserved or improved when using one or more recombinant HER2 ECD subdomains shortened by about 4 to 5 amino acids at the C-terminus.

[0082] The term "Fc domain" in this document is used to define the C-terminal domain of the immunoglobulin heavy chain. The Fc domain can be of various origins, such as mouse, rat, goat, or human. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. Unless otherwise indicated, the residue numbers in the immunoglobulin heavy chain herein refer to EU index numbers, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is explicitly incorporated herein by reference. "EU index as described in Kabat" refers to the residue number of the human IgG1 EU antibody.

[0083] As used herein, the term "detectable antibody" refers to an antibody linked to a reagent or detectable label capable of generating a detectable signal that can be used to evaluate the presence and / or quantity of the analyte to be detected (i.e., anti-HER2 antibody).

[0084] The term "label" or "detectable label" refers to any chemical group or portion that can be attached to a detectable antibody. Examples of detectable labels include luminescent labels (e.g., fluorescent, phosphorescent, chemiluminescent, bioluminescent, and electrochemiluminescent labels), radioactive labels, enzymes, particles, magnetic materials, electroactive materials, and the like. Alternatively, a detectable label may indicate its presence by participating in a specific binding reaction. Examples of such labels include haptens, antibodies, biotin, streptavidin, his tag, N-triacetic acid, glutathione S-transferase, glutathione, and the like.

[0085] The term "detection means" refers to a part or technique used to detect the presence of an antibody by means of a signal read out in the assay described herein. "Photoluminescence" is the process by which a material emits light after absorbing light (or electromagnetic radiation or EMR). Fluorescence and phosphorescence are two different types of photoluminescence. "Chemiluminescence" is a process by which a luminescent substance is produced through a chemical reaction. "Electrochemiluminescence" or "ECL" is the process by which a substance (e.g., the antibody of interest) emits light after being exposed to electrochemical energy in a suitable surrounding chemical environment.

[0086] As used herein, the term "ELISA" (also known as enzyme-linked immunosorbent assay) refers to a biochemical technique primarily used to detect the presence of antibodies in biological samples. For the purposes of this application, ELISA technology is used to detect and quantify anti-HER2 antibodies in a fixed dose combination. Typically, for ELISA-based assays, the capture reagent is fixed or immobilizable.

[0087] In this document, "potency" refers to the therapeutic activity or expected biological effect of a biotherapeutic drug. The potency of a biotherapeutic drug can be determined by measuring or quantifying the biological activity of the active ingredient of the biotherapeutic drug.

[0088] In this document, "bioactivity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen and produce a measurable biological response, which can be measured in vitro or in vivo. In one embodiment, bioactivity refers to the ability to bind to a capture agent in a binding assay as provided herein. In one embodiment, the binding of the anti-HER2 antibody in the FDC is related to the ability of the anti-HER2 antibody in the monoclonal antibody formulation to inhibit the proliferation of human breast cancer cell lines. The suitable human breast cancer cell line for testing pertuzumab is MDA-MB-175-VII. The suitable human breast cancer cell line for testing trastuzumab is BT-474.

[0089] The term “antibody” is used in the broadest sense and specifically includes monoclonal antibodies, multiclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that they exhibit the desired biological activity.

[0090] The "humanized" form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. Most humanized antibodies are human immunoglobulins (recipient antibodies), where residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and capability. In some cases, the backbone region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not present in the recipient or donor antibody. These modifications are performed to further improve antibody efficacy. Typically, humanized antibodies will include substantially all at least one (and usually two) variable domains, wherein all or substantially all highly variable loops correspond to highly variable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also, as needed, include at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. For further details, see Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992. Specifically, humanized HER2 antibodies include trastuzumab (HERCEPTIN®), as described in Table 3 of U.S. Patent 5,821,337 (expressly incorporated herein by reference) and as defined herein; and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

[0091] The term "intact antibody" in this article refers to an antibody that contains two antigen-binding regions and an Fc region. Preferably, an intact antibody has a functional Fc region.

[0092] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bivalent antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0093] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies in the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domains of the light chains are aligned with the first constant domain of the heavy chains, and the variable domains of the light chains are aligned with the variable domains of the heavy chains. It is believed that specific amino acid residues form interfaces between the variable domains of the light and heavy chains.

[0094] The term “hypervariant region” as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the complementarity-determining region (CDR) or the chain variable domain (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the hypervariable ring (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothi A See Lesk J. Mol. Biol. 196:901-917 (1987)). "Frame region" or "FR" residues are other variable domain residues besides the hypervariable region residues as defined herein.

[0095] Based on the amino acid sequence of the heavy chain constant domain, intact antibodies can be classified into different "types". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these types can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different types of antibodies are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.

[0096] "Naked antibody" is an antibody that has not been bound to a foreign molecule (such as a cytotoxic part or a radiolabeled molecule).

[0097] An "affinity-matured" antibody system has one or more alterations in one or more hypervariable regions compared to an unaltered parent antibody, which improve the antibody's affinity for the antigen. Better affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the industry. Marks et al. (Bio / Technology 10:779-783, 1992) describe affinity maturation achieved through shuffling of the VH and VL domains. Random mutagenesis of CDRs and / or framework residues is described in the following literature: Barbas et al., Proc Nat. Acad. Sci, USA91:3809-3813 (1994); Schier et al., Gene169:147-155 (1995); Yelton et al., J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

[0098] "Value" refers to a container suitable for containing liquid or lyophilized preparations. In one embodiment, the vial is a disposable vial, such as a 10 mL or 20 mL disposable vial with a stopper, such as a 10 mL disposable glass vial with a 20 mm stopper.

[0099] As used herein, “extraction” refers to the removal of a protein of interest (e.g., an antibody) from a cation exchange material, which is achieved by altering the ionic strength of the buffer surrounding the cation exchange material so that the buffer competes with the molecules for the charged points on the ion exchange material.

[0100] As used herein, the term “chromatography” refers to the process of separating a solute of interest (e.g., a protein of interest) from other solutes in a mixture by percolating a mixture with an adsorbent that more or less strongly adsorbs or retains the solute under specific process buffer conditions (due to solute properties such as pi, hydrophobicity, size and structure).

[0101] The terms “ion exchange” and “ion exchange chromatography” refer to a chromatography process in which an ionizable solute of interest (e.g., an antibody against FDC and its acidic and basic variants) interacts with an oppositely charged ligand attached (e.g., by covalent attachment) to a solid-phase ion exchange material under appropriate pH and conductivity conditions, such that the solute of interest interacts more or less nonspecifically with charged compounds than solute impurities or contaminants in the mixture.

[0102] "Ion exchange chromatography" specifically includes cation exchange (CEX) chromatography, anion exchange chromatography and mixed mode chromatography.

[0103] "Cation exchange material" or "CEX material" refers to a negatively charged solid phase having free cations for exchange with cations passing over or through the solid phase in an aqueous solution. Any negatively charged ligand attached to the solid phase suitable for forming a cation exchange material may be used, such as carboxylic acid esters, sulfonates, and others described below. Commercially available cation exchange materials include, but are not limited to, those having the following groups: sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S from GE Healthcare, SP Sepharose Fast Flow™, SP Sepharose High Performance, Toyopearl SP-650S and SP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic HyperD S, Trisacryl M and LS SP from Pall Technologies, and Spherodex LS SP); sulfoethyl-based groups (e.g., Fractogel SE from EMD, Poros S-10 and S-20 from Applied Biosystems); sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Poros HS-20 and HS from Applied Biosystems). 50); sulfoisobutyl-based groups (e.g., Fractogel EMD SO3 from EMD); sulfinylethyl-based groups (e.g., SE52, SE53, and Express-Ion S from Whatman); carboxymethyl-based groups (e.g., CM Sepharose Fast Flow from GE Healthcare, and Biochrom Labs Inc.).Hydrocell CM from BioRad, Macro-Prep CM from Pall Technologies, Ceramic HyperD CM, Trisacryl M CM, Trisacryl LS CM from Millipore, Matrx Cellufine C500 and C200 from Whatman, CM52, CM32, CM23 and Express-Ion C from Whatman, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh; sulfonic acid and carboxylic acid based groups (e.g., BAKERBOND Carboxy-Sulfon from JTBaker); carboxylic acid based groups (e.g., WP CBX from JT Baker, DOWEX MAC-3 from Dow Liquid Separations, Amberlite weak cation exchanger, DOWEX weak cation exchanger and Diaion weak cation exchanger from Sigma-Aldrich, and from EMD). Fractogel EMD COO-; sulfonic acid-based groups (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX fine-mesh strong acid cation exchange resin from Dow Liquid Separations, UNOsphere S and WP Sulfonic from JT Baker, Sartobind S membrane from Sartorius, Amberlite strong cation exchanger from Sigma-Aldrich, DOWEX strong cation exchanger, and Diaion strong cation exchanger); and orthophosphate-based groups (e.g., PI 1 from Whatman).

[0104] Depending on the chemical properties of the charged groups / substituents and the strength of the covalently bonded charged substituents, "ion exchange chromatography materials" can be classified as strong ion exchange materials or weak ion exchange materials. The "strong cation exchange materials" or "(SCX) materials" used in this article have sulfonic acid-based groups, such as sulfonates, sulfopropyl groups, sodium polystyrene sulfonate, or polyAMPS (poly(2-acrylamide-2-methyl-1-propanesulfonic acid)).

[0105] The "isoelectric point" or "pI" of a protein or antibody corresponds to the pH value at which the net charge of the protein or antibody is neutral. pI can be determined using standard experimental methods, such as isoelectric focusing or by calculation ("theoretical pI"). One example of a calculation method is the free online standard tool "ExPASy" (http: / / web.expasy.org / compute_pi / ), which calculates pI based on the amino acid sequence of the protein or antibody. The theoretical pI for trastuzumab is 8.4, and the theoretical pI for pertuzumab is 8.7.

[0106] The "mobile phase" is a liquid or gas that flows through the chromatography system to move the materials to be separated through the stationary phase at different rates. Preferably, the mobile phase is a liquid. In one example, the mobile phase may be a loading buffer ("mobile phase A") or an extraction buffer (mobile phase B).

[0107] "Loading buffer" provides conditions to ensure that the target molecules interact effectively with the ligands of the ion exchange chromatography material and are retained by the affinity medium as all other molecules flow through the column.

[0108] "Escape buffer" is used to first wash away unbound proteins and then release charged variants and natural antibodies at a higher concentration of autoligands.

[0109] The terms "major type antibody" or "natural antibody" used herein refer to the antibody amino acid sequence structure of the dominant antibody molecule in the composition. In a fixed-dose combination of two anti-HER2 antibodies, the two major type antibodies are part of the composition. Therefore, in one embodiment, the major type antibody system binds to both the extracellular subdomain II and the extracellular subdomain IV of HER2. In one embodiment, the major type antibody of FDC is pertuzumab and trastuzumab.

[0110] "Charge variants" are a type of variant of the main antibody class that has an overall charge different from that of the main antibody class. Examples of charge variants are acidic variants and basic variants.

[0111] "Acidic variants" are variants of major antibody species, which are more acidic than major antibody species. Compared to major antibody species, acidic variants have acquired a negative charge or lost a positive charge. These acidic variants can be resolved using methods that separate proteins based on charge (e.g., ion exchange chromatography). After separation by cation exchange chromatography, the acidic variants of major antibody species emerge earlier than the main peak. Acidic variants of pertuzumab and trastuzumab can be separated and quantified using the ion exchange chromatography method described herein. Examples of acidic pertuzumab variants include pertuzumab deacetylated at position 391 of the heavy chain asparagine (HC-Asn-391), pertuzumab Fc sialic acid variant, and pertuzumab lysine glycation variant. Examples of acidic trastuzumab variants are trastuzumab deacetinated at LC-Asn-30 and trastuzumab deacetinated at HC-Asn-55.

[0112] "Basic variants" are variants of major antibody species, which are more basic than major antibody species. Compared to major antibody species, basic variants have gained a positive charge or lost a negative charge. These basic variants can be resolved using methods that separate proteins based on charge (e.g., ion exchange chromatography). After separation by cation exchange chromatography, basic variants of major antibody species emerge later than the main peak. Basic variants of pertuzumab and trastuzumab can be separated and quantified using the ion exchange chromatography method described herein.

[0113] As used herein, the term "gradient" refers to a change in the properties of the mobile phase during the chromatography of a sample. In a "continuous gradient," one or more conditions of the mobile phase (e.g., pH, ionic strength, salt concentration, and / or mobile phase flow) are changed continuously, i.e., increased or decreased. This change can be linear, exponential, or asymptotic. In a "stepwise gradient," one or more conditions (e.g., pH, ionic strength, salt concentration, and / or chromatography flow) can be changed incrementally, i.e., gradually, compared to a linear change.

[0114] The term "RP-UHPLC" refers to reversed-phase ultra-high performance liquid chromatography. RP-HPLC stands for reversed-phase high performance liquid chromatography. HPLC uses polarity and interaction with the stationary phase of the column to separate compounds. Reversed-phase chromatography is a rinsing procedure used in liquid chromatography where the mobile phase is significantly more polar than the stationary phase.

[0115] The term "RP-HPLC phenyl column" as used herein refers to a column in which a hydrophobic phenyl group is present on the column packing material or resin (stationary phase). For example, a phenyl column exposes the material flowing through the column to unsubstituted phenyl groups. Phenyl columns contain, for example, short alkylphenyl ligands or diphenyl phases covalently bonded to a silicon dioxide surface. Some phenyl columns with phenyl groups have alkyl spacers between the phenyl group and the silicon dioxide surface. By increasing the length of the alkyl spacers, steric and aromatic selectivity can be enhanced. The differences between RP-HPLC phenyl columns lie in the number of aromatic groups (monophenyl-p-biphenyl), the length of the alkyl spacers between the silicon dioxide surface and the phenyl group, the nature of the substituents on the bonded ligands (usually methyl or spatially larger isobutyl groups), whether oxygen atoms are incorporated into the linker to activate the π-electron system in the aromatic ring, and whether the silicon dioxide stationary surface is capped. For example, RP-HPLC phenyl columns may have the following groups: ethylphenyl with methyl side groups and a capped silicon dioxide surface; phenylhexyl phase with methyl side groups of extended (hexyl) ligand spacers; ethylphenyl ligands with sterically protected (isobutyl) side groups; hexylbiphenyl with methyl side groups; biphenyl phase with methyl side groups; and oxygen-activated phenylethylphenyl phase with methyl side groups. HPLC columns with phenyl (e.g., monophenyl, biphenyl, diphenyl, phenylhexyl, phenylpropyl) modified stationary phases are readily available from most major column suppliers, such as: Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, Agilent Zorbax RRHD 300-Diphenyl, and Agilent AdvanceBio RP mAb Diphenyl.

[0116] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth.

[0117] "Advanced" cancer refers to cancer that has spread to the source site or outside the organ through local invasion ("locally advanced") or metastasis ("metastatic"). Accordingly, the term "advanced" cancer includes both locally advanced disease and metastatic disease.

[0118] "Metastatic" cancer refers to cancer that has spread from one part of the body (such as the breast) to another part of the body.

[0119] "Refractory" cancer refers to cancers that progress even when the patient is given antitumor agents (such as chemotherapy or biological therapy, such as immunotherapy). One example of refractory cancer is platinum-resistant cancer.

[0120] "Recurrent" cancer refers to cancer that has regrowth at the initial site or at a distant site after a response to an initial treatment (such as surgery).

[0121] "Locally recurrent" cancer refers to cancer that recurs in the same place as the previously treated cancer after treatment.

[0122] "Non-resectable" or "unresectable" cancers cannot be removed (resected) by surgery.

[0123] In this article, "early-stage breast cancer" refers to breast cancer that has not yet spread beyond the breast or axillary lymph nodes. This cancer is usually treated with neoadjuvant or adjuvant therapy.

[0124] "Neoadjuvant therapy" or "neoadjuvant treatment" or "neoadjuvant administration" refers to systemic therapy given prior to surgery.

[0125] “Adjunctive therapy” or “adjunctive treatment” or “adjunctive administration” refers to systemic therapy given after surgery.

[0126] In this document, "patient" or "individual" refers to a human patient. A patient may be a "cancer patient," that is, someone who has cancer, especially breast cancer, or is at risk of having one or more symptoms of cancer.

[0127] "Patient population" refers to a group of cancer patients. Such populations can be used to demonstrate statistically significant efficacy and / or safety of drugs (e.g., pertuzumab and / or trastuzumab).

[0128] A "relapsed" patient is one who has developed signs or symptoms of cancer after remission. Depending on the situation, the patient may have relapsed after adjuvant or neoadjuvant therapy.

[0129] A cancer or biological sample that “shows HER expression, amplification or activation” is one that shows (including overexpression) the HER receptor in diagnostic tests, has an amplified HER gene and / or otherwise shows activation or phosphorylation of the HER receptor.

[0130] Cancer or biological samples that "show HER activation" are those that show activation or phosphorylation of the HER receptor in diagnostic tests. This activation can be measured directly (e.g., by measuring HER phosphorylation using an ELISA) or indirectly (e.g., by gene expression analysis or by detecting HER heterodimers, as described herein).

[0131] Cancer cell lines exhibiting "HER receptor overexpression or amplification" have significantly higher levels of HER receptor protein or gene compared to non-cancerous cells of the same tissue type. This overexpression can be caused by gene amplification or increased transcription or translation. HER receptor overexpression or amplification can be determined in diagnostic or prognostic assays by assessing increased levels of HER protein present on the cell surface (e.g., via immunohistochemical IHC assay). Alternatively or additionally, the amount of HER-encoded nucleic acid in cells may be measured, for example, by in situ hybridization (ISH); including fluorescence in situ hybridization (FISH; see WO98 / 45479 published in October 1998) and chromogenic in situ hybridization (CISH; see, for example, Tanner et al., Am. J. Pathol. 157(5): 1467–1472 (2000); Bella et al., J. Clin. Oncol. 26: (May 20 Supplement; abstr 22147) (2008)); Southern blotting; or polymerase chain reaction (PCR) techniques, such as quantitative real-time PCR (qRT-PCR). HER receptor overexpression or amplification can also be studied by measuring exfoliated antigens (e.g., the HER extracellular domain) in biological fluids (e.g., serum) (see, for example, U.S. Patent No. 4,933,294, issued June 12, 1990; WO91 / 05264, published April 18, 1991; U.S. Patent No. 5,401,638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132: 73-80 (1990)). In addition to the above assays, those skilled in this technique can utilize various in vivo assays. For example, cells in a patient can be exposed to antibodies labeled with detectable markers (e.g., radiolabels), and radioactivity can be analyzed in situ, or biopsies obtained from patients pre-exposed to the antibodies can be analyzed.

[0132] "HER2 positive" cancers include cancer cells with HER2 levels higher than normal. Depending on the case, HER2 positive cancers have an immunohistochemical (IHC) score of 2+ or 3+; and / or are positive for in situ hybridization (ISH), fluorescence in situ hybridization (FISH), or chromogenic in situ hybridization (CISH), for example, with an ISH / FISH / CISH amplification ratio of ≥2.0.

[0133] “HER2-mutant” cancers include cancer cells with HER2-activating mutations (including kinase domain mutations), which can be identified, for example, by next-generation sequencing (NGS) or real-time polymerase chain reaction (RT-PCR). “HER2-mutant” cancers specifically include cancers with the following characteristics: insertion in HER2 exon 20, deletion around HER2 amino acid residues 755-759, any of the mutations G309A, G309E, S310F, D769H, D769Y, V777L, P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov 2013; 3:1-14), and the previously reported identical non-synonymous putative activating mutation (or insertion / deletion) found in two or more unique samples in the COSMIC database. For further details, see, for example, Stephens et al., Nature 2004;431:525-6; Shigematsu et al., Cancer Res 2005; 65:1642-6; Buttitta et al., Int J Cancer 2006; 119:2586-91; Li et al., Oncogene 2008; 27:4702-11; Sequist et al., J Clin Oncol 2010; 28:3076-83; Arcila et al., Clin Cancer Res 2012; 18:4910-8; Greulich et al., Proc Natl Acad SciU SA 2012; 109:14476-81; and Herter-Sprie et al., Front Oncol 2013;3:1-10.

[0134] In this document, "antitumor agent" refers to a drug used to treat cancer. Non-limiting examples of antitumor agents in this document include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, anti-hormonal compounds, cytokines, EGFR-targeting drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and antibodies, cytotoxic agents, apoptosis-inducing antibodies, COX inhibitors, farnesyltransferase inhibitors, antibodies binding to carcinoembryonic CA125, HER2 vaccines, Raf or ras inhibitors, liposomal doxorubicin, topotecan, taxane, dual tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib, and bevacizumab.

[0135] "Treatment" refers to therapeutic treatment and preventive or preventive measures. Those who need treatment include those who already have cancer and those who intend to prevent cancer. Therefore, the patients to be treated in this article may have been diagnosed with cancer or are susceptible to or prone to cancer.

[0136] The term "effective dose" refers to the amount of drug that is effective in treating a patient's cancer. An effective dose of drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow or better stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow or better stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. To some extent, the drug may prevent the growth of existing cancer cells and / or kill them; it may be growth-inhibiting and / or cytotoxic. An effective dose may prolong progression-free survival (e.g., as measured by the Response Evaluation Criteria for Solid Tumors, RECIST, or CA-125 changes), produce a target response (including partial response (PR) or complete response (CR), increase overall survival, and / or improve one or more cancer symptoms (e.g., as evaluated by FOSI).

[0137] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell damage. This term is intended to include radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu), chemotherapeutic agents, and toxins (e.g., small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof).

[0138] "Chemotherapy" refers to the use of chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents used in chemotherapy include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; TLK 286 (TELCYTATM); acetogenin (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronabinol, MARINOL®); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs HYCAMTIN®, CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin)); bryostatin; callystatin; CC-1065 (Including its synthetic analogues of adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (especially cryptophycin 1)And nostocin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine oxide. Hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines, such as annamycin, AD 32, alarubicin, daunorubicin, doxorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, valrubicin, KRN5500, menogaril, and dynemicin.(Including danendycin A, esperamicin, neocarzinostatin chromophores and related chromoprotein enediyne antiobiotic chromophores, aclacinomysin, actinomycin, autramycin, azoserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-xyloxy-L-leucine, ADRIAMYCIN® (doxorubicin) (Including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid) The following are listed as potential medications: nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogs, such as denopterin, pteropterin, and trimetrexate; and purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine.(thioguanine); pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenergics, such as aminoglutethimide and mitotane. Trilostane; folic acid supplements, such as leucovorin; aceglatone; antifolate antitumor agents, such as ALIMTA®, LY231514 (pemetrexed); dihydrofolate reductase inhibitors (e.g., methotrexate); antimetabolites (e.g., 5-fluorouracil (5-FU) and its prodrugs, such as UFT, S-1, and capecitabine); thymidylate synthase inhibitors; glycinamide ribonucleotide formyltransferase inhibitors (e.g., raltitrexed (TOMUDEXRM, TDX)); and dihydropyrimidine dehydrogenase inhibitors. Dihydropyrimidine dehydrogenase, such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazoquinone(diaziquone); eflornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; mushroom polysaccharide; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK7 polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs, such as cisplatin, oxaliplatin, and carboplatin.(carboplatin); vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinorelbine; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for combination therapy of oxaliplatin (ELOXATINTM) with 5-FU and folate.

[0139] This definition also includes anti-hormonal agents used to regulate or inhibit the effects of tumor hormones, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine. (1,3-dioxane-1,3-dioxane-1,3-cytosine analogues); antisense oligonucleotides, especially those that inhibit the expression of genes involved in signaling pathways related to abnormal cell proliferation (e.g., PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R)); vaccines, such as gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID® vaccines; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0140] "Taxane" is a chemotherapeutic agent that inhibits mitosis and interferes with microtubules. Examples of taxanes include paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, NJ); paclitaxel or nab-paclitaxel without cremophor, albumin-engineered nanoparticle formulation (ABRAXANETM; American Pharmaceutical Partners, Schaumberg, Illinois); and docetaxel (TAXOTERE®; Rhône-Poulenc Rorer, Antony, France).

[0141] "Anthracycline drugs" are a class of antibiotics derived from the fungus Streptomyces peucetius, including: daunorubicin, doxorubicin, epirubicin and any other anthracycline chemotherapeutic agents (including those listed above).

[0142] "Chemotherapy based on anthracyclines" refers to a chemotherapy regimen consisting of or including one or more anthracyclines. Examples include, but are not limited to, 5-FU, epirubicin and cyclophosphamide (FEC); 5-FU, doxorubicin and cyclophosphamide (FAC); doxorubicin and cyclophosphamide (AC); epirubicin and cyclophosphamide (EC); dose-dense doxorubicin and cyclophosphamide (ddAC) and the like.

[0143] For the purposes of this document, "carboplatin-based chemotherapy" means a chemotherapy regimen consisting of or including one or more carboplatins. An example is TCH (docetaxel / TAXOL®, carboplatin, and trastuzumab / HERCEPTIN®).

[0144] "Aromatase inhibitors" inhibit aromatase, which regulates the production of estrogen in the adrenal glands. Examples of aromatase inhibitors include: 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In one embodiment, the aromatase inhibitor herein is letrozole or anastrozole.

[0145] "Antimetabolite chemotherapy" uses drugs that are structurally similar to metabolites but cannot be effectively used in the body. Many antimetabolite chemotherapy therapies interfere with the production of nucleic acids, RNA, and DNA. Examples of antimetabolite chemotherapy agents include gemcitabine (GEMZAR®), 5-fluorouracil (5-FU), capecitabine (XELODATM), 6-mercaptopurine, methotrexat, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine (CYTOSAR-U®), dacarbazine (DTIC-DOME®), azocytosine, deoxycytosine, pyrimidines, fludarabine (FLUDARA®), cladrabine, and 2-deoxy-D-glucose.

[0146] "Chemotherapy-resistant" cancer means that the cancer patient progresses while receiving chemotherapy (i.e., the patient is "chemotherapy-refractory"), or the patient progresses within 12 months (e.g., within 6 months) after completing the chemotherapy regimen.

[0147] The term "platin" is used herein to refer to platinum-based chemotherapy, including but not limited to cisplatin, carboplatin and oxaliplatin.

[0148] The term "fluoropyrimidine" is used in this document to refer to antimetabolite chemotherapy, including but not limited to capecitabine, fluorouridine and fluorouracil (5-FU).

[0149] The “fixed” or “uniform” dose of the therapeutic agent in this article refers to the dose administered to a human patient without taking into account the patient’s weight (WT) or body surface area (BSA). Therefore, the fixed or uniform dose is not provided in mg / kg or mg / m2, but in the absolute amount of the therapeutic agent.

[0150] II. Determination

[0151] Combining therapeutic monoclonal antibodies (mAbs) into fixed-dose combinations (FDCs) increases the complexity of the drug and presents challenges for the characterization and control of product quality. This challenge is exacerbated when co-coated antibodies have similar physicochemical properties (e.g., similar isoelectric points, sequence similarity, and insignificant size differences). Furthermore, each co-coated antibody can exhibit heterogeneity in size, charge, and post-translational modifications during manufacturing. For these reasons, it is necessary to characterize and understand the interactions between mAbs in a fixed-dose combination. This article describes analytical methods for determining the critical quality attributes (CQA) of a fixed-dose combination of two anti-HER2 antibodies.

[0152] In one aspect, these assays are suitable for analyzing a fixed-dose combination of two anti-HER2 antibodies, trastuzumab and pertuzumab. Trastuzumab and pertuzumab share greater than 93% sequence identity (difference of only 30 Da) and both have a molecular weight of approximately 148 kDa. Furthermore, the two antibodies have very similar isoelectric points, bind to the same target (HER2), and exhibit synergistic effects in vivo. Due to these structural and functional similarities, most commonly used known analytical methods cannot be applied to this combination formulation. Additionally, the assays developed for the testing strategy take into account that the trastuzumab-pertuzumab fixed-dose combination is provided at two different doses, namely a loading dose and a maintenance dose, the difference being the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance.

[0153] (i) Efficacy determination

[0154] Potency is included in the control system for the release and stability testing of biological therapeutics (including therapeutic monoclonal antibodies). Potency monitors the cumulative effect of product quality attributes on biological activity, which can potentially affect safety and efficacy; that is, higher potency may cause safety issues, while lower potency may raise efficacy considerations. Ideally, a potency assay would represent the product's mechanism of action (i.e., the associated therapeutic activity or expected biological effect). According to the U.S. Food and Drug Administration (FDA) "Guidance for Industry on Potency Tests for Cellular and Gene Therapy Products," the traditional approach to evaluating the potency of biological products is to develop quantitative bioassays (bioassays) that measure the product activity in relation to a specific ability to achieve a given result. Bioassays can measure potency by evaluating the active ingredient of a product in a living biological system. Bioassays may include in vivo animal studies, in vitro organ, tissue, or cell culture systems, or any combination of such assays. A widely used example of a bioassay for measuring or quantifying potency is cell-based assays. This study evaluated the suitability of two different cell-based assays (specifically designed to measure cell growth inhibition and antiproliferative activity of pertuzumab or trastuzumab) for controlling the bioactivity of a fixed-dose pertuzumab-trastuzumab combination. This evaluation confirmed that these assays are not suitable for the fixed-dose combination due to limitations that hinder the control of variations in the product quality of the individual antibodies in the combination formulation. Because of the nature of the combination formulation of two antibodies that bind to the same receptor and inhibit similar signaling pathways, no alternative HER2-expressing cell lines can overcome these limitations.

[0155] For trastuzumab and pertuzumab, which bind to the same receptor and act on similar signaling pathways in target cells, their effects on downstream signaling, gene expression, and proliferation in HER2-expressing target cells are modulated by their binding activity to specific epitopes on HER2. Therefore, potential molecular changes in antibodies affecting their efficacy in inhibiting HER2-driven cell growth can be observed at the binding level. This hypothesis has been evaluated in comparative studies using selected product variants (charge and size variants, and CDR affinity mutants), as illustrated in the examples presented herein. This study confirmed that binding differences (as detected by the binding assays provided) reflected the observed changes in antiproliferative activity in most of the tested product variants (except for size variants). Considering the interference in antiproliferative assays and the ability of the binding assays provided to detect single antibody quality changes affecting efficacy, the novel binding assays provided can be considered the best possible assay for controlling for changes in product quality affecting target binding and HER2 signaling.

[0156] In one embodiment, the pertuzumab-trastuzumab FDC drug was tested by a binding assay that specifically measures the binding of HER2 to pertuzumab or trastuzumab to determine potency. Both trastuzumab and pertuzumab target HER2, but they bind to different and non-overlapping epitopes on the extracellular domain (ECD) of HER2: trastuzumab recognizes subdomain IV (juxtamembranous region), while pertuzumab recognizes subdomain II (dimerization region) (Rocca A, Andreis D, Fedeli A et al., Pharmacokinetics, Pharmacodynamics and Clinical Efficacy of Pertuzumab in the Treatment of Breast Cancer. Expert Opin Drug Metab Toxicol 2015;11:1647-63.). Trastuzumab binds to the HER2 subdomain IV by blocking its homodimerization, thereby inhibiting ligand-independent HER2 signaling (Junttila TT, Akita RW, Parsons K et al., Ligand-independent HER2 / HER3 / PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 2009;15:429-40.), and prevents the proteolytic cleavage of its ECD, thereby preventing subsequent constitutive activation of related intracellular signaling pathways. (Molina MA, Codony-Servat J, Albanell J et al., Trastuzumab (Herceptin), a humanized anti-HER2 receptor monoclonal antibody, inhibits basal and activated HER2 ectodomain cleavage in breast cancer cells. Cancer Research 2001;61:4744-9). Therefore, trastuzumab inhibits the proliferation of human tumor cells that overexpress HER2, as demonstrated in in vitro assays and in animals.The binding of pertuzumab to HER2 subdomain II blocks ligand-dependent heterodimerization of HER2 with other HER family members (including EGFR, HER3, and HER4). (Franklin MC, Carey KD, Vajdos FF et al., Insights into ErbB signaling from the structure of the ErbB2 pertuzumab complex. Cancer Cell 2004;5:317-28; Adams CW, Allison DE, Flagella K et al., Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 2006;55:717-27; Diermeier-Daucher S, Hasmann M, Brockhoff G. Flow cytometric FRET analysis of erbB receptor interaction on a cell by cell basis. Ann NY Acad Sci.) 2008;1130:280-6.). Therefore, pertuzumab inhibits ligand-induced intracellular signaling, thereby inducing cell growth arrest and apoptosis in human tumor cells that overexpress HER2.

[0157] Pertuzumab and trastuzumab bind to different and non-overlapping epitopes on the HER2 ECD without competing with each other, and have complementary mechanisms that disrupt HER2 signaling. When pertuzumab and trastuzumab are administered in combination, this enhances in vitro and in vivo antiproliferative activity (Scheuer W, Friess T, Burtscher H et al., Strongly enhanced antitumor activity of trastuzumab and pertuzumab combination treatment on HER2-positive human xenograft tumor models. Cancer Res 2009;69:9330-6.). In one embodiment, two different HER2 binding assays are used to determine the antiproliferative activity and HER2 signaling of the FDC drug, which ensures control over the quality of each of the two antibodies in the pertuzumab-trastuzumab FDC drug.

[0158] In one embodiment, a binding assay for a fixed-dose combination (FDC) of two anti-HER2 antibodies is provided, comprising: a. contacting the FDC with a capture reagent, wherein the capture reagent is a modified HER2 ECD; b. contacting the sample with a detectable antibody; c. quantifying the amount of antibody bound to the capture reagent using a detection method for the detectable antibody.

[0159] A fixed-dose combination of two anti-HER2 antibodies is contacted with and incubated together with a capture reagent, thereby capturing or binding the anti-HER2 antibody of interest to make it detectable in the detection step. The capture reagent is a modified HER2 ECD comprising one or more recombinant HER2 ECD subdomains. In one embodiment, the modified HER2 ECD is a genetically engineered protein or peptide comprising one or more recombinant HER2 ECD subdomains. In one embodiment, the HER2 ECD is modified so that one of the anti-HER2 antibodies to be evaluated in the FDC can bind to it, while the second anti-HER2 antibody in the FDC does not bind to it. This is achieved by deleting the HER2 ECD subdomain bound by the second anti-HER2 antibody or by replacing that subdomain with a structurally compact subdomain that does not bind either anti-HER2 antibody. Structurally similar subdomains can be any subdomain that does not disrupt the three-dimensional conformation of the modified HER2 ECD when included in the modified HER2 ECD. Examples of structurally similar subdomains are the corresponding subdomains of EGFR, HER3, or HER4. Preferably, the modified HER2 ECD has a three-dimensional configuration that closely mimics the native HER2 ECD. The subdomain can be full-length or shortened by a few amino acids at the N-terminus or C-terminus. The inventors of this invention have discovered that the integrity of the three-dimensional structure of the HER2 ECD can be preserved or improved when using one or more recombinant HER2 ECD subdomains shortened by about 4 to 5 amino acids at the C-terminus.

[0160] In one embodiment, the modified HER2 ECD is fused to a peptide or protein to facilitate the immobilization of the capture reagent onto a solid substrate. Examples of suitable peptides or proteins are biotin, bovine serum albumin (BSA), and Fc domains. In a modified HER2, the extracellular domain is fused to the Fc domain. In one embodiment, the Fc domain is derived from a species different from the species whose Fc domain is being analyzed in the anti-HER2 antibody. For example, if the anti-HER2 antibody being analyzed contains a human Fc domain, the capture reagent should contain a non-human Fc domain, such as the Fc domain of mice, porcupines, rats, rabbits, etc. In one embodiment, the Fc domain of the recombinant HER2 ECD subdomain is a mouse Fc domain. In one embodiment, the Fc domain contains SEQ ID NO. 35.

[0161] In the next step, the sample containing the capture reagent and the captured anti-HER2 antibody is incubated together with a detectable antibody. Upon contact with any anti-HER2 antibody of interest, the detectable antibody binds to the antibody of interest. In the next step, a detection method is used to detect the label on the detectable antibody and thereby detect the presence or amount of the anti-HER2 antibody of interest present in the FDC.

[0162] In one embodiment, the fixed-dose combination comprises an antibody binding to HER2 extracellular subdomain II and an antibody binding to HER2 extracellular subdomain IV. In one embodiment, the antibody binding to HER2 extracellular subdomain II is pertuzumab. In one embodiment, the antibody binding to HER2 extracellular subdomain IV is trastuzumab. In one embodiment, the fixed-dose combination comprises pertuzumab and trastuzumab. In one embodiment, the fixed-dose combination further comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In a preferred embodiment, the hyaluronidase is rHUPH20. The pertuzumab-trastuzumab FDC drug is provided at two different doses, namely a loading dose (LD) and a maintenance dose (MD). The LD and MD have the same total protein content and differ in the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance. In one embodiment, a binding assay is used to analyze the LD of the pertuzumab-trastuzumab FDC. In one embodiment, a binding assay is used to analyze the pertuzumab-trastuzumab FDC comprising 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further comprises 2000 U / mL rHuPH20. In one embodiment, a binding assay is used to analyze the MD of the pertuzumab-trastuzumab FDC. In one embodiment, a binding assay is used to analyze the pertuzumab-trastuzumab FDC comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further comprises 2000 U / mL rHuPH20.

[0163] In one embodiment, the binding of pertuzumab and trastuzumab is determined in two separate binding assays.

[0164] The pertuzumab binding assay determines the bioactivity as the ability of pertuzumab to specifically bind to its epitopes in a recombinant HER2 capture reagent. In one embodiment, the binding of pertuzumab is quantified. In one such embodiment, the capture reagent comprises HER2 extracellular subdomain II or a portion thereof. In one embodiment, the capture reagent comprises human HER2 extracellular subdomain II. In one embodiment, the capture reagent comprises SEQ ID NO. 23 or SEQ ID No: 2.

[0165] In one embodiment, the modified HER2 ECD includes HER2 ECD subdomains I, II, and III, or portions thereof. In one embodiment, the modified HER2 ECD includes human HER2 ECD subdomains I, II, and III, or portions thereof. In one embodiment, the modified HER2 ECD does not include subdomain IV. The inventors have discovered that when the recombinant subdomain III, which has been truncated at the C-terminus, is included, a modified HER2 ECD having a three-dimensional configuration similar to that of the natural HER2 ECD can be produced. In one such embodiment, the modified HER2 ECD includes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34. In one embodiment, the modified HER2 ECD includes SEQ ID NO. 24. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 24.

[0166] In one embodiment, recombinant HER2 extracellular subdomains I, II, and III are fused to the Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In any of the above embodiments, the capture reagent used to evaluate pertuzumab binding does not contain HER2 subdomain IV. In one embodiment, the capture reagent contains SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 25. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 26. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 27.

[0167] In one embodiment, the binding of trastuzumab is quantified. In one such embodiment, the capture agent comprises recombinant HER2 extracellular subdomain IV or a portion thereof. In one such embodiment, the capture agent comprises human recombinant HER2 extracellular subdomain IV. In one embodiment, the capture agent comprises SEQ ID NO.28 or SEQ ID No: 4.

[0168] In one embodiment, the capture agent comprises recombinant HER2 extracellular subdomains I, III, and IV. In one embodiment, the capture agent comprises human HER2 extracellular subdomains I, III, and IV. In one embodiment, the capture agent comprises recombinant HER2 extracellular subdomains I, III, and IV and EGFR subdomain II. In one embodiment, the capture agent comprises recombinant human HER2 extracellular subdomains I, III, and IV and recombinant human EGFR subdomain II. The inventors have discovered that when recombinant HER2 extracellular subdomain I and recombinant HER2 extracellular subdomain IV (both truncated at the C-terminus) are included, a modified HER2 ECD with a three-dimensional conformation similar to that of a natural HER2 ECD can be produced. In one embodiment, the modified ECD comprises SEQ ID NO: 33, SEQ ID NO: 3, and SEQ ID NO: 28. In one embodiment, the modified ECD includes SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 28.

[0169] In one embodiment, the modified HER2 ECD includes SEQ ID NO. 29. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 29.

[0170] In one embodiment, recombinant HER2 extracellular subdomains I, III, and IV, and EGFR subdomain II are fused to the Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In any of the foregoing embodiments, the capture reagent used to evaluate trastuzumab binding does not contain HER2 subdomain II. In one embodiment, the capture reagent contains SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 30. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 31. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 32.

[0171] In any of the above embodiments, the detectable antibody includes a label that allows it to be detected by various means. Such labels include directly detectable portions, such as fluorescent dyes, chemiluminescent labels, and radioactive labels; and portions that must be reacted or derivatized for detection, such as enzymes. Examples of such markings include radioactive isotopes 32P, 14C, 125I, 3H, and 131I; luciferases, such as rare earth chelates or luciferin and their derivatives; rose red and its derivatives; ruthenium; dansyl sulfadiazine; umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; 2,3-dihydrophthalic anhydride; HRP; alkaline phosphatase; β-galactosidase; glucosyl amylase; lysozyme; carbohydrate oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, used in conjunction with enzymes employing hydrogen peroxide dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase); biotin (which can be obtained via, for example, avidin, streptoavidin, streptoavidin-HRP). And streptavidin-β-galactosidase and MUG detection); rotation labeling; phage labeling; stable free radicals; and the like.

[0172] A preferred label for detectable antibodies is horseradish peroxidase (HRP). Receptors used for HRP are generally classified into different categories, including chromogenic (e.g., aminoethylcarbazole (AEC), 3,3'-diaminobenzidine tetrahydrochloride (DAB), a combination of chloronaphthol and diaminobenzidine (CN / DAB), tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 2,2'-azobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)), fluorescent (e.g., ADHP), and chemiluminescent (e.g., enhanced chemiluminescence (ECL)) receptors, depending on whether they produce colored, fluorescent, or luminescent derivatives, respectively. ABTS is the preferred receptor.

[0173] In one embodiment, the antibody targeting the F(ab')2 portion of human IgG can be detected. In one embodiment, the antibody targeting the F(ab')2 portion of an anti-HER2 antibody can be detected.

[0174] In one embodiment, the binding assay is an enzyme-linked immunosorbent assay (ELISA). In an ELISA, a capture reagent is attached to a solid substrate. The solid phase used for immobilization can be any inert carrier or loading agent that is substantially insoluble in water and can be used for immunoassay, including carriers in the form of surfaces, particles, porous matrices, etc. Examples of common carriers include flakes, SEPHADEX® gels, polyvinyl chloride, plastic beads, and assay plates or tubes (including 96-well microplates) made of polyethylene, polypropylene, polystyrene, and the like, as well as particulate materials (e.g., filter paper, agarose, cross-linked polydextrose, and other polysaccharides). Alternatively, the capturing reagent may be immobilized using a reactive, water-insoluble matrix (e.g., cyanogen bromide-activated carbohydrates and reactive acceptors) as described in U.S. Patents 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440. In a preferred embodiment, the immobilized capturing reagent is coated onto a microplate, and specifically, the preferred solid-phase system used is suitable for porous microplates capable of analyzing several samples simultaneously. The preferred microplate is a plate with a highly charged polystyrene surface that has a high affinity for molecules with polar or hydrophilic groups, exhibiting a high binding capacity for proteins. The most preferred are MICROTEST® or MAXISORP® 96-well ELISA plates, such as those sold as NUNC MAXISORP® or IMMULON®.

[0175] Preferably, the 96-well plate is coated with a capture reagent for at least 30, 40, 50, 60 minutes, about 20 to 80 minutes, or about 30 to 60 minutes. More preferably, the 96-well plate is coated with the capture reagent at a temperature of about 4-20°C, more preferably about 2-8°C. The plates can be stacked and coated before the assay, and then the assay can be performed simultaneously for several samples in a manual, semi-automatic, or automatic manner (e.g., by using a robot).

[0176] The amount of capture reagent used is large enough to obtain a good signal, but should not be excessive compared to the maximum expected amount of the antibody of interest in the sample. In one embodiment, the concentration of the coating reagent is about 0.5 µg / mL to 5 µg / mL, preferably about 1 µg / mL to 1.5 µg / mL.

[0177] The coated plate is then typically treated with a blocking agent that nonspecifically binds to and saturates the binding sites to prevent unnecessarily excessive binding of free ligands to excess sites on the plate wells. Examples of suitable blocking agents for this purpose include, for example, gelatin, bovine serum albumin (BSA), ovalbumin, casein, and skim milk. The blocking treatment is typically performed at ambient temperature for about 1-4 hours, about 1-3 hours, and preferably about 1-1.5 hours.

[0178] After coating and blocking, the standard or the FDC sample to be analyzed is added to the coated plate in the form of a standard dilution. In one embodiment, incremental concentrations of pertuzumab-trastuzumab FDC (standard, product control and sample to be analyzed) are added to the coated plate.

[0179] The incubation conditions for the FDC sample and the immobilized capture reagent are selected to maximize assay sensitivity and minimize dissociation, and to ensure that the anti-HER2 antibody to be evaluated in the FDC sample binds to the immobilized capture reagent. Preferably, the incubation is carried out at a relatively constant temperature in the range of about 0°C to about 40°C, more preferably at room temperature or about room temperature. The incubation time typically does not exceed about 10 hours. Preferably, the incubation time is carried out at room temperature or about room temperature for about 0.5 to 3 hours, and more preferably about 1 to 1.5 hours, to maximize the binding of the anti-HER2 antibody to be evaluated in the FDC sample to the capture reagent.

[0180] The immobilized capture reagent, which is conjugated with any anti-HER2 antibody, is preferably in contact with the detectable antibody at a temperature of about 20-40°C, more preferably at room temperature, wherein the exact temperature and time of contact depend primarily on the detection method used.

[0181] In another embodiment, the binding assay is electrochemiluminescence (ECL).

[0182] In one embodiment, a binding assay is used to analyze the potency of an anti-HER2 antibody. Therefore, in one embodiment, the binding assay further includes the step of: d. correlating the amount of antibody bound to the capture reagent with the biological activity of the antibody.

[0183] In one embodiment, the dose-response curve generated for the sample is compared with the dose-response curve for the standard. In one embodiment, the potency of the standard is quantified by correlating the results obtained in a binding assay with the bioactivity of the isolated antibody in a cell-based assay.

[0184] In one embodiment, nonlinear 4-parameter dose-response curves generated for the sample and the standard are compared. After evaluating the similarity criteria between the standard dose-response curve and the sample dose-response curve, the relative potency of the sample is calculated based on the concentration shift between the fitted standard dose-response curve and the fitted sample dose-response curve, using 4-parameter parallel line analysis.

[0185] In one embodiment, the binding assay is used for the fractional release of a fixed-dose combination of pertuzumab and trastuzumab. In one embodiment, the binding assay is used to determine the shelf life of the fixed-dose combination of pertuzumab and trastuzumab. In such an embodiment, the binding assay described above is used to analyze the pertuzumab-trastuzumab FDC at several time points during storage.

[0186] (ii) Analysis of charge variants

[0187] In one embodiment, a method is provided for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, the method comprising evaluating the amount of charge variants of pertuzumab and trastuzumab in the composition. In one embodiment, the fixed-dose composition further comprises hyaluronidase. In one embodiment, the method is ion exchange chromatography. Ion exchange chromatography (IEX) is widely used for detailed characterization of therapeutic proteins and can be considered a powerful and reliable technique for qualitative and quantitative assessment of charge heterogeneity. Ion exchange high-performance liquid chromatography (IE-HPLC, IEC) separates molecules in solution based on charge heterogeneity. Separation is achieved by the reversible adsorption of charged solute molecules onto ion exchange groups of opposite charge fixed in a column packing material. The adsorption of molecules to the solid support is driven by ionic interactions between the two components. The strength of the interaction depends on the number and position of charges on the molecules and the stationary phase. IEX is typically a effluent method in which specifications are specifically set for the distribution of each acidic, major, and basic substance for mAb. These charged substances can be considered product-related impurities that can affect efficacy. Furthermore, it is one of the few methods that can characterize proteins in their natural conformation because no denaturing agents are added. IEX can also be used as an identification method for certain biological agents and is a routine test for stability and shelf-life verification.

[0188] Analyzing the distribution of charge variants in a fixed-dose combination of two anti-HER2 antibodies (such as trastuzumab and pertuzumab) with very similar isoelectric points requires a specific ion exchange chromatography protocol to properly separate all relevant substances.

[0189] In one embodiment, a method is provided for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, the method comprising evaluating the amount of charge variants of pertuzumab and trastuzumab in the composition. In one embodiment, the fixed-dose combination further comprises hyaluronidase. In one embodiment, the method is ion exchange chromatography. In a specific embodiment, the method is cation exchange chromatography. In cation exchange chromatography, as applied to a pertuzumab / trastuzumab fixed-dose combination (FDC), positively charged molecules are retained on a negatively charged stationary phase. Acidic substances are flushed out with a shorter retention time than basic substances.

[0190] After column equilibration and sample application, the FDC anti-HER2 antibodies pertuzumab and trastuzumab adsorbed onto the column ligands. The column was then washed to remove unadsorbed proteins, and extraction was performed by varying the ionic strength of the mobile phase while maintaining the pH within a predefined range. In one embodiment, the pH was maintained at a constant value.

[0191] The ionic strength is altered by applying a gradient of increasing salt concentration, which can be a stepwise or continuous gradient. The inventors have found that the pH ranges of the loading buffer (mobile phase A) and the extraction buffer (mobile phase B) are critical when analyzing charge variants of the FDCs of two anti-HER2 antibodies, pertuzumab and trastuzumab. The predefined pH range of the loading buffer (mobile phase A) (pH 7.5-7.65) and the predefined pH range of the extraction buffer (mobile phase B) (pH 7.5-7.7) are used to optimally separate the charge variants. In one embodiment, the pH is maintained at a constant value. In one embodiment, the constant pH value of the loading buffer is 7.5, 7.55, 7.6, or 7.65. In one embodiment, the constant pH value of the extraction buffer is 7.5, 7.55, 7.6, 7.65, or 7.7.

[0192] After rinsing, the column is then reequilibrated using loading buffer (mobile phase A).

[0193] In one embodiment, the pertuzumab-trastuzumab fixed-dose combination is contacted with a cation exchange material, and the charge variant and the natural antibody are extracted using a salt gradient while the pH of the mobile phase is maintained within a predefined range. In one embodiment, the salt gradient is a continuous salt gradient. In one embodiment, the pH of the mobile phase of the loading buffer (mobile phase A) is between pH 7.5 and pH 7.65. In one embodiment, the pH of the mobile phase of the extraction buffer (mobile phase B) is between pH 7.5 and pH 7.7.

[0194] In one embodiment, the salt gradient is a sodium chloride gradient. In another embodiment, the salt gradient is a sodium chloride gradient and the pH of the mobile phase of the extraction buffer (mobile phase B) is between pH 7.5 and pH 7.7.

[0195] In one embodiment, a method is provided for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, the method comprising: a. binding the antibody to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibody using an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0196] In one embodiment, a salt gradient is used to perform the extraction in step b. In one embodiment, the salt gradient is a continuous salt gradient. In one embodiment, the salt gradient is a sodium (Na+) gradient. Therefore, in one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium ions (Na+). In one embodiment, the sodium gradient is a sodium chloride (NaCl) gradient. In one embodiment, the extraction buffer contains NaCl. Suitable buffers for loading and extraction are MES (2-ethanesulfonic acid), ACES (N-(2-acetylamino)-2-aminoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-hexahydropyrazine ethanesulfonic acid), phosphate buffer, MOPS (3-(N-morpholino)propanesulfonic acid), TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), CAPSO (N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane), PIPES (hexahydropyrazine-N,N′-bis(2-ethanesulfonic acid)), and TPP (Tris, phosphate, hexahydropyrazine). ACES and HEPES are preferred buffers.

[0197] In one embodiment, the sodium chloride concentration of the extraction buffer (mobile phase B) is about 180-220 mM NaCl, about 200 mM NaCl, about 180 mM NaCl, about 190 mM NaCl, about 210 mM NaCl, or about 220 mM NaCl.

[0198] In one embodiment, the ion exchange material is a cation exchange material. In further optimizing the method of the present invention, the inventors found that the separation of charge variants is improved when using a strong cation exchange column material. In a preferred embodiment, the method is performed using a non-porous SCX column with sulfonic acid groups and using Na+ as the relative ion for extraction. Therefore, in one embodiment, the cation exchange material has sulfonic acid groups. In such an embodiment, the cation exchange material is a strong cation exchanger (SCX) column with sulfonic acid groups and the extraction buffer contains sodium. In such an embodiment, the extraction buffer contains sodium ions. In one embodiment, the SCX column is non-porous. Preferred cation exchange columns that can be used include: YMC Bio Pro SP-F column, MabPac SCX-10, Waters BioResolve SCX mAb, Sepax Proteomix SCX-NP1.7, or non-porous Agilent Bio SCX.

[0199] In one embodiment, steps A and b of the above method are performed at a temperature of 32°C to 40°C or about 36°C.

[0200] In one embodiment, ion exchange chromatography is performed using a loading of approximately 50 µg to 149 µg or approximately 51 µg to 153 µg of total protein. In one embodiment, ion exchange chromatography is performed using a loading dose of pertuzumab-trastuzumab FDC with a total protein content of approximately 50 µg to 149 µg. In one embodiment, ion exchange chromatography is performed using a maintenance dose of pertuzumab-trastuzumab FDC with a total protein content of approximately 51 µg to 153 µg. In one embodiment, the total protein loaded onto the ion exchange chromatography plate is approximately 100 µg.

[0201] In one embodiment, a method is provided for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, the method comprising: a. binding an antibody to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. flushing the antibody using an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7; c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0202] In one embodiment, acidic variants, natural forms, and basic variants of trastuzumab and pertuzumab in a fixed-dose combination are selectively detected.

[0203] In one embodiment, ion exchange chromatography is performed using a fixed-dose combination of pertuzumab and trastuzumab that have been digested with carboxypeptidase B before being loaded onto the chromatography column.

[0204] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab and trastuzumab FDC contains approximately 2000 U / mL rHuPH20. The pertuzumab-trastuzumab FDC is provided at two different doses, namely a loading dose (LD) and a maintenance dose (MD). The LD and MD have the same total protein content and differ in the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance. In one embodiment, the method can be used to determine the charge variant of the loading dose of pertuzumab and trastuzumab FDC. In one embodiment, the charge variants of pertuzumab and trastuzumab are determined simultaneously, i.e., in the same method. In one embodiment, the method can be used to analyze the charge variants of a pertuzumab-trastuzumab FDC containing 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further contains 2000 U / mL rHuPH20. In one embodiment, the method can be used to determine the charge variants of the maintenance dose of pertuzumab and trastuzumab FDC. In one embodiment, the charge variants of pertuzumab and trastuzumab are determined simultaneously, i.e., in the same method. In one embodiment, the method can be used to analyze the charge variants of a pertuzumab-trastuzumab FDC containing 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further contains 2000 U / mL rHuPH20.

[0205] In one embodiment, the natural antibody and its acidic and basic variants are extracted in a 1–100% (solvent B) salt gradient for at least 44 minutes. In one embodiment, the salt gradient is increased from 1% solvent B to 47% solvent B over 43 minutes. In one embodiment, the salt gradient is increased from about 1.8 mM NaCl to 103.4 mM NaCl. In another embodiment, the salt gradient is increased from about 2 mM NaCl to about 94 mM NaCl.

[0206] In one embodiment, the mobile phase of ion exchange chromatography comprises ACES buffer. In one embodiment, mobile phase A and mobile phase B comprise ACES buffer. In one embodiment, ion exchange chromatography solvent A comprises about 10-50 mM, about 15-25 mM, about 18-22 mM, or about 20 mM ACES. In one embodiment, ion exchange chromatography solvent B comprises about 10-50 mM, about 15-25 mM, about 18-22 mM, or about 20 mM ACES and about 180-220 mM NaCl. In one embodiment, solvent B comprises about 20 mM ACES.

[0207] (ii) Quantity / Protein Content Determination

[0208] UV spectrophotometry is a typical method for determining the total protein content of pharmaceutical samples. However, for a fixed-dose combination (FDC) of two anti-HER2 antibodies, a different approach is required because conventional methods cannot individually and quantitatively analyze the protein content of each anti-HER2 antibody in the FDC. Different chromatographic methods have been tested, such as hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC). Reversed-phase chromatography has proven to be the most suitable method for individually and quantitatively analyzing the protein content of pertuzumab-trastuzumab FDC.

[0209] Reversed-phase ultra-high performance liquid chromatography (RP-UHPLC, RPC) separates molecules in solution based on hydrophobicity. Separation is achieved through the reversible, hydrophobic adsorption of molecules on a nonpolar stationary phase in a column. The adsorption of molecules to the solid support is driven by the hydrophobic / nonpolar interaction between the two components. The strength of the interaction depends on the number and position of functional groups on the molecule and the stationary phase. In reversed-phase chromatography, nonpolar molecules are ejected from the stationary phase with a longer residence time than polar molecules. Because the two anti-HER2 antibodies, trastuzumab and pertuzumab, have greater than 93% sequence identity and differ by only 30 Da in total, a stable method was developed that provides reliable overall resolution and peak separation without significant sample residue (i.e., the residue should not exceed 0.2% in subsequent analyses). Furthermore, the assay strategy developed for testing considers that the trastuzumab-pertuzumab fixed-dose combination is provided with two different doses, namely a loading dose and a maintenance dose, the difference being the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance. The inventors have discovered that phenyl-based columns yield particularly good results at specific temperatures, and that the most critical parameters for stabilizing the method are column temperature and flow rate. Phenyl-based RP-UHPLC columns are known in the industry and can have the following groups: ethylphenyl with methyl side groups and a capped silica surface; phenylhexyl phase with methyl side groups of extended (hexyl) ligand spacers; ethylphenyl ligands with sterically protective (isobutyl) side groups; hexylbiphenyl with methyl side groups; biphenyl phase with methyl side groups; and oxygen-activated phenylethylphenyl phase with methyl side groups. HPLC columns with stationary phases modified with phenyl groups (e.g., monophenyl, biphenyl, diphenyl, phenylhexyl, phenylpropyl) are readily available from most major column suppliers. An example of a phenyl column that can be used in this study is the Agilent Zorbax RRHD 300-Diphenly column. In one embodiment, this column is a 2.1 × 100 mm column.

[0210] This article provides a method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, comprising: a. providing an RP-HPLC phenyl column; b. loading the fixed-dose combination (FDC) of the two anti-HER2 antibodies onto the RP-HPLC column; c. separating the two anti-HER2 antibodies at a flow rate of 0.2–0.4 mL / min, wherein the column temperature is 64°C to 76°C.

[0211] The principle of RP-HPLC separation is based on the hydrophobic association between peptide solutes and hydrophobic ligands on the surface of the chromatography resin. RP-HPLC columns are typically part of a UHPLC system, which is equipped with an online vacuum degasser, an autosampler with a sample cooler, a column heater, and a UV / VIS detector. Examples of suitable UHPLC systems are the Waters Aquity and the Thermo Ultimate 3000 RS.

[0212] The FDCs of two anti-HER2 antibodies are loaded onto a column by injecting samples into an RP-HPLC system. Typically, the samples are diluted to concentrations such as approximately 0.5–5 mg / mL or 1 mg / mL. The inventors have found that a sample concentration of 1.0 mg / mL allows for good detection of minor substances without saturating the detector signal. In one embodiment, a formulation buffer is used to dilute the sample. In one embodiment, the formulation buffer contains L-histidine, L-histidine sulfate monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20. By using a formulation buffer as a diluent, the risk of altering the sample and reference solutions due to the use of a different diluent is eliminated. No interference from the formulation buffer to the RP-HPLC method has been observed. In one embodiment, the injection volume is 0.5–100 µL, 1–50 µL, 5–10 µL, or 10 µL. In one embodiment, the injection volume is 10 µL. In another embodiment, the total protein load on the column is 10 µg.

[0213] Proteins bind to an aqueous mobile phase on an RP-HPLC column and are flushed out of the column by increasing the hydrophobicity of the mobile phase. The proteins are then separated based on the hydrophobicity. In one embodiment of a method for analyzing the protein content of two fixed-dose combinations (FDC) of anti-HER2 antibodies, a water-2-propanol / acetonitrile gradient is used to achieve the separation in step c). In one such embodiment, the protein binds to a column containing an aqueous phase (extraction agent A) of water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and is then flushed with an organic phase containing acetonitrile at increasing concentrations. In one such embodiment, the organic phase (extraction agent B) contains 2-propanol:acetonitrile:extraction agent A (70:20:10). Due to the phenyl-based column type, improved specificity is achieved, and the novel substance is detected only using 2-propanol, but not using pure acetonitrile. The different specificities achieved are due to the interaction between the phenyl-based column and the analyte via classical hydrophobic interactions as well as other π-π interactions. It has been shown in the literature that pure acetonitrile hinders these interactions, while 2-propanol does not (Yang, M., Fazio, S., Munch, D. & Drumm, P. Impact of methanol and acetonitrile on separations based on π–π interactions with a reversed-phase phenyl column. Journal of Chromatography A 1097, 124-129). However, considering the high viscosity of 2-propanol and the associated increased back pressure, 20% acetonitrile was added to reduce the back pressure in the system.

[0214] In one embodiment, the aqueous mobile phase comprises 70% extractant A and 30% extractant B, wherein extractant A comprises water: 2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and extractant B comprises 2-propanol:acetonitrile:extractant A (70:20:10). In one such embodiment, the organic phase (extractant B) is increased to 55% extractant A and 45% extractant B. In one embodiment, the gradient is increased to 45% extractant B over 15 minutes.

[0215] In one embodiment, the organic phase (extractant B) is increased to 10% extractant A and 90% extractant B. In one embodiment, the gradient is increased to 90% extractant B over 20 minutes.

[0216] Flow rates of 0.4 mL / min and 0.2 mL / min were tested and found to have no significant effect on method performance. In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the flow rate in step c) was approximately 0.3 mL / min.

[0217] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the antibody system is separated within 10 to 20 minutes. In another embodiment, the antibody system is separated within 15 minutes. In one embodiment, the antibodies are separated over 15 minutes at a flow rate of 0.3 mL / min.

[0218] In addition to the loading and rinsing (separation) steps, RP-HPLC purification may also include other steps such as equilibration, washing, and regeneration. In one embodiment, the RP-HPLC phenyl column is equilibrated using 70% rinsing agent A and 30% rinsing agent B, wherein rinsing agent A comprises water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and rinsing agent B comprises 2-propanol:acetonitrile:rinsing agent A (70:20:10). In one embodiment, the RP-HPLC phenyl column is washed using 10% rinsing agent A and 90% mobile phase B, wherein rinsing agent A comprises water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and rinsing agent B comprises 2-propanol:acetonitrile:rinsing agent A (70:20:10).

[0219] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the column temperature was 70°C ± 2°C. Compared to room temperature, a column temperature of 70°C produced higher reproducibility, eliminated tailing effects, exhibited lower system back pressure, and generally produced better resolution and separation. Several column temperatures were tested, and 70°C showed an improved peak shape without reaching the maximum temperature allowed by the system and column type. Temperatures of 64°C - 76°C and 66°C - 74°C were tested respectively and found to have no significant effect on method performance.

[0220] In one embodiment of the method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the phenyl column is selected from the group consisting of: Agilent Zorbax RRHD 300-Diphenyl column, Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, BioResolve RP mAb Polyphenyl, and Agilent AdvanceBio RP mAb Diphenyl. In one embodiment, the phenyl column is an Agilent Zorbax RRHD 300-Diphenyl column. In another embodiment, the base column is a BioResolve RP mAb Polyphenyl column.

[0221] In one embodiment, the protein is detected by UV light. In one embodiment, the detection wavelength is 280 nm.

[0222] In one embodiment, the fixed-dose combination comprises pertuzumab and trastuzumab. In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab and trastuzumab FDC contains approximately 2000 U / mL rHuPH20. The pertuzumab-trastuzumab FDC is provided at two different doses, namely a loading dose (LD) and a maintenance dose (MD). The LD and MD have the same total protein content and differ in the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance. In one embodiment, this method can be used to determine the protein content of the loading dose of the pertuzumab and trastuzumab FDC. In one embodiment, the protein content of pertuzumab and trastuzumab is determined simultaneously, i.e., using the same method. In one embodiment, this method is used to analyze the protein content of a pertuzumab-trastuzumab FDC containing 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further contains 2000 U / mL rHuPH20. In one embodiment, this method can be used to determine the protein content of a maintenance dose of pertuzumab and trastuzumab FDC. In one embodiment, the protein content of pertuzumab and trastuzumab is determined simultaneously, i.e., using the same method. In one embodiment, this method is used to analyze the protein content of a pertuzumab-trastuzumab FDC containing 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further comprises 2000 U / mL rHuPH20.

[0223] III. Anti-HER2 Antibody and its Composition

[0224] (i) Anti-HER2 antibody

[0225] The HER2 antigen intended for antibody production may be, for example, a soluble form containing the extracellular domain of or a portion thereof of the HER2 receptor containing the desired epitope. Alternatively, antibodies may be generated using cells expressing HER2 at their cell surface (e.g., NIH-3T3 cells converted to overexpress HER2; or cancer cell lines, such as SK-BR-3 cells, see Stancovski et al., PNAS (USA) 88:8691-8695 (1991)). Other forms of the HER2 receptor that may be used for antibody production are known to those skilled in the art.

[0226] Various methods are available in the industry for preparing the monoclonal antibodies described herein. For example, the monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), by means of a recombinant DNA method (US Patent No. 4,816,567).

[0227] The anti-HER2 antibodies pertuzumab and trastuzumab used in this invention are commercially available.

[0228] U.S. Patent No. 6,949,245 describes the generation of an exemplary humanized HER2 antibody that binds to HER2 and blocks ligand activation of the HER receptor.

[0229] Humanized HER2 antibodies specifically include trastuzumab, as described in Table 3 of U.S. Patent 5,821,337, which is expressly incorporated herein by reference and is as defined herein; and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

[0230] The humanized antibody described herein may, for example, include non-human hypervariable residues incorporated into the human variable heavy chain domain, and may further include frame region (FR) substitutions at positions selected from the group consisting of 69H, 71H, and 73H, using the variable domain numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody includes FR substitutions at two or all of positions 69H, 71H, and 73H.

[0231] The exemplary humanized antibodies of interest herein comprise a variable heavy chain domain complementarity-determining residue GFTFTDYTMX (SEQ ID NO: 17), wherein X is preferably D or S; DVNPNSGGSIYNQRFKG (SEQ ID NO: 18); and / or NLGPSFYFDY (SEQ ID NO: 19), and, where appropriate, amino acid modifications to these CDR residues, for example, wherein such modifications substantially maintain or improve the antibody's affinity. For example, antibody variants used in the methods of this invention may have about one to about seven or about five amino acid substitutions in the aforementioned variable heavy chain CDR sequence. These antibody variants can be prepared by affinity maturation, as described below, for example.

[0232] For example, in addition to the variable heavy chain domain (CDR) residues mentioned in the preceding paragraph, humanized antibodies may also include the variable light chain domain complementarity-determining residues KASQDVSIGVA (SEQ ID NO:20); SASYX1X2X3, wherein X1 is preferably R or L, X2 is preferably Y or E, and X3 is preferably T or S (SEQ ID NO:21); and / or QQYYIYPYT (SEQ ID NO:22). These humanized antibodies may, as appropriate, include amino acid modifications to the aforementioned CDR residues, for example, wherein such modifications substantially maintain or improve the antibody's affinity. For example, the antibody variant of interest may have about one to about seven or about five amino acid substitutions in the aforementioned variable light chain CDR sequence. These antibody variants can be prepared by affinity maturation, as described below, for example.

[0233] This application also considers affinity-matured antibodies that bind to HER2. The parental antibody may be a human antibody or a humanized antibody, such as one containing the variable light chain sequence and / or variable heavy chain sequence (i.e., containing the VL and / or VH of pertuzumab) of SEQ ID No. 7 and 8, respectively. The affinity-matured variant of pertuzumab preferably binds to the HER2 receptor with an affinity superior to that of murine 2C4 or pertuzumab, for example, about two or four times to about 100 or about 1000 times the modified affinity, as evaluated by ELISA. Exemplary variable heavy chain CDR residues used for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two, three, four, five, six, or seven of these residues. Examples of variable light chain CDR residues used for modification include combinations of two to three, four, five, or up to about ten of the residues, such as L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or two or more of these residues.

[0234] The humanization of murine 4D5 antibodies to generate their humanized variants (including trastuzumab) is described in the following literature: U.S. Patents 5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and 6,800,738, and Carter et al., PNAS (USA), 89:4285-4289 (1992). HuMAb4D5-8 (trastuzumab) binds to the HER2 antigen with three times the tightness of the murine 4D5 antibody and has secondary immunomodulatory function (ADCC) to allow the humanized antibody to have targeted cytotoxic activity in the presence of human effector cells. HuMAb4D5-8 contains variable light chain (VL) CDR residues incorporated into the VLκ subgroup I common frame and variable heavy chain (VH) CDR residues incorporated into the VH subgroup III common frame. The antibody further includes frame region (FR) substitutions at positions 71, 73, 78, and 93 (Kabat numbers of FR residues) in VH; and at position 66 (Kabat number of FR residues) in VL. Trastuzumab contains a non-A allotype human γ1 Fc region.

[0235] Consider various forms of humanized antibodies or affinity-matured antibodies. For example, humanized antibodies or affinity-matured antibodies may be antibody fragments. Alternatively, humanized antibodies or affinity-matured antibodies may be complete antibodies, such as complete IgG1 antibodies.

[0236] (ii) Pertuzumab composition

[0237] In one embodiment of a HER2 antibody composition, the composition comprises a mixture of a natural pertuzumab antibody and one or more variants thereof. A preferred embodiment of the natural pertuzumab antibody herein comprises the variable light chain amino acid sequence and the variable heavy chain amino acid sequence of SEQ ID No. 7 and 8, and preferably comprises the light chain amino acid sequence of SEQ ID No. 11 and the heavy chain amino acid sequence of SEQ ID No. 12. In one embodiment, the composition comprises a mixture of a natural pertuzumab antibody and a variant of its amino acid sequence comprising an amino-terminal leader extension. Preferably, the amino-terminal leader extension is located on the light chain of the antibody variant (e.g., on one or both light chains of the antibody variant). The primary HER2 antibody or antibody variant may be a full-length antibody or an antibody fragment (e.g., the Fab fragment of the F(ab=)2 fragment), but preferably both are full-length antibodies. The antibody variants described herein may contain an amino-terminal leader extension on either or more of their heavy or light chains. Preferably, the amino-terminal leader extension is located on one or both light chains of the antibody. The amino-terminal leader extension preferably contains or is composed of VHS-. The presence of the amino-terminal leader extension in the composition can be detected by various analytical techniques, including but not limited to N-terminal sequence analysis, charge heterogeneity assays (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, etc. The amount of antibody variant in the composition is generally between the amount constituting the detection limit for any assay (preferably N-terminal sequence analysis) used to detect the variant and the amount less than that of the major antibody class. Typically, about 20% or less (e.g., about 1% to about 15%, or 5% to about 15%) of the antibody molecules in the composition contain an amino-terminal leader extension. Quantitative N-terminal sequence analysis or cation exchange assays (preferably using high-resolution, weak cation exchange columns, such as PROPAC WCX-10™ cation exchange columns) are preferred for determining these percentage amounts. In addition to amino-terminal leader extension variants, other amino acid sequence alterations in major antibody classes and / or variants should also be considered, including but not limited to antibodies containing C-terminal lysine residues on one or both heavy chains, deamination antibody variants, etc.

[0238] Furthermore, the main types of antibodies or variants may further include glycosylation changes, and non-limiting examples include antibodies containing a G1 or G2 oligosaccharide structure attached to the Fc region, antibodies containing a carbohydrate moiety attached to a light chain (e.g., attached to one or two light chains of the antibody, or one or two carbohydrate moieties attached to one or more lysine residues, such as glucose or galactose), antibodies containing one or two non-glycosylated heavy chains, or antibodies containing sialylated oligosaccharides attached to one or two heavy chains, etc.

[0239] The composition can be recovered from genetically modified cell lines (e.g., Chinese hamster ovary (CHO) cell lines expressing HER2 antibodies) or can be obtained by peptide synthesis.

[0240] For more information on the exemplary pertuzumab composition, see U.S. Patent Nos. 7,560,111 and 7,879,325 and US 2009 / 0202546A1.

[0241] (iii) Trastuzumab composition

[0242] Trastuzumab compositions typically comprise a mixture of major antibody species (containing the light chain and heavy chain sequences of SEQ ID NO: 13 and 14, respectively) and their variant forms, particularly acidic variants (including deacetylated variants). Preferably, the amount of such acidic variants in the composition is less than about 25%, less than about 20%, or less than about 15%. See U.S. Patent No. 6,339,142. For information on trastuzumab forms that can be resolved by cation exchange chromatography, see also Harris et al., J. Chromatography, B752:233-245 (2001). These forms include peak A (Asn30 deacetylated to Asp in two light chains); peak B (Asn55 deacetylated to isoAsp in one heavy chain); peak 1 (Asn30 deacetylated to Asp in one light chain); peak 2 (Asn30 deacetylated to Asp in one light chain and Asp102 isomerized to isoAsp in one heavy chain); peak 3 (main peak form or major antibody); peak 4 (Asp102 isomerized to isoAsp in one heavy chain); and peak C (Asp102 is converted to succinylimine (Asu) in one heavy chain).

[0243] (iv) Trastuzumab-pertuzumab components in fixed-dose combinations

[0244] Examples of this invention disclose extensive research on various charge variants found in the trastuzumab-pertuzumab fixed-dose combination. Acceptance criteria were established based on clinical experience and hypothetical effects on bioactivity / PK and safety / immunogenicity morphology. The compositions presented herein are considered to have the bioactivity and PK required for safe biologics without increasing the risk of immunogenicity or safety.

[0245] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 28% of pertuzumab natural antibody, at least 16% of trastuzumab natural antibody and less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0246] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 38% of pertuzumab natural antibody, at least 16% of trastuzumab natural antibody and less than 9% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0247] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 21% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30 and a trastuzumab variant deacetylated at HC-Asn-55, at least 28% of pertuzumab natural antibody, at least 23% of trastuzumab natural antibody and less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0248] In one embodiment, ion exchange chromatography is used to analyze a composition comprising pertuzumab, trastuzumab, and their charge variants. In one embodiment, ion exchange chromatography is used according to any of the foregoing embodiments to analyze a composition comprising pertuzumab, trastuzumab, and their charge variants. In one embodiment, the percentage of natural antibodies and charge variants is equal to the peak area determined by ion exchange chromatography according to any of the embodiments described above, wherein (i) the pertuzumab variant deacetaminated at HC-Asn-391, the pertuzumab FC sialic acid variant, the pertuzumab lysine glycation variant, the trastuzumab deacetaminated at LC-Asn-30, and the trastuzumab deacetaminated at HC-Asn-55 are ejected at peaks 1 to 3 and the percentage of such variants in the composition is equal to the sum of peak areas 1 to 3, (ii) the pertuzumab natural antibody is ejected at peak 4 and the percentage of the pertuzumab natural antibody in the composition is equal to the peak area of ​​peak 4, and (iii) the trastuzumab natural antibody is ejected at peak 7. (iv) Trastuzumab monoisomeric to isaspartic acid at one heavy chain is proposed by Zhongchong and the percentage of this variant in the composition is equal to the peak area of ​​peak 7.

[0249] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of the total peak area of ​​peaks 1 to 3, at least 28% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 16% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 12% of the peak area of ​​peak 8, as determined by the method described in any of the embodiments above. In one aspect, the method comprises the steps of: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0250] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0251] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0252] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0253] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0254] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0255] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 23% of the total peak area of ​​peaks 1 to 3, at least 38% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 16% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 9% of the peak area of ​​peak 8, as determined by the method described in any of the embodiments above. In one aspect, the method comprises the steps of: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0256] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0257] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0258] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0259] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0260] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0261] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 21% of the total peak area of ​​peaks 1 to 3, at least 28% of the peak area of ​​peak 4 (pertuzumab natural antibody), at least 23% of the peak area of ​​peak 7 (trastuzumab natural antibody), and less than 12% of the peak area of ​​peak 8, as determined by the method described in any of the embodiments above. In one aspect, the method comprises the steps of: a. binding the antibodies to an ion exchange material using a loading buffer, wherein the pH of the loading buffer is between about pH 7.5 and about pH 7.65; b. eluting the antibodies with an extraction buffer, wherein the pH of the extraction buffer is between about pH 7.5 and about pH 7.7.

[0262] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0263] In one embodiment, step b is performed using a salt gradient. In one embodiment, the extraction buffer contains sodium. In one embodiment, the extraction buffer contains sodium chloride.

[0264] In one embodiment, the method for evaluating the above-mentioned fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps: c. selectively detecting charge variants of pertuzumab and trastuzumab in the composition.

[0265] In one embodiment, the method is performed at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0266] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0267] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 22% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30, and a trastuzumab variant deacetylated at HC-Asn-55, at least 29.2% of pertuzumab natural antibody, at least 21.8% of trastuzumab natural antibody, and less than 5% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0268] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 22% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30, and a trastuzumab variant deacetylated at HC-Asn-55, at least 39.4% of pertuzumab natural antibody, at least 21.8% of trastuzumab natural antibody, and less than 4.1% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0269] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises less than 19.8% of an acidic pertuzumab variant selected from HC-Asn-391 deacetylated variant, Fc sialic acid variant and lysine glycosylated variant, a trastuzumab variant deacetylated at LC-Asn-30, and a trastuzumab variant deacetylated at HC-Asn-55, at least 29.2% of pertuzumab natural antibody, at least 31% of trastuzumab natural antibody, and less than 5% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0270] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0271] In another aspect of the present invention, the composition provided herein can be obtained by a method comprising the following steps: a. adding a predetermined amount of pertuzumab to a mixing vessel; b. adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or in a 1:2 ratio of trastuzumab to pertuzumab; c. adding rHuPH20.

[0272] In one embodiment, a 1:1 trastuzumab / pertuzumab ratio can produce a composition comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, a 1:2 trastuzumab / pertuzumab ratio can produce a composition comprising 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, rHuPH20 is added to the composition to achieve a final concentration of 2000 U / mL rHuPH20.

[0273] IV. Recombinant HER2 extracellular domain

[0274] The inventors have discovered that by including a recombination subdomain III that has been truncated at the C-terminus, a modified HER2 ECD lacking subdomain IV can be produced, having a three-dimensional configuration similar to that of the natural HER2 ECD. In one such embodiment, the modified HER2 ECD comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34. In one embodiment, a modified HER2 ECD comprising SEQ ID NO. 24 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 24 is provided.

[0275] In one embodiment, recombinant HER2 extracellular subdomains I, II, and III are fused to the Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In one embodiment, a modified HER2 ECD comprising SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 25 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 26 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 27 is provided.

[0276] In one embodiment, a modified ECD comprising SEQ ID NO: 33, SEQ ID NO: 3, and SEQ ID NO: 4 is provided. In one embodiment, the modified ECD comprises SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3, and SEQ ID NO: 4.

[0277] The inventors have discovered that by including a recombination subdomain I that has been truncated at the C-terminus and replacing the HER2 ECD subdomain II with the EGFR subdomain II, a modified HER2 ECD lacking subdomain II can be produced, having a three-dimensional configuration similar to that of the natural HER2 ECD. In one embodiment, a modified HER2 ECD comprising SEQ ID NO. 29 is provided. In one embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 29 is provided.

[0278] In one embodiment, recombinant HER2 extracellular subdomains I, III, and IV, and EGFR subdomain II are fused to the Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In any of the foregoing embodiments, the capture reagent used to evaluate trastuzumab binding does not contain HER2 ECD subdomain II. In one embodiment, a recombinant HER2 extracellular domain comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32 is provided. In one embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 30 is provided. In one embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 31 is provided. In one embodiment, a modified HER2 ECD is provided that has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 32.

[0279] Recombinant HER2 extracellular domains can be generated and purified using methods known in the art. In one embodiment, a method for preparing recombinant HER2 extracellular domains is provided, wherein the method comprises culturing a host cell containing nucleic acid encoding the recombinant HER2 extracellular domain under conditions suitable for expressing the recombinant HER2 extracellular domain, and, where appropriate, recovering the recombinant HER2 extracellular domain from the host cell (or host cell culture medium). During the recombinant production of the recombinant HER2 extracellular domain, the nucleic acid encoding the recombinant HER2 extracellular domain is isolated and inserted into one or more vectors for further selection and / or expression in host cells. These nucleic acids can be isolated and sequenced using conventional procedures or generated by recombinant methods or obtained by chemical synthesis.

[0280] Host cells used for the selection or expression of vectors encoding the recombinant HER2 extracellular domain include prokaryotic or eukaryotic cells as described herein. For example, the recombinant HER2 extracellular domain can be generated in bacteria. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523. After expression, the recombinant HER2 extracellular domain can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0281] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable selection or expression hosts for vectors encoding the recombinant HER2 extracellular domain. Suitable host cells for expressing the recombinant HER2 extracellular domain are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells. Plant cell cultures can also be used as hosts. Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines include: monkey kidney CV1 cell lines transformed from SV40 (COS-7); human fetal kidney cell lines (such as 293 or 293T cells described in the following literature, e.g., Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); juvenile hamster kidney cells (BHK); mouse supporting cells (such as TM4 cells described in the following literature, e.g., Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (such as those described in the following literature, e.g., Mather, (As described in JP et al., Annals NYAcad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl.Acad.Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. In one aspect, the host cell is, for example, Chinese hamster ovary (CHO) cells.

[0282] V. Set

[0283] The present invention also provides a kit for binding antibodies that bind to HER2 extracellular subdomain II in a specific quantitative fixed-dose combination (FDC), the fixed-dose combination containing a first antibody and a second anti-HER2 antibody that bind to HER2 extracellular subdomain II, the kit comprising: (a) a container containing proteins comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 34 as capture reagents; and (b) a description of the quantitative binding of antibodies to HER2 extracellular subdomain II.

[0284] In one embodiment, the capture reagent comprises SEQ ID NO: 24. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 24.

[0285] In one embodiment, the capture reagent comprises SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 25. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 26. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 27.

[0286] In one embodiment, the description further includes a description relating the binding of a first antibody to the extracellular subdomain II of HER2 to its efficacy.

[0287] In one embodiment, the second antibody binds to an epitope different from that of the first antibody. In one embodiment, the second antibody binds to the HER2 extracellular subdomain IV.

[0288] In one embodiment, the first antibody is pertuzumab. In one embodiment, the second antibody is trastuzumab.

[0289] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab and trastuzumab FDC contains approximately 2000 U / mL rHuPH20.

[0290] The present invention also provides a kit for binding of an antibody to HER2 extracellular subdomain IV in a specific quantitative fixed-dose combination (FDC), the fixed-dose combination containing an antibody to HER2 extracellular subdomain IV and a second anti-HER2 antibody, the kit comprising: (a) a container containing proteins comprising SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 4 as a capture reagent; and (b) a description of the quantitative binding of the antibody to HER2 extracellular subdomain IV.

[0291] In one embodiment, the capture reagent comprises SEQ ID NO: 29. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 29.

[0292] In one embodiment, the capture reagent comprises SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 30. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 31. In one embodiment, the capture reagent has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 32.

[0293] In one embodiment, the description further includes a description of establishing a relationship between the binding of an antibody to the extracellular subdomain IV of HER2 and its efficacy.

[0294] In one embodiment, the second antibody binds to an epitope different from that of the first antibody. In one embodiment, the second antibody binds to HER2 extracellular subdomain II.

[0295] In one embodiment, the first antibody is trastuzumab. In one embodiment, the second antibody is pertuzumab.

[0296] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab and trastuzumab FDC contains approximately 2000 U / mL rHuPH20.

[0297] VI. Manufacturing Method

[0298] In one embodiment, a method for preparing a composition is provided, comprising: (1) producing a fixed-dose composition comprising pertuzumab, trastuzumab and one or more variants thereof, and (2) subjecting the composition thus produced to an analytical assay to assess the amount of the variant, wherein the variant comprises: (i) pertuzumab deacetinated at HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumab lysine glycation variant, trastuzumab deacetinated at LC-Asn-30, trastuzumab deacetinated at HC-Asn-55, (ii) a natural pertuzumab antibody, (iii) a natural trastuzumab antibody, and (vi) trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0299] In one embodiment, the variant comprises (i) less than 23% of the following variants: pertuzumab deacetinated at HC-Asn-391, pertuzumab FC sialic acid variant and pertuzumab lysine glycation variant, trastuzumab deacetinated at LC-Asn-30, and trastuzumab deacetinated at HC-Asn-55; (ii) at least 28% of pertuzumab natural antibody; (iii) at least 16% of trastuzumab natural antibody; and (iv) less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0300] In one embodiment, the variant comprises (i) less than 23% of the following variants: pertuzumab deacetinated at HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumab lysine glycation variant, trastuzumab deacetinated at LC-Asn-30, and trastuzumab deacetinated at HC-Asn-55; (ii) at least 38% of natural pertuzumab antibody; (iii) at least 16% of natural trastuzumab antibody; and (iv) less than 9% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0301] In one embodiment, the variant comprises (i) less than 21% of the following variants: pertuzumab deacetinated at HC-Asn-391, pertuzumab FC sialic acid variant and pertuzumab lysine glycation variant, trastuzumab deacetinated at LC-Asn-30, and trastuzumab deacetinated at HC-Asn-55; (ii) at least 28% of pertuzumab natural antibody; (iii) at least 23% of trastuzumab natural antibody; and (iv) less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0302] In one embodiment, the analytical assay is ion exchange chromatography. In one embodiment, the analytical assay is ion exchange chromatography as described in any of the embodiments above. In one embodiment, the percentages are equal to the peak areas measured by ion exchange chromatography of any of the embodiments described above, wherein (i) the pertuzumab variant deamined at HC-Asn-391, the pertuzumab FC sialic acid variant, the pertuzumab lysine glycation variant, the trastuzumab deamined at LC-Asn-30, and the trastuzumab deamined at HC-Asn-55 are ejected in peaks 1 to 3 and the percentage of such variants in the composition is equal to the sum of the peak areas 1 to 3, (ii) the pertuzumab natural antibody is ejected in peak 4 and the percentage of the pertuzumab natural antibody in the composition is equal to the peak area of ​​peak 4, and (iii) the trastuzumab natural antibody is ejected in peak 7 and the percentage of the trastuzumab natural antibody in the composition is equal to the peak area of ​​peak 7. (iv) Trastuzumab monoisomeric to isaspartic acid at one heavy chain is extracted in peak 8 and the percentage of this variant in the composition is equal to the peak area of ​​peak 8.

[0303] In one embodiment, the amounts of the following other variants are analyzed in the analytical assay: (v) pertuzumab with N-terminal VHS on both the heavy and light chains, pertuzumab with C-terminal lysine on the heavy chain, trastuzumab with HC-Asn-392 deamination, trastuzumab with lysine glycosylation, and trastuzumab with increased Fc sialic acid content.

[0304] In one embodiment, the analytical assay is ion exchange chromatography. In one embodiment, the analytical assay is ion exchange chromatography of any of the embodiments described above. In one embodiment, the percentage is equal to the peak area measured by ion exchange chromatography of any of the embodiments described above, wherein (vii) pertuzumab with N-terminal VHS on both the heavy and light chains, pertuzumab with C-terminal lysine on the heavy chain, trastuzumab with HC-Asn-392 deamination, trastuzumab with lysine glycosylation, and trastuzumab with increased Fc sialic acid content are flushed out in peaks 5-6, thereby the percentage of such variants in the composition is equal to the peak area of ​​peaks 5-6.

[0305] In one embodiment, the following other variants are analyzed in an analytical assay: (vi) trastuzumab monoisomerized to succinimide at one heavy chain and trastuzumab with Fc oxidation.

[0306] In one embodiment, the analytical assay is ion exchange chromatography. In one embodiment, the analytical assay is ion exchange chromatography of any of the embodiments described above. In one embodiment, the percentage is equal to the peak area measured by ion exchange chromatography of any of the embodiments described above, wherein (vi) trastuzumab monoisomerized to succinimide at one heavy chain and trastuzumab with Fc oxidation.

[0307] It is extracted in peaks 9-10. Therefore, the percentage of such variants in the composition is equal to the peak area of ​​peaks 9-10.

[0308] In one embodiment, the method is used to prepare a composition further comprising rHuPH20. In one embodiment, the composition comprises 2000 U / ml rHuPH20. In one embodiment, the method is used to prepare a composition comprising 40-60 mg / mL trastuzumab and 60-80 mg / mL pertuzumab. In one embodiment, the composition comprises 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, the composition comprises 60 mg / mL trastuzumab and 60 mg / mL pertuzumab.

[0309] In one embodiment, step (1) of the preparation method described above includes the following steps: a. adding a predetermined amount of pertuzumab to a mixing vessel; b. adding trastuzumab at a ratio of 1:1 to pertuzumab or at a ratio of 1:2 to pertuzumab; c. adding rHuPH20.

[0310] In one embodiment, a 1:1 trastuzumab / pertuzumab ratio can produce a composition comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, a 1:2 trastuzumab / pertuzumab ratio can produce a composition comprising 40 mg / mL trastuzumab and 80 mg / mL pertuzumab.

[0311] In one embodiment, rHuPH20 is added to the composition to achieve a final concentration of 2000 U / ml rHuPH20.

[0312] VII. Selecting patients for the treatment

[0313] Detection of HER2 expression or amplification can be used to select patients for treatment with this invention. Several FDA-approved commercial assays can be used to identify cancer patients who are HER2-positive, HER2-expressing, HER2-overexpressing, or HER2-amplified. These methods include HERCEPTEST® (Dako) and PATHWAY® HER2 (immunohistochemical (IHC) assays) and PathVysion® and HER2 FISH pharmDx™ (FISH assays). For information on the validation and performance of each assay, users should refer to the package insert of the specific assay kit.

[0314] For example, HER2 expression or overexpression can be analyzed by IHC, such as using HERCEPTEST® (Dako). Paraffin-embedded tissue sections from tumor biopsies can undergo IHC testing and meet the following HER2 protein staining intensity criteria: Score 0: No staining observed or membrane staining observed in less than 10% of tumor cells. Score 1+: Weak / virtually imperceptible membrane staining detected in more than 10% of tumor cells. Only the cell membrane is stained. Score 2+: Weak to moderate complete membrane staining observed in more than 10% of tumor cells. Score 3+: Moderate to strong complete membrane staining observed in more than 10% of tumor cells.

[0315] Tumors with a HER2 overexpression score of 0 or 1+ can be characterized as HER2 negative, while tumors with a score of 2+ or 3+ can be characterized as HER2 positive.

[0316] Tumors that overexpress HER2 can be graded based on the immunohistochemical score corresponding to the number of HER2 molecules expressed per cell, and can be determined biochemically: 0 = 0-10,000 copies / cell, 1+ = at least about 200,000 copies / cell, 2+ = at least about 500,000 copies / cell, 3+ = at least about 2,000,000 copies / cell.

[0317] HER2 overexpression in grade 3+ breast cancer can lead to ligand-independent activation of tyrosine kinase (Hudziak et al., Proc. Natl. Acad. Sci. USA, 84:7159-7163 (1987)), which occurs in approximately 30% of breast cancers, and in these patients, recurrence-free survival and overall survival are reduced (Slamon et al., Science, 244:707-712 (1989); Slamon et al., Science, 235:177-182 (1987)).

[0318] The presence of HER2 protein overexpression is highly correlated with gene amplification; therefore, alternatively or additionally, in situ hybridization (ISH) assays (e.g., fluorescence in situ hybridization (FISH)) for detecting gene amplification can be used to select patients suitable for treatment according to the present invention. FISH assays (e.g., INFORM™ (sold by Ventana, Arizona) or PathVysion® (Vysis, Illinois)) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the extent of HER2 amplification in the tumor (if present).

[0319] Most commonly, archived paraffin-embedded tumor tissue is used and any of the methods described above is used to confirm HER2 positivity.

[0320] Preferably, HER2-positive patients with a 2+ or 3+ IHC score and / or who are FISH or ISH positive are selected for treatment according to the present invention. Patients with a 3+ IHC score and who are FISH / ISH positive are particularly suitable for treatment according to the present invention.

[0321] HER2 mutations associated with HER2-targeted therapy responsiveness have also been identified. These mutations include, but are not limited to, insertions in HER2 exon 20, deletions around HER2 amino acid residues 755-759, any of the following mutations: G309A, G309E, S310F, D769H, D769Y, V777L, P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov 2013; 3:1-14), and previously reported identical nonsynonymous putative activating mutations (or insertions / deletions) found in two or more unique samples in the COSMIC database.

[0322] For alternative assays and examples for screening patients for pertuzumab therapy, see also U.S. Patent No. 7,981,418.

[0323] Table 1: Sequence illustrate SEQ ID NO picture HER2 domain 1 1 HER2 Domain II 2 1 HER2 domain III 3 1 HER2 Domain IV 4 1 2C4 Variable Light Chain 5 2A 2C4 Variable Heavy Chain 6 2B 574 / Pertuzumab Variable Light Chain 7 2A 574 / Pertuzumab Variable Heavy Chain 8 2B Human V L Common framework 9 2A Human V H Common framework 10 2B Pertuzumab light chain 11 3A Pertuzumab heavy chain 12 3B Trastuzumab light chain 13 4A Trastuzumab heavy chain 14 4B Variant pertuzumab light chain 15 5A Variant pertuzumab heavy chain 16 5B GFTFTDYTMX 17 DVNPNSGGSIYNQRFKG 18 NLGPSFYFDY 19 KASQDVSIGVA 20 SASYX 1 X 2 X 3 twenty one QQYYIYPYT twenty two Recombinant HER2 extracellular domain II twenty three Capture agent for binding to ECD domain II anti-HER2 antibodies twenty four Capture agent for anti-HER2 antibodies that bind to ECD domain II linked to the Fc domain. 25 Capture agent for anti-HER2 antibodies that bind to ECD domain II linked to the Fc domain. 26 Capture agent for anti-HER2 antibodies that bind to ECD domain II linked to the Fc domain. 27 Recombinant HER2 extracellular domain IV 28 Capture agent for binding to ECD domain IV anti-HER2 antibodies 29 Capture agent for anti-HER2 antibodies that bind to the ECD domain IV linked to the Fc domain. 30 Capture agent for anti-HER2 antibodies that bind to the ECD domain IV linked to the Fc domain. 31 Capture agent for anti-HER2 antibodies that bind to the ECD domain IV linked to the Fc domain. 32 Recombinant HER2 extracellular domain I 33 Recombinant HER2 extracellular domain III 34 Mouse Fc domain 35 EGFR ECD Subdomain II 36

[0324] Table 2 - List of abbreviations and definitions of terms abbreviation definition ABTS 2,2'-Metazolylbis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt ACES N-(2-acetylamino)-2-aminoethanesulfonic acid BSA Bovine serum albumin CDR Complementary Determinant Zone CoA Analysis Certificate CpB Carboxypeptidase B DPBS Dulbecco's phosphate-buffered saline EC 50 Half of the maximum effective concentration ECD extracellular domain EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunoassay FDC Fixed dose combination FDC Pharmaceuticals Pertuzumab-Trastuzumab fixed-dose combination drug for subcutaneous injection HER Human epidermal growth factor receptor HER2 Human epidermal growth factor receptor 2 HPLC High performance liquid chromatography HRP Horseradish peroxidase HSR Hill slope ratio IE-HPLC Ion exchange high performance liquid chromatography IgG Immunoglobulin G iv For intravenous injection LAD Lower asymptote deviation LC-MS Liquid chromatography-mass spectrometry LD Loading dose mAB monoclonal antibodies MD Maintenance dose NA not applicable OD Optical density rHuPH20 Recombinant human hyaluronidase RP-UHPLC Reversed-phase ultra-high performance liquid chromatography R 2 Measurement coefficient SC subcutaneous U unit UAD Upper asymptote deviation UV Ultraviolet light WFI Water for Injection

[0325] Example 1: Pertuzumab-Trastuzumab FDC

[0326] Pertuzumab and trastuzumab (the two active ingredients of FDC drugs LD and MD) are recombinant humanized monoclonal antibodies targeting the IgG1 subclass of the HER2 extracellular domain. rHuPH20 (the third active ingredient of FDC drugs) is a transiently active enzyme (recombinant human hyaluronidase) used as a local penetration enhancer to allow subcutaneous delivery of therapeutic agents that are traditionally delivered intravenously.

[0327] FDC provides sterile, colorless to light brownish solutions for subcutaneous injection. They are preservative-free. Two formulations are available as described below:

[0328] Loading Dosage: FDC Drug LD

[0329] At target pH 5.5, each 20 mL single-dose vial contains 1200 mg (nominal) pertuzumab, 600 mg (nominal) trastuzumab, and 2000 U / mL hyaluronidase (rHuPH20, α-hyaluronidase). The drug is formulated to contain 80 mg / mL pertuzumab and 40 mg / mL trastuzumab. The excipients used in the formulation are L-histidine, L-histidine monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0330] Maintenance dose: FDC Drug MD

[0331] At target pH 5.5, each 15 mL single-dose vial contains 600 mg (nominal) pertuzumab, 600 mg (nominal) trastuzumab, and 2000 U / mL hyaluronidase (rHuPH20, α-hyaluronidase). The drug is formulated to contain 60 mg / mL pertuzumab and 60 mg / mL trastuzumab. The excipients used in the formulation are L-histidine, L-histidine monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0332] Example 2: The potency of pertuzumab-trastuzumab FDC determined by cell-based assays

[0333] This method determines the potency of pertuzumab and trastuzumab by measuring their ability to inhibit the proliferation of MDA-MB-175-VII or BT-474 cells, respectively. In a typical assay, MDA-MB-175-VII or BT-474 cells were seeded into 96-well microplates and incubated overnight in a humidified incubator at 37°C and 5% CO2. After incubation, the culture medium was removed, and reference standards, assay controls, and samples at different concentrations were added to the plates. The plates were then incubated for 3 days, and the relative number of viable cells was indirectly quantified using the redox dye Alamar Blue.

[0334] Fluorescence was measured using excitation at 530 nm and emission at 590 nm.

[0335] AlamarBlue dye is blue and non-fluorescent in its oxidized state, but it is reduced to a highly fluorescent pink form by the intracellular environment of the cell. The changes in color and fluorescence are proportional to the number of viable cells. The results (expressed in RFU form) were plotted against antibody concentration, and a parallel line analysis program was used to estimate the antiproliferative activity of FDC samples relative to reference standards.

[0336] Cell-based assays are selectively sensitive to one or more antibodies in the FDC drug, but not to both, as shown in Figures 7A and 7B. Individually, trastuzumab showed antiproliferative activity against BT-474 cells but not against MDA-MD-175 VII cells; pertuzumab showed antiproliferative activity against MDA-MB-175 VII cells, but its activity against BT-474 cells strongly shifted to higher concentrations. The difference in sensitivity between the two cell lines may be based on different levels of HER2 expression (high expression in BT-474 and moderate expression in MDA-MB-175 VII), rather than differences in HER2 affinity. Additionally, HER3 expression and other potential parameters related to overall antiproliferative activity (e.g., the presence or absence of HER3 endogenous ligand regulatory proteins) can also contribute to differences in sensitivity. Furthermore, in cell-based assays of drug substances, the presence of one antibody can affect the response of another, thereby masking potential quality changes occurring in one or the other antibody. Pertuzumab and trastuzumab have complementary mechanisms of action that disrupt HER2 signaling, resulting in higher antiproliferative activity when both are present (Figures 8A and 8B). Although trastuzumab alone cannot inhibit the proliferation of MDA-MB-175 VII cells in the pertuzumab antiproliferative assay (Figures 7A and 7B), its addition to pertuzumab shifts the trastuzumab dose-response curve to a lower EC50 value, reflecting the higher potency when trastuzumab and pertuzumab are combined (Figure 8A). Therefore, no slight quality change of pertuzumab in the FDC drug was detected in the MDA-MB-175 VII antiproliferative assay. Similar observations were obtained for pertuzumab in the BT-474 antiproliferative assay, but less pronounced (Figure 8B). In addition, slight quality changes of antibodies along opposite directions can produce 100% efficacy.

[0337] To confirm that no substantial quality changes of any antibody in the FDC drug were detected in the antiproliferative assay, pertuzumab and trastuzumab HER2 affinity mutants (HC S55A and L C91A mutations, respectively) with directional changes in CDR were tested in the pertuzumab and trastuzumab antiproliferative assays (Figures 9A and 9B). The significantly reduced affinity of the mutants for HER2 was associated with a reduction in their antiproliferative activity in individual cell-based assays. Adding pertuzumab to the trastuzumab mutant (or vice versa) partially restored the dose-response curve shape and thereby restored the antiproliferative activity.

[0338] In summary, based on selective sensitivity, complementary mechanisms, and the masking effect observed in antiproliferative assays, these assays are considered unsuitable for detecting changes in the activity of either antibody in a combination drug formulation. These limitations prevent antiproliferative assays from being used to determine and control the bioactivity of FDC drugs. Therefore, two selective ELISAs unaffected by these cross-interferences have been designed to control changes in the binding activity of two antibodies in FDC drugs. ELISA selectivity is ensured by using different binding epitopes of the HER2 receptor as the primary binding targets.

[0339] Example 3: Efficacy of pertuzumab in FDC as measured by ELISA

[0340] Two separate ELISAs were used to control the efficacy of FDC drugs. Here, the ELISA used to control the bioactivity of the pertuzumab component of the FDC drug is described.

[0341] Pertuzumab is a monoclonal IgG1 antibody targeting HER2, specifically the extracellular subdomain II of HER2. Upon binding, pertuzumab blocks HER2 activation by preventing heterodimerization between HER2 and ligand-activating members of the HER receptor family. This inhibits downstream signaling pathways in HER2-overexpressing cells.

[0342] Pertuzumab ELISA determines the bioactivity by the ability of pertuzumab to specifically bind to the epitope (i.e., subdomain II) of recombinant HER2. Figure 6 illustrates a schematic diagram of the capture reagents used in pertuzumab ELISA and trastuzumab ELISA (see Example 6 for details).

[0343] Binding is measured using a secondary antibody that binds to peroxidase. The dose-response curves generated for the samples and standards provide a quantitative basis. For ELISA, the actual protein content of pertuzumab (rather than the total actual protein content of the FDC drug) is considered in the preparation of the dilution buffer. Pertuzumab ELISA is used for both LD and MD of the FDC drug.

[0344] Equipment and Materials: 96-well immunoassay plates (e.g., Maxisorp ELISA), absorbance plate reader with 4-parameter data reduction software and parallelism analysis software (e.g., SoftMaxPro), computerized microplate washer.

[0345] Reagent: Pertuzumab coating reagent: Recombinant HER2 extracellular domains I, II, and III fused to mouse Fc; domain IV excluding (containing trastuzumab epitopes) (SEQ ID NO: 27). Detection antibody: HRP-bound goat anti-human antibody (specific to the F(ab')2 portion of human IgG) (e.g., Jackson ImmunoResearch). 1X DPBS, calcium and magnesium-free purified water, e.g., Milli-Q. BSA, Component V, Tween 20 ABTS, Receptor solution, concentrated phosphate (85%).

[0346] Solution Note: The formulation is for the nominal amount of reagent and can be adjusted proportionally according to the assay requirements. Washing Buffer: 1X DPBS, 0.05% Tween 20 Assay Diluent: 1X DPBS, 0.05% Tween 20, 0.5% BSA Fifth Component Spreading Solution: Pertuzumab spreading reagent (1 µg / mL) in 1X DPBS Detection Antibody: 0.8 mg / mL HRP-bound Goat Anti-Human Antibody Detection Solution: The detection solution is prepared by diluting the detection antibody (0.8 mg / mL) in the assay diluent to a concentration of 16 ng / mL. Prepare fresh before use. Stop Solution: 1 M Phosphate Reference Standard FDC MD Reference Standard

[0347] Coating plate – Transfer 100 µL of coating solution into each well of the microplate. – Incubate the coated plate at 2°C–8°C for 30–60 minutes.

[0348] Blocking Plate – Remove excess plating solution by washing all plating plates three times with 300 µL / well wash buffer. – Block all plates by adding 100 µL of assay diluent to each well. – Incubate the plates at ambient temperature with gentle shaking for 60–90 minutes. – Wash the blocking plate three times with 300 µL / well wash buffer.

[0349] Sample Transfer – Transfer 100 µL / well of FDC reference standard, product control, and sample dilution to the wells of the immunoassay plate. – Incubate the plate at ambient temperature with gentle shaking for 60–90 minutes.

[0350] Detection – Transfer 100 µL of detection solution (16 ng / mL) to each well of the plate. – Incubate the plate at ambient temperature with gentle shaking for 30–90 minutes. – Wash the plate three times with 300 µL / well of washing buffer.

[0351] Substrate Transfer and Measurement – ​​Transfer 100 µL / well of ABTS substrate solution to each well of the plate. – Incubate the plate at ambient temperature with gentle shaking for 20–35 minutes. – To terminate the reaction, transfer 100 µL / well of stop solution to each well of the plate. – Mix the plate by gently stirring for at least 1 minute. – Within 30 minutes, measure the OD value at 405 nm (reference wavelength 490 nm) using an absorbance reader.

[0352] Evaluation – Calculate the OD value for each well as follows: OD (405 nm) - OD (490 nm) Where: OD (405 nm): Detection absorbance at 405 nm, OD (490 nm): Reference absorbance at 490 nm – Average the OD values ​​of parallel measurements to determine the average OD. – Generate dose-response curves for standards, product controls, and samples by plotting the average OD (y) against pertuzumab antibody concentration (in ng / mL) (x). – Apply nonlinear regression using the following 4-parameter equation: Where: A: Lower asymptote B: Hill slope C: EC50 value D: Lower asymptote – Calculate the R² for the standard curve, product control curve, and sample curve. – The standard ΔOD is calculated as follows: Standard ΔOD = (Average maximum OD of standards) - (Average minimum OD of standards) – The maximum OD is determined as follows: The maximum OD value is the maximum OD value obtained in all parallel determinations of the dose-response curve at 405 nm.

[0353] Efficacy Calculation – Using 4-parameter parallel line analysis, a set of common Hill slopes, lower asymptotes, and lower sub-asymptotes are calculated for the standard and sample (or product control) curves. – The equations for the obtained curves of the standard and sample (or product control) are: Where: A = Common lower asymptote B = Common Hill slope C Standard = Standard EC50 value D = Common lower asymptote ρ = Efficacy of the sample and product control relative to the reference standard – Relative efficacy is calculated as follows: Relative efficacy = ρ × Activity of the reference standard

[0354] Assignment of Reference Standard Validity

[0355] The pertuzumab potency of FDC drugs is based on the pertuzumab protein content, not the total protein content of the FDC drug. Therefore, the measured potency is independent of the ratio of the two molecules in the FDC drug, and a single-molecule reference standard can be used to determine the potency of the MD and LD samples of the FDC drug. The FDC MD reference standard is selected as the potency reference standard.

[0356] Details of the potency assignment for the FDC MD reference standards are provided below: - The potency is set to 1.00 × 10⁴ U / mg. - Pertuzumab potency was determined by ELISA against the commercial pertuzumab IV reference standard anti2C4907-2. - Trastuzumab potency was determined by ELISA against the commercial trastuzumab SC reference standard G005.03EP1.

[0357] Results: The binding of pertuzumab in a fixed-dose pertuzumab-trastuzumab combination was analyzed in a pertuzumab ELISA assay. Representative dose-response curves are shown in Figure 10.

[0358] Example 4: Specificity of Pertuzumab ELISA

[0359] To evaluate the specificity of the pertuzumab ELISA in Example 3, two replicates were tested on a single plate at the highest assay concentration for formulation buffer and structure-related molecules. If interference from structure-related molecules was observed (the average of parallel assays being three times the lower asymptote mean of the reference standard dose-response curve), the assay was extended to the full dose-response curve and a reportable result was determined (n = 1).

[0360] These results confirm that the pertuzumab ELISA is specific for pertuzumab: both rHuPH20 FDC LD and MD formulation buffers showed no interference with the assay, thus confirming that the assay is suitable for analyzing FDC drug samples formulated in these matrices. Structure-related molecules (including trastuzumab, except pertuzumab, see below) do not interfere with the pertuzumab ELISA. This is demonstrated by the following results: the mean OD value of parallel assays was less than three times the mean OD value of the lower asymptote of the dose-response curve of the reference standard. As expected, pertuzumab SC drug substance and IV pertuzumab formulated in FDC drug formulations showed pertuzumab ELISA interference, due to their binding to the same HER2 domain II.

[0361] The results are shown in Table 3.

[0362] Table 3: ELISA Specificity of Pertuzumab sample Name / Description Detectable reaction Relative effectiveness (%) Average OD value of parallel measurements IV. Pertuzumab yes 92 NA SC pertuzumab yes 93 NA IV Trastuzumab no NA NA SC Trastuzumab no NA NA Trastuzumab - Aitansin no NA NA Buffer solution containing rHuPH20 FDC LD no NA 0.07 Buffer solution containing rHuPH20 FDC MD no NA 0.07 Epoetin β no NA 0.07 PEG-2A Interferon Alpha-2a no NA 0.07 Interferon α-2a no NA 0.07 Methoxylated polyethylene glycol-epotene β no NA 0.08 Atoruzumab no NA 0.07 Ocrelizumab no NA 0.07 Rituximab IV no NA 0.09 Bevacizumab no NA 0.07 Tocilizumab SC no NA 0.07 Etrolizumab no NA 0.07 Etrolizumab no NA 0.07 Vorhyaluronidase Alfa no NA 0.07 Lebrikizumab no NA 0.07 Satralizumab no NA 0.07 PEG-Fegricans no NA 0.07 Filgrastim no NA 0.08 Polatuzumab no NA 0.07 The average value of the asymptote below the dose-response curve of the reference standard. NA NA 0.08 3 × Minimum value of reference standard NA NA 0.23

[0363] Example 5: Robustness of Pertuzumab ELISA

[0364] The robustness of pertuzumab ELISA was evaluated by deliberately varying the assay parameters, which are potential sources of variation in practice. Robustness results were assessed by comparing the obtained dose-response curve parameters, system suitability and similarity criteria, and methodological procedures. Overall robustness results are summarized in Table 4.

[0365] Table 4: Robustness results of pertuzumab ELISA. Stability parameters Test conditions result Coating reagent concentration 0.5, 1, 1.5 μg / mL The assay tolerates coating reagent concentrations between 1 and 1.5 μg / mL. 10 Coating and cultivation time 30, 60, 90 minutes The assay showed tolerance to coating and incubation times between 30 and 60 minutes. Interruption of cultivation time 30, 60, 90 minutes The assay showed tolerance to incubation periods ranging from 60 to 90 minutes. FDC Drug Development Time 30, 60, 90 minutes This assay measures tolerance to FDC drug incubation time between 60 and 90 minutes. 15 antibody batch testing 3 batches This assay tolerance is derived from the use of different batches of detection antibodies by Jackson ImmunoResearch. Detecting antibody incubation time 30, 60, 90 minutes The assay tolerates antibody incubation times ranging from 30 to 90 minutes. Plasma culture time 20, 30, 40 minutes The assay showed tolerance to substrate incubation times between 20 and 35 minutes. 20 Use of different reading instruments SpectraMax M5, i3x This assay is resistant to use with both SpectraMax M5 and i3x molecular devices.

[0366] Example 6: Efficacy of trastuzumab in FDC as measured by ELISA

[0367] Two ELISA-like methods were used to control the efficacy of FDC drugs. This section describes the ELISA control of the bioactivity of the trastuzumab component of FDC drugs.

[0368] Trastuzumab is a monoclonal IgG1 antibody targeting HER2, specifically the extracellular subdomain IV of HER2. Upon binding, trastuzumab blocks HER2 activation by preventing HER2 homodimerization and causing the detachment of the HER2 extracellular domain.

[0369] This will inhibit the downstream signaling pathways of HER2-overexpressing cells.

[0370] Trastuzumab ELISA determines the bioactivity by the ability of trastuzumab to specifically bind to the epitope (i.e., subdomain IV) of recombinant HER2. Figure 6 illustrates a schematic diagram of the capture reagent used in trastuzumab ELISA.

[0371] Binding is measured using a secondary antibody that binds to peroxidase. The dose-response curves generated for the samples and standards provide a quantitative basis. For ELISA, the actual protein content of trastuzumab (rather than the total actual protein content of the FDC drug) is considered in the preparation of the dilution buffer. Trastuzumab ELISA is used for both LD and MD of the FDC drug.

[0372] The reagents, buffers, and procedures are as described in Example 3, except for the coating reagent and coating solution: Trastuzumab coating reagent: recombinant HER2 extracellular domains I, III, and IV fused to mouse Fc; domain II is replaced by the structurally relevant domain II (SEQ ID NO: 32) in EGFR that cannot bind pertuzumab. Coating solution: trastuzumab coating reagent (1 µg / mL) in 1X DPBS.

[0373] Evaluation – Calculate the OD value for each well as follows: OD (405 nm) - OD (490 nm) Where: OD (405 nm): Detection absorbance at 405 nm, OD (490 nm): Reference absorbance at 490 nm – Average the OD values ​​of parallel measurements to determine the average OD. – Generate dose-response curves for standards, product controls, and samples by plotting the average OD (y) against trastuzumab antibody concentration (expressed in ng / mL) (x). – Apply nonlinear regression using the following 4-parameter equation: Where: A: Lower asymptote B: Hill slope C: EC50 value D: Lower asymptote – Calculate the R² for the standard curve, product control curve, and sample curve. – The standard ΔOD is calculated as follows: Standard ΔOD = (Average maximum OD of standards) - (Average minimum OD of standards) – The maximum OD is determined as follows: The maximum OD value is the maximum OD value obtained in all parallel determinations of the dose-response curve at 405 nm.

[0374] Efficacy Calculation – Using 4-parameter parallel line analysis, a set of common Hill slopes, lower asymptotes, and lower sub-asymptotes are calculated for the standard and sample (or product control) curves. – The equations for the obtained curves of the standard and sample (or product control) are: Where: A = Common lower asymptote B = Common Hill slope C Standard = Standard EC50 value D = Common lower asymptote ρ = Efficacy of the sample and product control relative to the reference standard – Relative efficacy is calculated as follows: Relative efficacy = ρ × Activity of the reference standard

[0375] Assignment of Reference Standard Validity

[0376] The trastuzumab potency of FDC drugs is based on the trastuzumab protein content, not the total protein content of the FDC drug. Therefore, the measured potency is independent of the ratio of the two molecules in the FDC drug, and a single-molecule reference standard can be used to determine the potency of the MD and LD samples of the FDC drug. The FDC MD reference standard is selected as the potency reference standard.

[0377] Details of the potency assignment for the FDC MD reference standards are provided below: The potency was set to 1.00 × 10⁴ U / mg. Pertuzumab potency was determined by ELISA against the commercial pertuzumab IV reference standard anti2C4907-2. Trastuzumab potency was determined by ELISA against the commercial trastuzumab SC reference standard G005.03EP1.

[0378] Results: The binding of trastuzumab in the pertuzumab-trastuzumab fixed-dose combination was analyzed in a trastuzumab ELISA assay. Representative dose-response curves are shown in Figure 11.

[0379] Example 7: Specificity of Trastuzumab ELISA

[0380] To evaluate the specificity of trastuzumab ELISA, two replicates were tested on a single plate at the highest assay concentration for formulation buffer and structure-related molecules. If interference from structure-related molecules was observed (the average of parallel assays being three times the lower asymptote mean of the reference standard dose-response curve), the assay was extended to the full dose-response curve and a reportable result was determined (n = 1).

[0381] Results confirmed that the trastuzumab ELISA is specific for trastuzumab: both rHuPH20 FDC LD and MD formulation buffers showed no interference with the assay, thus confirming that the assay is suitable for analyzing FDC drug samples formulated in these matrices. Related molecules (including pertuzumab, excluding trastuzumab, see below) did not interfere with the trastuzumab ELISA. This is demonstrated by the following results: the mean OD value of parallel assays was less than three times the mean OD value of the lower asymptote of the dose-response curve of the reference standard. As expected, trastuzumab (IV and SC) and trastuzumab emtansine showed interference with the trastuzumab ELISA, due to their binding to the same HER2 domain IV.

[0382] The results are shown in Table 5.

[0383] Table 5: ELISA Specificity of Trastuzumab sample Name / Description Detectable reaction Relative effectiveness (%) Average OD value of parallel measurements IV Trastuzumab yes 88 NA SC Trastuzumab yes 92 NA Trastuzumab - Aitansin yes 76 NA IV. Pertuzumab no NA NA SC pertuzumab no NA NA Buffer solution containing rHuPH20 FDC LD no NA 0.03 Buffer solution containing rHuPH20 FDC MD no NA 0.03 Epoetin β no NA 0.03 PEG-2A Interferon Alpha-2a no NA 0.03 Interferon α-2a no NA 0.04 Methoxylated polyethylene glycol-epotene β no NA 0.04 Atoruzumab no NA 0.03 Ocrelizumab no NA 0.03 Rituximab IV no NA 0.03 Bevacizumab no NA 0.03 Tocilizumab SC no NA 0.03 Etrolizumab no NA 0.03 Etrolizumab no NA 0.03 Vorhyaluronidase Alfa no NA 0.03 Lebrikizumab no NA 0.03 Satralizumab no NA 0.03 PEG-Fegricans no NA 0.03 Filgrastim no NA 0.04 Polatuzumab no NA 0.03 The average value of the asymptote below the dose-response curve of the reference standard. NA NA 0.04 3 × Minimum value of reference standard NA NA 0.11

[0384] Example 8: Robustness of Trastuzumab ELISA

[0385] The robustness of trastuzumab ELISA was evaluated by deliberately varying the assay parameters, which are potential sources of variation in practice. Robustness results were assessed by comparing the obtained dose-response curve parameters, system suitability and similarity criteria, and methodological procedures. Overall robustness results are summarized in Table 6.

[0386] Table 6: Robustness results of trastuzumab ELISA. Stability parameters Test conditions result Coating reagent concentration 0.5, 1, 1.5 μg / mL The assay tolerates coating reagent concentrations between 1 and 1.5 μg / mL. 10 Coating and cultivation time 30, 60, 90 minutes The assay showed tolerance to coating and incubation times between 30 and 60 minutes. Interruption of cultivation time 30, 60, 90 minutes The assay showed tolerance to incubation periods ranging from 60 to 90 minutes. FDC Drug Development Time 30, 60, 90 minutes This assay measures tolerance to FDC drug incubation time between 60 and 90 minutes. antibody batch testing 3 batches This assay tolerance is derived from the use of different batches of detection antibodies by Jackson ImmunoResearch. 15 Detecting antibody incubation time 30, 60, 90 minutes The assay tolerates antibody incubation times ranging from 30 to 90 minutes. Plasma culture time 20, 30, 40 minutes The assay showed tolerance to substrate incubation times between 20 and 35 minutes. Use of different reading instruments SpectraMax M5, i3x This assay is resistant to use with both SpectraMax M5 and i3x molecular devices. 20

[0387] Example 9: Development of IEC used to analyze FDC charge variants

[0388] Various ion exchange chromatography protocols have been tested to separate FDC charge variants. The following parameters have been tested: column type, buffer type and concentration, salt concentration, flow rate, injection volume, pH, column temperature, and gradient pattern.

[0389] The research and development testing method is to separate the following peaks / peak groups and determine their relative abundance (expressed as total peak area %): - Sum of peaks 1-3 - Peak 4 (pertuzumab main peak) - Sum of peaks 5-6 - Peak 7 (trastuzumab main peak) - Peak 8 - Sum of peaks 9-10

[0390] An FDC IE-HPLC method has been developed and optimized to achieve optimal separation of pertuzumab charge variants and trastuzumab charge variants. The expected charge variants can be extrapolated by analyzing SC trastuzumab and SC pertuzumab separately. Experiments were performed individually and in combination with SC perjeta (batch: GB0005, c = 120 mg / mL) and SC Herceptin (batch: P0003, c = 120 mg / mL).

[0391] In the first step, registration IE-HPLC methods for individual molecules of IV Pertuzumab and IV / SC Herceptin are tested. These methods are disclosed, for example, in Zephania W. Kwong Glover et al., Compatibility and Stability of Pertuzumab and Trastuzumab Admixtures in ivInfusion Bags for Coadministration, Pharmaceutical Biotechnology, Vol. 2, No. 3, pp. 794-812, March 1, 2013, DOI: https: / / doi.org / 10.1002 / jps.23403. In these methods, a weak cation exchange column (WCX-10) is used (see Methods 1 and 2 in Table 7). In the next step, the ProPac WCX-10 column is tested using operating conditions successfully used for another mAb product with a similar pI value to pertuzumab / trastuzumab (see Method 3 in Table 7). In the next step, a strong cation exchange column was used, and different buffer solutions and pH values ​​were tested. The parameters used and the results are summarized in Table 7 below.

[0392] In the next step, different columns were screened. The optimal resolution system was achieved using a strong cation exchange column (Mab PAC SCX-10). Different buffer solutions and pH values ​​were tested (Methods 4 to 6). The parameters used and the results are summarized in Table 7. Another series was performed based on Method 6, which yielded the best results (see Table 8).

[0393] Result

[0394] Method 1: When using Method 1 to analyze pertuzumab-trastuzumab FDC under conditions, the peak separation was not satisfactory for the requirements of product release assay: the separation between peak 7 (trastuzumab main peak) and peak 8 (trastuzumab IsoAsp102) was poor and the basic pertuzumab region overlapped with the trastuzumab main peak.

[0395] Method 2: When using Method 2 to analyze pertuzumab-trastuzumab FDC under conditions, the peak separation is not satisfactory for the requirements of product release assay: the basic pertuzumab region completely overlaps with the main trastuzumab peak (peak 7) and peak 8, which is therefore unacceptable.

[0396] Method 3: The main peaks of trastuzumab and pertuzumab were both separable, with only minor overlap observed in the basic pertuzumab region. However, the acidic trastuzumab region overlapped with the pertuzumab main peak.

[0397] Method 4: The basic pertuzumab region overlaps with the main peak of trastuzumab and the IsoAsp102 peak (peak 8) of trastuzumab.

[0398] Method 5: 1: pH 7.5: Fully separate the two main peaks and peak 8. The alkaline pertuzumab region and the trastuzumab main peak have only a small overlap.

[0399] Method 5:2: pH 8.0: Sufficient peak 8, but the overlap between the alkaline pertuzumab region and the trastuzumab main peak is stronger than that of Method 5 using pH 7.5.

[0400] Method 6: Thoroughly separate all substances of interest.

[0401] Table 7: Development of IEC schemes used to analyze FDC charge variants. Method 1 Method 2 Method 3 Method 4 Method 5 Method 6 tubular ProPac WCX-10 MabPac SCX-10 Solvent A 20mM MES + 1mM Na2EDTA 20 mM ACES 10mM NaHPO4 20mM MES + 1mM Na2EDTA 20mM HEPES 20 mM ACES Solvent B Buffer A+ 250mM NaCl Buffer A + 200 mM NaCl Buffer A + 100mM NaCl Buffer A+ 250mM NaCl Buffer A+ 200 mM NaCl Buffer A + 200 mM NaCl pH pH 6.0 pH 7.5 pH 7.5 pH 6.0 1: pH 7.5 2: pH 8.0 pH 7.5 gradient Within 65 minutes, 18% to 58% B Within 40 minutes, 8% to 45% B Within 30 minutes, 15% to 55% B Within 65 minutes, 18% to 58% B 1: Within 40 minutes, 1% to 47% B 2: Within 40 minutes, 0% to 47% B Within 40 minutes, 8% to 45% B Column temperature 34°C 35°C 23°C 34°C 40°C 35°C Flow rate 0.8 mL / min 0.5 mL / min 0.8 mL / min 0.8 mL / min 0.8 mL / min 0.5 mL / min Injection volume 50 µg protein 100 µg protein 50 µg protein 50 µg protein 100 µg protein 100 µg protein

[0402] Table 8: Development of the IEC scheme used to analyze FDC charge variants. Investigate the test parameters of Method 6 above. Method 6 Method 6A Method 6B Method 6C Method 6D tubular MabPac SCX-10 Solvent A 20 mM ACES 20 mM ACES 20 mM ACES 20 mM ACES 20 mM ACES Solvent B Buffer A + 200 mM NaCl Buffer A + 200 mM NaCl Buffer A + 200 mM NaCl Buffer A + 200 mM NaCl Buffer A + 200 mM NaCl pH pH 7.5 pH 6.8 pH 7.5 pH 7.5 pH 7.5 gradient Within 40 minutes, 8% to 45% B Within 40 minutes, 8% to 45% B Within 30 minutes, 3% to 40% B Within 30 minutes, 0% to 35% B Within 40 minutes, 1% to 47% B Column temperature 35°C 35°C 35°C 40°C 40°C Flow rate 0.5 mL / min 0.5 mL / min 1.0 mL / min 0.8 mL / min 0.8 mL / min Injection volume 100 µg protein 100 µg protein 100 µg protein 100 µg protein 100 µg protein

[0403] Based on the HPLC parameters described in Table 8 above, several experimental designs (Design of Experiments, DoE) were implemented. The following parameters were tested: gradient flow rate (0.5 – 1.0 mL / min), pH values ​​of mobile phase A and mobile phase B (7.4-7.6 and 6.8 respectively), NaCl concentration in mobile phase B (100-300 mM), and column temperature (25-40℃).

[0404] Based on the analysis of data obtained within the framework of these experimental designs, the following test parameters demonstrate optimal results: Extraction reagent A: 20 mM ACES, pH 7.5; Extraction reagent B: 20 ​​mM ACES, 200 mM NaCl, pH 7.5; Column: MabPac SCX-10, BioLC, 4 × 250 mm; Column temperature: 40℃; Flow rate: 0.8 mL / min; Injection volume: 10 μL (100 μg protein); Gradient: 1% to 47% B over 40 min.

[0405] To analyze the robustness of this research method, a DoE-based experimental factor design was performed. Therefore, the following parameters varied in the matrix: ACES concentration: 18–22 mM; NaCl concentration: 180–220 mM; Column temperature: 36–44℃; Flow rate: 0.7–0.9 mL / min; pH: 7.4–7.6; Injection volume: 8–12 µL (80–120 µg).

[0406] The results of these experiments show that the test method is relatively stable within the test range for the trastuzumab main peak (peak 7) and peak 8. However, the purity values ​​obtained for the pertuzumab main peak (peak 4) and the intermediate region (the region between the pertuzumab main peak and the trastuzumab main peak) were highly variable. This variability mainly depends on pH and column temperature. The statistical evaluation of this experiment yielded the following settings for the final method: Extraction reagent A: 20 mM ACES, pH 7.6; Extraction reagent B: 20 ​​mM ACES, 200 mM NaCl, pH 7.6; Column temperature: 36°C; Flow rate: 0.8 mL / min; Injection volume: 10 μL (100 μg protein); Gradient: 1% to 47% B over 40 min.

[0407] Evaluate possible alternatives for determining charge heterogeneity in pertuzumab / trastuzumab FDC variants. Among these alternatives, assess the suitability of different column types and pH gradient methods.

[0408] Several separation attempts were also carried out using a ProPac WAX-10 bio LC (4×250mm) weak anion exchange column under the following chromatographic conditions: The following extractants A and B were prepared and tested: 1. A = 20mM CAPSO (pH 10.0), B = 20mM CAPSO + 250mM NaCl (pH 10.0) 2. A = 20mM hexahydropyrazine (pH 10.0), B = 20mM hexahydropyrazine + 250mM NaCl (pH 10.0) 3. A = 20mM trisma (pH 10.5), B = 20mM trisma + 250mM NaCl (pH 10.5) 4. A = 20mM trisma (pH 8.0), B = 20mM trisma + 250mM NaCl (pH 8.0) 5.A = 20mM phosphate (pH 11.0), B = 20mM phosphate + 250mM NaCl (pH 11.0) Column temperature: 30°C Flow rate: 0.8 mL / min; 1.0 mL / min Injection volume: 5 μL (50 μg protein) Gradient 1: 0 to 100% B over 60 min Gradient 2: 0 to 100% B over 40 min

[0409] For all the conditions tested on the weak anion exchange column, the substance of interest was not retained on the column and was flushed out along with the injection peak, thus demonstrating that these experimental conditions are not suitable for isolating the charge variant of pertuzumab / trastuzumab FDC.

[0410] Experiment on pH-gradient separation mode

[0411] The suitability of the pH gradient-based IEC method was evaluated as a possible alternative to the salt gradient method. A strong cation exchange column (MabPac SCX-10 column) was used with the following HPLC test parameters: Extractant A: 10 mM Tris, 10 mM phosphate, 10 mM hexahydropyrazine, pH 6.0; Extractant B: 10 mM Tris, 10 mM phosphate, 10 mM hexahydropyrazine, pH 11.0; Extractant C: 100 mM NaCl; Extractant D: pure water. Column: MabPac SCX-10, BioLC, 4 × 250 mm; Column temperature: 35°C; Flow rate: 0.5 mL / min; Injection volume: 10 μL (10 μg protein); Gradient: 10% to 50% B over 45 min (see details below). Equipment: Waters Alliance

[0412] Extractants C and D were combined to provide constant salt concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM NaCl, respectively. Therefore, the ratio of extractant C to extractant D varied from 0% extractant C / 50% extractant D (0 mM NaCl) to 50% extractant C / 0% extractant D (50 mM NaCl). SC Parjet (batch: GB0005, c = 120 mg / mL) and SC Herceptin (batch: P0003, c = 120 mg / mL) were used individually and in combination to perform experiments. Test samples were diluted to a final concentration of 1 mg / mL using 90% extractant A / 10% extractant B.

[0413] Optimal separation was obtained using 40 mM NaCl; however, under these conditions, the peaks emerged very early, and the two main peaks exhibited a wider shape and a smaller height.

[0414] An experimental design (DoE) was implemented using a salt concentration of 40 mM NaCl while simultaneously varying the gradient pattern and flow rate. However, a statistical model could not be established, demonstrating a lack of robustness in the investigated method, even with minor modifications to the test conditions.

[0415] Based on the experiments conducted above, the most critical parameters for the IEC method appear to be: 1. pH value, 2. column type, 3. column temperature, and 4. gradient pattern. These parameters have a significant impact on resolution. On the other hand, buffer type and concentration, salt concentration, flow rate, and injection volume have a smaller impact on resolution.

[0416] Example 10: IEC used to analyze FDC charge variants

[0417] Purpose and Principle

[0418] IE-HPLC separates proteins present in pharmaceuticals based on their charge properties in the dissolved state. This separation is based on the interaction between the surface charge of the protein and the charged groups present on the column stack surface. In the cation exchange HPLC used in this analytical procedure, acidic substances are first eluted in a salt gradient, followed by more basic substances. The same method was applied to the LD and MD of FDC pharmaceuticals. The LD and MD of FDC pharmaceuticals were tested using FDC MD reference standards.

[0419] Equipment and Materials: HPLC system equipped with a UV detector (Waters Alliance 2695 / e2695 with 2487 / 2489 detector or equivalent); HPLC column (Thermo Scientific MAbPac SCX-10, 4 mm-250 mm, particle size: 10 µm or equivalent).

[0420] Drug product dilution buffer: 20 mM L-histidine / L-histidine monohydrochloride, 105 mM trehalose, 100 mM sucrose, 10 mM methionine, 0.04% [w / v] polysorbate 20, pH 5.5 ± 0.2; Mobile phase A: 20 mM ACES, pH 7.60 ± 0.05; Mobile phase B: 20 ​​mM ACES, 200 mM sodium chloride, pH 7.60 ± 0.05; CpB solution: 1 mg / mL CpB (in mobile phase A).

[0421] Preparation of sample solution: Dilute FDC drug with mobile phase A to prepare a sample solution with a total protein concentration of about 10 mg / mL and a CpB of about 0.08 mg / mL.

[0422] Preparation of blank solution: Dilute the drug dilution buffer in the same manner as the sample.

[0423] CpB Digestion: Incubate the reference standard solution, sample solution, and blank solution at 37℃ ±2℃ for 20±5 minutes. Store the sample at 10℃ ±4℃ until analysis and complete the HPLC analysis within 24 hours.

[0424] Before injecting the first sample, flush the column with 99% mobile phase A until a stable baseline is obtained. If necessary, inject a reference solution for column conditioning purposes until a visual assessment of the chromatography shows at least two consecutive injections exhibiting a continuous pattern.

[0425] Operating Parameters: Detection wavelength: 280 nm; Injection volume: 10 µL; Flow rate: 0.8 mL / min; Column temperature: 36℃ ± 2℃; Automatic sampler temperature: 10℃ ± 4℃; Run time: 60 min

[0426] Gradient Time (min) Shifting phase A (%) Shifting phase B (%) 0 99 1 3 99 1 43 53 47 44 0 100 50 0 100 51 99 1 60 99 1

[0427] The injection protocol shall be performed in the following order: 1. Mobile phase A 2. Blank solution 3. Reference standard 4. Samples (maximum 10 samples) 5. Reference standard 6. Blank solution Note: If more than 10 samples are to be analyzed, each set of 10 samples shall be injected together with the reference standard solution.

[0428] Result:

[0429] An IE-HPLC method for FDC drugs has been developed and optimized to achieve optimal separation of pertuzumab charge variants and trastuzumab charge variants. Because pertuzumab (pI 8.7) and trastuzumab (pI 8.4) have similar isoelectric points, IE-HPLC cannot completely separate all charge variants of the two antibody molecules (see Figure 13). All important charge variants of individual molecules in FDC drugs can be controlled because all relevant peaks can be separated. The reported measurement parameters for FDC drugs are the sum of peaks 1-3, peak 4 (pertuzumab main peak), the sum of peaks 5-6, peak 7 (trastuzumab main peak), peak 8, and the sum of peaks 9-10. An example chromatogram is shown in Figure 12.

[0430] Example 11: HPLC Robustness and Repeatability Study

[0431] Different experiments were performed to assess the robustness of the analytical procedure in Example 10 for different input variables. These input variables were, in particular: column temperature (32°C, 36°C, 40°C), flow rate (0.7 mL / min, 0.8 mL / min, 0.9 mL / min), pH of mobile phases A and B (pH 7.5–7.7), and sodium chloride concentration in mobile phase B (180 mM to 220 mM).

[0432] The analysis pattern and results obtained after changing the parameters of interest will be compared with the analysis pattern and results based on the target parameters. The relative difference between the results will be calculated using the sum of peaks 4, 7, and 1-3, and the relative peak area of ​​peak 8 (expressed as area %). These results meet the acceptance criteria, thus confirming that the procedure is appropriately stable for its intended purpose.

[0433] For peaks 4, 7, the sum of peaks 1-3, and peak 8, the repeatability of the analytical procedure is shown within the following ranges: – Injected protein of 50 µg to 149 µg of FDC drug LD, covering 50% to 149% of the nominal working volume (100 µg protein) – Injected protein of 51 µg to 153 µg of FDC drug MD, covering 51% to 153% of the nominal working volume (100 µg protein).

[0434] Example 12: Stability Indicator Properties

[0435] The method described in Example 10 was used to test unstressed and stressed FDC drug MD and LD samples, demonstrating the procedure's ability to separate, identify antibodies, and determine their purity under different stress conditions. The following stress conditions were tested: thermal stress, forced oxidation, high-pH (pH 7.4) stress, low-pH (pH 4) stress, and light stress. Impurities and related substances with different charges were separated. Compared to the unstressed samples (Figures 12 and 13), the chromatography of the stressed samples showed an increase in the sum of peaks 1–3 and peak 8 (data not shown). In conclusion, the procedure indicates stability.

[0436] Example 13: The potency of trastuzumab and pertuzumab charge variants and CDR affinity mutants in FDC was measured by ELISA.

[0437] Confirmed the ability of ELISA to reflect the antiproliferative activity of charge variants and CDR affinity mutants:

[0438] Pertuzumab and trastuzumab HER2 affinity mutants as described above were tested in antiproliferative assays and ELISA (Figures 9 and 14, respectively). The absence or shift of dose-response curves to higher concentrations and failure to meet similarity criteria (parallelism of antiproliferative assays and ELISA, and upper asymptote bias, respectively) observed in HER2 affinity mutants confirmed a significant reduction in similar potency.

[0439] To evaluate charge variants, supportive technique batches of FDC drugs containing trastuzumab or pertuzumab MD formulation buffer were used to exclude the cross-interference mentioned above in cell-based assays.

[0440] All IE-HPLC fractions showed similar potency in cell-based assays and ELISAs (considering the accuracy of individual methods), except for peak 9 (trastuzumab with increased Fc Met261 oxidation). Although this Fc oxidation at Met261 should not affect the target binding activity of CDR, this variant showed reduced potency in trastuzumab ELISA (73% vs. 91% in cell-based assays).

[0441] It cannot be definitively determined whether the fractionation process of the isoform, present in only very small amounts, contributed to this finding, or whether the power values ​​of the two assays can truly be considered different. However, the trastuzumab ELISA can be considered conserved in this regard because it indicates a reduction in power not reflected by cell-based antiproliferative assays.

[0442] The ability of ELISA to control the bioactivity of known bioactive product variants is comparable to that of antiproliferative assays, as detailed below: In both assays, the trastuzumab deacetylated product variants HC Asn55 / isoAsp55 and LC Asn30 / Asp30 in peak 1 showed reduced activity. In both assays, the trastuzumab product variant with succinimide at the Asp102 position in each heavy chain and with increased FcMet oxidation in peak 10 showed reduced activity. In both assays, as expected, all other IE-HPLC fractions (including peaks 4 and 7, corresponding to the main peaks of pertuzumab and trastuzumab, respectively) showed unchanged activity between 80% and 120%.

[0443] Furthermore, although similar peak 8 potency was obtained in cell-based assays and ELISA, it has been confirmed that no known negative impact of HC IsoAsp102 on the potency of trastuzumab IV was observed in this study. The isomerization of HC Asp102 to IsoAsp at one heavy chain of trastuzumab extracted in IE-HPLC peak 4 corresponds to IE-HPLC peak 8 in the FDC drug. Other studies conducted during the development of SC trastuzumab regarding the effect of the HC Asp102 / isoAsp102 form on antiproliferative activity showed that the effect of SC trastuzumab was less significant than that of IV trastuzumab.

[0444] This can help optimize formulation (e.g., pH changes) and increase the stability of SC trastuzumab. Finally, it should be noted that the control of this variant in FDC drugs should be maintained via the defined acceptance criteria of IE-HPLC.

[0445] Table 9: Association between binding activity and antiproliferative activity of trastuzumab and pertuzumab sample FDC Drug MD Preparation Buffer (Batch: GTK0003) Contains Pertuzumab FDC Drug MD Preparation Buffer (Batch: GTK0004) Contains Trastuzumab The relative potency (%) of pertuzumab as measured by ELISA The relative potency (%) of pertuzumab MDA-MB-175 VII as determined by antiproliferative analysis The relative potency (%) of trastuzumab as measured by ELISA The relative potency (%) of trastuzumab as determined by BT-474 antiproliferative assay IE-HPLC fraction (non-force-dependent) a Peak 1 88 equivalent b 75 61 Peak 2 95 84 87 90 Peak 3 93 112 95 114 Peak 4 113 107 86 83 Peak 5 109 109 89 109 Peak 6 81 equivalent b 102 116 Peak 7 NA c NA c 101 109 Peak 8 NA c NA c 100 105 Peak 9 NA c NA c 73 91 Peak 10 NA c NA c 71 73 a. For fraction characterization, refer to Example 14. b. Provide a qualitative estimate relative to the reference standard, as the dose-response curves of the sample and the reference standard are not similar and therefore relative potency cannot be reported (n ≥ 3 plate results). c. FDC drug IE-HPLC peaks 7 to 10 contain only the trastuzumab isoform.

[0446] Example 14: Characterization of Charge Aberrations

[0447] The charge variants of FDC drugs separated and isolated by the IE-HPLC method were characterized (Figure 13). In addition, the charge variants of individual antibodies in the FDC formulation at the time of release were separated by the same IE-HPLC method and characterized (Figure 13).

[0448] Perform comprehensive peak characterization studies using the following methods to confirm the charge variants of FDC drugs: LC-MS / MS for evaluating trypsin antibody peptides at chemical degradation sites; borate affinity chromatography combined with HILIC for assessing the degree of lysine glycosylation; and 2-AB labeling for analyzing Fc glycosylation.

[0449] LC-MS Peptide Localization:

[0450] As described by Schmid et al. in 2018, LC-MS / MS peptide localization and quantification of relevant amino acid modifications were performed (Schmid I, Bonnington L, Gerl M et al., Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions. Commun Biol 2018;1:28). In short, all samples were denatured with 8 mol / L guanidine hydrochloride (pH 6.0) and reduced with dithiothreitol at 50 °C for 1 h.

[0451] Samples were buffer-exchanged (0.02 mol / L histidine salt, pH 6.0) and further digested with trypsin at 37°C for 18 h. Peptide separation was performed on a BEH C18 column in an ACQUITY UPLC system. Online mass spectrometry was performed using a Synapt G2 HDMS Q-ToF mass spectrometer. Relative quantification of modified peptides was performed using GRAMS AI software.

[0452] Borate affinity chromatography:

[0453] Boronate affinity chromatography was performed using a TSKgel Boronate-5PW affinity column. Separation was performed at 280 nm on an HPLC system equipped with UV detection using an extraction buffer consisting of 100 mmol / L Hepes, 70 mmol / L Tris, 200 mmol / L NaCl, and 500 mmol / L sorbitol (pH 8.6). Peak integration and saccharification quantification were performed as described in the following literature (Fischer S, Hoernschemeyer J, Mahler HC. Glycation during storage and administration of monoclonal antibody formulations. Eur J Pharm Biopharm. 2008;70:42–50.).

[0454] Glycan Analysis:

[0455] To evaluate Fc glycosylation, samples were buffer-exchanged with ammonium formate buffer (pH 8.6) and incubated with PNGase F at 45°C for 1 h. Glycan 2-AB labeling was then performed at 65°C for 2 h. The labeled glycan structures were subjected to HILIC separation and fluorescence detection for subsequent peak integration and glycan quantification, as described in the literature (Reusch D, Haberger M, Maier B et al., Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods. MAbs. 2015;7:167-79.)

[0456] Results and Conclusions:

[0457] All charge variants (relative abundance ≥1%) found in individual pertuzumab and trastuzumab molecules in the FDC formulation were also detected in the FDC drug product. No new charge variants were detected in the FDC drug product compared to the individual antibodies in the FDC formulation at release and after storage. Table 10 summarizes these findings.

[0458] Table 10: IE-HPLC Peak Characterization Results of FDC Drugs sample IE-HPLC peak 1 IE-HPLC peak 2 IE-HPLC peak 3 IE-HPLC peak 4 IE-HPLC peak 5 Pertuzumab - Trastuzumab FDC MD (Batch: GTK0002) Deamination at the following sites: HC-Asn-55 and LC-Asn-30 (Trastuzumab); Lys-glycated (pertuzumab) Deamination at the following sites: HC-Asn-55 and LC-Asn-30 (Trastuzumab); Fc sialic acid and Lys glycation (pertuzumab) Deamination at the following sites: LC-Asn-30 (Trastuzumab); Deamination (trace amounts) of HC-Asn-391 and HC-Asn-327 Fc sialic acid and Lys glycation (pertuzumab) Deamination at the following sites: Isomerization of LC-Asn-30 and HC-Asp-102 (Trastuzumab); IE-HPLC main peak (pertuzumab) Lys glycation (trastuzumab); N-terminal VHS and pyroglutamic acid salts (pertuzumab) on both heavy and light chains Pertuzumab in FDC Pharmaceutical MD formulation Lys glycation Fc sialic acid and Lys glycation Low-level deamination of HC-Asn-391 and HC-Asn-327 Fc sialic acid and Lys glycation IE-HPLC main peak N-terminal VHS and pyroglutamic acid on both heavy and light chains Trastuzumab in FDC Pharmaceutical MD formulation Deamination at the following sites: HC-Asn-55 and LC-Asn-30 Deamination at the following sites: HC-Asn-55 and LC-Asn-30 Deamination at the following sites: LC-Asn-30 Deamination at the following sites: Isomerization of LC-Asn-30 and HC-Asp-102 Lys glycation

[0459] Table 10 (continued): IE-HPLC peak characterization results of FDC drugs sample IE-HPLC peak 6 IE-HPLC peak 7 IE-HPLC peak 8 IE-HPLC peak 9 IE-HPLC peak 10 Pertuzumab - Trastuzumab FDC MD (Batch: GTK0002) Deamination at the following sites: HC-Asn-392 and HC-Asn-328 (trace), Fc sialic acid and Lys glycosylation (Trastuzumab); N-terminal VHS light chain and C-terminal lysine and proline amide at the heavy chain (pertuzumab) IE-HPLC main peak (trastuzumab) Heterogeneity at the following locations: HC-Asp-102 (trastuzumab) Increased Fc oxidation (trastuzumab) Heterogeneity at the following locations: HC-Asp-102 and the increased Fc oxidation (trastuzumab) Pertuzumab in FDC Pharmaceutical MD formulation N-terminal VHS light chain and C-terminal lysine and proline amide at the heavy chain NA NA NA NA Trastuzumab in FDC Pharmaceutical MD formulation Deamination at the following sites: HC-Asn-392 and HC-Asn-328 (trace), Fc sialic acid and Lys glycosylation IE-HPLC main peak (trastuzumab) Heterogeneity at the following locations: HC-Asp-102 (trastuzumab) Increased Fc oxidation (trastuzumab) Heterogeneity at the following locations: HC-Asp-102 and the increased Fc oxidation (trastuzumab)

[0460] The sum of peaks 1-3 contains acidic variants of pertuzumab (HC-Asn-391 deacetylated, FC sialic acid and lysine glycosylated) and trastuzumab (LC-Asn-30 deacetylated and HC-Asn-55).

[0461] Peak 4 contains the major charge variant of pertuzumab (i.e., the natural antibody) and a lower amount of acidic trastuzumab variant (LC-Asn-30 deamination and HC-Asp-102 isomerization).

[0462] The sum of peaks 5-6 contains pertuzumab basic variant (N-terminal VHS on the heavy and light chains and C-terminal lysine on the heavy chain) and trastuzumab acidic variant (HC-Asn-392 deamination, lysine glycosylation and increased Fc sialic acid content).

[0463] Peak 7 contains the major charge variant of trastuzumab (i.e., the natural antibody) and does not show overlap with the pertuzumab variant.

[0464] Peak 8 contains trastuzumab monoisomerized to isaspartic acid (at one heavy chain) with HC-Asp-102 and does not show overlap with the charge variant of pertuzumab.

[0465] The sum of peaks 9-10 contains a trastuzumab charge variant with increased FC oxidation (at HC-Met-255 and -431) and HC-Asp-102 isomerization, and does not show overlap with pertuzumab variants.

[0466] All abundant charge variants found in pertuzumab SC and trastuzumab SC drug substances were also detected in the FDC drug substance. No new charge variants were detected in the FDC drug substance material at release and after storage. All key charge variants of individual molecules in the FDC drug substance can be controlled.

[0467] No additional co-extraction or increase in existing peak co-extraction was observed or anticipated during stability testing. This was because the stress-induced charge variants of pertuzumab and trastuzumab shifted to earlier and later extraction times, respectively: the peak pattern of stressed pertuzumab shifted to the acidic region of the chromatography, while the peak pattern of stressed trastuzumab shifted to the basic region.

[0468] Example 15: FDC Components

[0469] The sum of peaks 1-3 of FDC drugs as measured by IE-HPLC

[0470] The sum of peaks 1-3 of FDC drugs consists of the following variants: Acidic variants of pertuzumab (HC Asn391 deacetylation, Fc sialic acid and lysine glycosylation). Deacetylation of Asn327 in pertuzumab, which was only observed in trace amounts in IE-HPLC peak characterization studies of FDC drugs. Acidic variants of trastuzumab (mainly LC Asn30 and HC Asn55 deacetylation). Compared with LC Asn30, HC Asn55 showed lower degradation susceptibility under biological and physiological conditions (Schmid I, Bonnington L, Gerl M et al., Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions. Commun Biol 2018;1:28).

[0471] The FDC (Factory Direct Distribution) drug shelf-life acceptance criteria are validated based on clinical experience and the anticipated impact on PK / biological activity and safety / immunogenicity. The proposed acceptance criteria are suitable for controlling product quality and cover the potential impact of drug substances and drug handling and storage.

[0472] For FDC drugs, the following end-of-life acceptance criteria are established: Sum of peaks 1-3: ≤ 23.0 area % (LD) / ≤ 21.0 area % (MD). These acceptance criteria are established based on clinical experience and hypothetical impacts on bioactivity / PK and safety / immunogenicity patterns. Extensions beyond current clinical experience are considered reasonable due to low impact on bioactivity and PK and no risk to immunogenicity / safety.

[0473] Safety and Immunogenicity Considerations: Since the acidic variants found in pertuzumab and trastuzumab materials are typically found in modifications of IgG antibodies, any increase in the content of acidic variants within the acceptance guidelines is not expected to represent a new form and is not expected to increase the risk of toxicity or the incidence of ADA. This is supported by the low incidence of ADA and favorable safety profiles observed in FDC drug clinical studies. During the pivotal study period, aged clinical study materials with a maximum area % LD and area % MD of 18.7% FDC drug were administered to patients. No new acidic variants were generated during storage or handling. Furthermore, it has been disclosed that degradation of solvent-accessible residues in trastuzumab occurs at conserved Fc sites (deamination of HC Asn387, Asn392, and Asn393) and CDR sites (primarily LC Asn30 deamination and HC Asp102 isomerization), and typically occurs in vivo (within days) significantly faster than biological processes and real-time storage conditions (Schmid et al., 2018). The degradation of the same Fc Asn deamination sites in endogenous human antibodies was significantly higher than observed in liquid formulations stored at 5°C. Therefore, it is inferred that such degradation does not increase the safety / immunogenic risk in patients (Liu YD, van Enk JZ, Flynn GC. Human antibody Fc deamidation in vivo. Biologicals 2009;37:313-22). This conclusion also applies to pertuzumab deamination, where only one deamination site was detected in this peak region and it is located in the Fc region (HC Asn391).

[0474] In summary, the potential increase of 4.3% area of ​​peaks 1-3 exceeding the patient exposure value is not expected to alter the immunogenicity and safety profile of the product.

[0475] Bioactivity Considerations: Based on the maximum clinical experience at 18.7 area % (LD) and 16.5 area % (MD) for the acid variants of pertuzumab and trastuzumab (sum of peaks 1-3), specification limits of 23.0 area % (LD) and 21.0 area % (MD) could reduce the binding activity of pertuzumab and trastuzumab by up to approximately 4% (based on the efficacy obtained by ELISA values ​​described in Table 8). A 4% change in bioactivity is not considered impactful. Therefore, if the sum of peaks 1-3 is present at the specification limits, efficacy is expected to be maintained.

[0476] PK Considerations: Antibody Fc is involved in clearance (Jefferis R. Antibody therapeutics: isotype and glycoform selection. Expert Opin Biol Ther 2007;7:1401-13.); therefore, deamination in the CDR is not expected to affect PK. Although it is known that charge properties affect the PK behavior of antibodies, a single negative charge introduced by deamination should not affect PK (Khawli et al., 2010). Notably, only a low degree of Fc deamination alteration was observed during FDC drug stability testing (IE-HPLC peak 3: pertuzumab HC Asn391; IE-HPLC peak 6: trastuzumab HC Asn392). Therefore, if the sum of peaks 1-3 exists within the specification limits, PK is not expected to be affected.

[0477] Peak 4 of FDC drug measured by IE-HPLC

[0478] Peak 4 of the FDC drug is part of the parameters reported by the IE-HPLC method and constitutes the expected major charge isotype of pertuzumab. Its inclusion in the specification ensures consistent product purity.

[0479] The acceptance criteria for drug substances and drug products release and stability testing are based on other reported measurement parameters of IE-HPLC, taking into account manufacturing experience and stability effects. The FDC drug acceptance criteria of ≥38 area % (LD) and ≥28 area % (MD) at the end of the shelf life ensure product purity and proper control of the manufacturing process.

[0480] The sum of peaks 5-6 of the FDC drug as measured by IE-HPLC

[0481] The sum of peaks 5-6 of FDC drugs is composed of the following variants: Basic variants of pertuzumab (N-terminal VHS, N-terminal pyroglutamic acid, and C-terminal lysine and proline acetamide on the heavy and light chains); Acidic variants of trastuzumab (HC Asn392 deamination, lysine glycosylation, and increased FC sialic acid content).

[0482] The sum of peaks 5-6 is not controlled in FDC drug release or stability tests because historical data have shown that the basic variants of pertuzumab and the acidic variants of trastuzumab remain unchanged during drug manufacturing and storage and therefore cannot be regarded as stability indicators.

[0483] Peak 7 of FDC drug measured by IE-HPLC

[0484] Peak 7 of the FDC drug is part of the output of the IE-HPLC method and constitutes the expected major charge isoform of trastuzumab. Its specification ensures consistent product purity. The acceptance criteria for drug release and stability testing are set based on other reported measurement parameters of IE-HPLC, taking into account manufacturing experience and stability effects. The FDC drug acceptance criteria of ≥ 16.0 area % (LD) and ≥ 23.0 area % (MD) at the end of the shelf life ensure proper control of product quality and manufacturing process.

[0485] Peak values ​​of FDC drugs measured by IE-HPLC

[0486] The peak 8 of FDC drug is composed of trastuzumab monoisomerized from HC Asp102 to isaspartic acid (at one heavy chain) and does not exhibit co-extraction with pertuzumab charge variant.

[0487] Peak 8 can be controlled in FDC drug release and stability tests.

[0488] Based on clinical experience and anticipated impacts on PK / biological activity and safety / immunogenicity, the FDC (Fulfilled Drug Distribution) end-of-life acceptance criteria for pharmaceutical products are validated at ≤ 9.0 area % (LD) / ≤ 12.0 area % (MD). The proposed acceptance criteria are suitable for controlling product quality and cover the potential impacts of pharmaceutical substances and pharmaceutical handling and storage.

[0489] Safety and Immunogenicity Considerations: Since the acidic variants found in trastuzumab material are typically found in modifications of IgG antibodies, any increase in the content of any acidic variant within the acceptance guidelines is unlikely to represent a new form and is unlikely to increase the risk of toxicity or the incidence of ADA. FDC drugs are generally safe and well-tolerated. The safety profile is comparable to that of IV pertuzumab + IV trastuzumab (P + H IV), with a lower incidence of ADA (≤ 5%) and no clinical consequences related to PK, efficacy, or safety.

[0490] During pivotal studies, aged clinical trial materials with up to 6.4% area of ​​the FDC drug's LD peak 8 and 9.4% area of ​​the FDC drug's MD peak 8 were administered to patients. No new charge variants were generated during storage or handling. Furthermore, for trastuzumab, it has been disclosed that degradation of solvent-accessible residues is located in conserved Fc sites (deamination of HC Asn387, Asn392, and Asn393) and CDR sites (primarily LC Asn30 deamination and HC Asp102 isomerization), and typically occurs in vivo (within days) significantly faster than biological processes and real-time storage conditions (Schmid et al., 2018). The degradation of the same Fc Asn deamination sites in endogenous human antibodies was significantly higher than observed in liquid formulations stored at 5°C. Therefore, it can be inferred that such degradation does not increase the safety / immunogenic risk for patients (Liu et al., 2009). The potential 2.5% area increase of peak 8 (HC Asp102 isomerization to isaspartic acid) exceeding the patient exposure value is not expected to change the immunogenicity of the product.

[0491] Bioactivity considerations: Compared to the reference standard, enriched peak 8 (92% peak purity, mainly containing the monoisomerization of HC Asp102 to isostearic acid at one heavy chain) exhibits similar activity to trastuzumab (100% binding activity). Therefore, if peak 8 is present within the specification limits, it is expected to maintain the efficacy of the FDC drug.

[0492] PK Considerations: In the CDRs of pertuzumab and trastuzumab, aspartate isomerization to isostaspartate does not change the charge and is not expected to affect PK. Therefore, single aspartate isomerization of trastuzumab HC Asp102 should not affect PK.

[0493] The sum of peaks 9-10 of FDC drugs as measured by IE-HPLC

[0494] The sum of peaks 9-10 of the FDC drug consists of trastuzumab monoisomerized to succinimide (at one heavy chain) and does not show overlap with the charge variant of pertuzumab. Additionally, a low degree of trastuzumab Fc oxidation was detected in these peaks. Due to the low level, it is not expected to have any impact. Since succinimide (the sum of peaks 9-10) is in equilibrium with peaks 8 (iso-Asp) and 7 (Asp), it is indirectly controlled via the acceptance criteria for peaks 8 and 7.

[0495] Therefore, the sum of peaks 9-10 in the control system does not need to be accepted by the criteria.

[0496] Example 16: Production of FDC Components

[0497] Transfer the pertuzumab SC drug substance from the drug substance storage container to a steam-sterilized stainless steel dispensing vessel. Multiple batches of pertuzumab SC drug substance can be combined for drug manufacturing.

[0498] The target amount of trastuzumab is defined based on the amount of pertuzumab added to the mixing vessel (depending on the mass, density, and pertuzumab content of the transferred pertuzumab SC drug substance). The trastuzumab SC drug substance is then added (based on density and trastuzumab content) to the mixing vessel. Multiple batches of trastuzumab SC drug substance can be combined for FDC drug manufacturing.

[0499] Based on the volume (depending on mass and density) of the pertuzumab SC and trastuzumab SC drug substances added to the mixing vessel, add the required amount of thawed rHuPH20 to the mixing vessel (based on the rHuPH20 solution content and activity). Multiple rHuPH20 batches can be combined for drug manufacturing.

[0500] After all components have been transferred to the mixing vessel, the solution is then homogenized by mixing.

[0501] Example 17: Development of RP-UPHLC for determining FDC content

[0502] Equipment

[0503] Equivalent instruments and appropriate operating conditions may be used. HPLC System: HPLC system equipped with data acquisition software (with an online vacuum degasser) Detector: UV / visible absorbance detector or photodiode array detector Membrane Filter: 0.2 μm filter (e.g., Corning catalog number: 430049) Column: TSK-Gel G3000SWXL, 7.8 × 300 mm, 5 μm (Tosoh Bioscience, catalog number: 08541); or BioSuite 250, 7.8 × 300 mm, 5 μm (Waters, catalog number: 186002165)

[0504] Reagents: Purified water (Milli-Q treated water), Trifluoroacetic acid (TFA) (Fluka, Cat. Nr. 40967), Acetonitrile (Merck, Cat. Nr. 1.00030.2500), Anhydrous L-histidine (Sigma, Cat. Nr. H8000), Sucrose (Merck, Cat. Nr. 1.07687), L-methionine (Sigma, Cat. Nr. 64319), Glacial acetic acid (Merck, Cat. Nr. 1.00063.1000), Polysorbate 20 (Sigma, Cat. Nr. 93773). Solvent A: 0.1% TFA in Milli-Q water. Solvent B: 0.1% TFA in acetonitrile. Reagent buffer: 20 mM acetate-histidine, 240 mM... Sucrose, 10 mM methionine, and polysorbate 20, 0.02% [w / v], pH 5.7 ± 0.2. Dilution buffer: 20 mM histidine acetate, pH 5.5. Column storage solution: 60% acetonitrile (v / v). Sample solution: Dilute the sample to approximately 10 mg / mL using formulation buffer. Dilute the 10 mg / mL test sample solution to approximately 1 mg / mL using dilution buffer. Blank: Inject formulation buffer and dilution buffer undiluted. Flow rate: 0.4 mL / min. Maximum pressure: 400 bar / 6000 psi. Wavelength: 280 nm. Run time: 29 min. Column temperature setting: 60°C. Autosampler temperature setting: ≤ 10°C. Injection volume: Sample and reference standard: 25 μg protein (nominal). Blank and mobile phase: Injection volume is the same as the reference standard.

[0505] Gradient Time (minutes) Solvent A (%) Solvent B (%) 0.0 64 36 2.0 64 36 20.0 40 60 20.5 5 95 21.0 5 95 22.0 64 36 29.0 64 36

[0506] Although this method effectively separates the peaks of pertuzumab and trastuzumab, the main remaining problems of the above method become more apparent. Trace amounts of Herceptin / pertuzumab were still detected after injecting five blank samples (formulation buffer). Therefore, further method development is needed. Different chromatography techniques were tested, and reversed-phase chromatography (RPC) was selected as the most suitable method for protein content analysis. Several parameters were evaluated for method accuracy and repeatability.

[0507] The Influence of String Type on Separation

[0508] Different types of columns for pertuzumab / trastuzumab FDC testing.

[0509] Table 11: Columns tested for RP-UHPLC protein content method and their respective test temperatures column Column temperature [°C] Resolution BEH300 C4 70 -- 80 + 90 ++ Agilent AdvanceBio RP mAb Diphenyl 70 -- 80 + 90 ++ Zorbax 300SB C3 70 + 80 ++ Poroshell 300 SB-C8 70 -- 80 - 90 - Hamilton PRP 70 / 80 / 90 --- Baker Wide Pore C4 70 / 80 / 90 -- Baker Wide Pore C18 70 / 80 --- Grace Aquapore RP-300 70 / 80 / 90 - Agilent PLRP-S 300 80 / 90 + Agilent Zorbax RRHD 300-Diphenyl 70 +++ 80 ++ 90 ++

[0510] Several potential columns for pertuzumab / trastuzumab FDC were identified. For example, BEH300C4 showed good separation but required a high column temperature (90°C). Agilent AdvanceBio RP mAb and Agilent Zorbax RRHD 300-Diphenyl showed similar separation, but with lower overall resolution. The optimal column was determined to be the Agilent Zorbax RRHD 300-Diphenyl (2.1 × 100 mm) column, which showed low residue and improved separation of the two antibodies compared to the initial method.

[0511] DoE (Experimental Design) of Agilent Zorbax RRHD 300-Diphenyl Column

[0512] The mobile phase, flow rate, gradient, and column chamber temperature were tested on an Agilent Zorbax RRHD 300-Diphenyl (2.1 × 100 mm) column. The DoE for developing reversed-phase protein content methods was set using MODDE®. A summary of the factors tested within the DoE range is listed in Table 12.

[0513] Table 12: Factors used for DoE screening as determined by the results. name abbreviation unit type set up accuracy flow flow mL / min Quantitative 0.6-0.8 mL / min 0.005 mL / min temperature temperature ℃ Quantitative 70-90℃ 0.5℃ Duration Keep Min Quantitative 0-3min 0.05min gradient Grad Min Quantitative 10-20 min 0.3min start start %B Quantitative 20-30%B 0.05%B

[0514] Evaluation of "Trastuzumab / Pertuzumab split":

[0515] In summary, the resolution of reversed-phase chromatography methods for protein determination is strongly influenced by flow rate and gradient length. Lower flow rates and longer gradient lengths improve resolution. Column chamber temperature and starting conditions have a weak but insignificant effect on the method. A temperature of 70°C and a relatively high starting condition of 30% B have been shown to produce optimal results. Increasing the holding time has no effect on resolution.

[0516] Evaluation of the "sum of secondary forms":

[0517] The total effect of secondary concentrations depends heavily on the initial concentration (high) and the column temperature (low). Flow rate and gradient time have only a minor effect. Hold time alone is insignificant, but it exhibits a certain effect when combined with flow rate and column temperature.

[0518] Evaluation of "Trastuzumab height ratio":

[0519] To achieve a higher height-to-weight ratio (i.e., no additional shoulder on the trastuzumab peak), the temperature must be lowered. Flow rate analysis is not definitive. Ideally, gradient time and initiation conditions should be in the higher range. Similarly, additional hold time does not demonstrate an effect.

[0520] Evaluation of "Pertuzumab USP tail":

[0521] To reduce the tailing of the pertuzumab main peak, the flow rate should be increased and the gradient and initiation conditions reduced. Similarly, additional hold time does not show an effect.

[0522] Based on the DoE results, select the following parameters: Flow rate: 0.8 mL / min; Wavelength: 280 nm; Column temperature: 70°C; Automatic sampler temperature: 10°C; Run time: 20 min

[0523] Table 13: DoE Gradient Time [min] %B 0 30 20 50 25 90 26 30 30 30

[0524] Based on these results, the column temperature, gradient and flow rate were further optimized.

[0525] The effect of column temperature on separation

[0526] High temperatures in reversed-phase chromatography can significantly affect peak separation, tailing effect, and system pressure. Three different temperatures were selected for testing on an Agilent Zorbax RRHD 300 Diphenyl column. Temperature tests were performed within the DoE range (results not shown). In summary, as the column chamber temperature increased, the residence time shifted towards earlier extraction. This was expected, as the extractant viscosity and secondary column interaction decreased with increasing temperature. However, the overall resolution decreased with increasing column chamber temperature. Therefore, the optimal column chamber temperature within the experimental range was 70°C.

[0527] The Influence of Gradient Pattern on Separation

[0528] Gradients have a significant impact on analyte separation. For protein determination by reversed-phase chromatography, four major gradients were tested on an Agilent Zorbax RRHD 300 Diphenyl column (see Table 14). To directly compare gradients, the column chamber temperature was always set at 70°C and the flow rate at 0.6 mL / min. The gradient must be re-evaluated after the final flow rate has been set. The final gradient for the RP protein determination method is listed in gradient 5 of Table 14. The initial DoE gradient (Table 13) was changed to achieve optimal separation and equilibration time using a new flow rate (0.3 mL / min).

[0529] Table 14: Patterns of the 5 selected gradients. Gradients 1-4 were selected using a flow rate of 0.6 mL / min, while gradient 5 was selected using half the flow rate (0.3 mL / min). Gradient 1 Gradient 2 Gradient 3 Gradient 4 Gradient 5 Time [min] %B Time [min] %B Time [min] %B Time [min] %B Time [min] %B 0 30 0 20 0 20 0 30 0 30 10 50 3 20 20 50 3 30 15 45 15 90 13 50 25 90 twenty three 50 20 90 16 30 18 90 26 20 28 90 twenty one 30 twenty one 30 19 20 31 20 29 30 30 30 twenty four 20 34 30

[0530] Observation:

[0531] All five test gradients demonstrated adequate protein retention and optimized starting conditions (between 20-30% B). Any starting condition within this range is suitable for pertuzumab / trastuzumab FDC separation. However, to shorten the gradient and run time, a 30% starting condition was chosen. Considering the gradient time, the tested range (10-20 min) was optimized. Since the gradient time is also significantly affected by the flow rate, a separation time of 15 min at a flow rate of 0.3 mL / min was ultimately selected.

[0532] Using a 10-minute separation time, especially with a 30% B gradient steepness, both antibodies were flushed within a window of only 1-2 minutes. However, a 20-minute separation and a 20% B gradient steepness produced a wider flush pattern and a lower intensity detector signal.

[0533] The final 15 min separation time was combined with a 15% B gradient steepness. Together with a slow flow rate (0.3 mL / min), it demonstrated good baseline separation of the two antibodies without excessive loss of signal intensity.

[0534] The effect of flow rate on separation

[0535] Finally, the optimal flow rate must be determined. A faster flow rate generally means earlier extraction, but may result in a loss of resolution. Initial experiments were performed using flow rates of 0.6 or 0.8 mL / min. It was subsequently found that lower flow rates were more beneficial for this particular RP protein content method. Four different flow rates (0.3 mL / min to 0.6 mL / min) were tested on samples containing a single mAb using an Agilent Zorbax RRHD 300 Diphenyl column. For direct comparison, the column chamber temperature was always set at 70°C and all separations were performed using the smallest gradients listed in Table 13.

[0536] Lowering the flow rate produces a narrower peak shape and higher signal intensity. Residence time is shifted later. Split (especially for side peaks) improves as the flow rate decreases; therefore, a flow rate of 0.3 mL / min is ideal for this method. Setting the gradient run time to 30 min and achieving sufficient column rebalancing at 0.3 mL / min demonstrates this approach.

[0537] Based on these experiments, the most critical parameters for this method are column type, column temperature, and flow rate. Using a phenyl-based column improves resolution without leaving any residue. Temperatures of 64℃-76℃ and 66℃-74℃ were tested and found to have no significant impact on method performance. Within the robustness testing range of the Phase III and BLA / MAA method validation, flow rates of 0.4 mL / min and 0.2 mL / min were tested and found to have no significant impact on method performance.

[0538] Example 18: RP-UPHLC determination of the content in FDC

[0539] Note: Equivalent instruments, appropriate operating conditions, and solvents, chemicals and reagents of equivalent quality may be used.

[0540] The contents of pertuzumab and trastuzumab in FDC drugs were determined by UV detection using RP-UHPLC. Pertuzumab and trastuzumab were separated based on their differences in hydrophobicity. The contents of pertuzumab and trastuzumab were calculated from an external calibration curve generated by injecting different volumes of FDC reference standards into each analytical sequence. The same method was applied to the LD and MD of FDC drugs. Measurements were taken for each dosage form against the corresponding reference standards.

[0541] Equipment and Materials: UHPLC system equipped with a UV detector (Thermo Ultimate 3000 RS or equivalent system); UHPLC column (Agilent Zorbax RRHD 300-Diphenyl, 2.1 mm × 100 mm, particle size: 1.8 µm or equivalent particle size).

[0542] Reagents: 2-propanol acetonitrile (TFA), anhydrous L-histidine, L-histidine monohydrochloride monohydrate, sucrose, trehalose, L-methionine, polysorbate 20, sodium hydroxide, hydrochloric acid, purified water (e.g., MilliQ).

[0543] Solution

[0544] Drug Product Dilution Buffer

[0545] 20 mM L-histidine / L-histidine monohydrochloride, 105 mM trehalose, 100 mM sucrose, 10 mM methionine, 0.04% (w / v) polysorbate 20, pH 5.5 ±0.2

[0546] Moving Phase A

[0547] 2% (v / v) 2-propanol, 0.1% (v / v) TFA / water

[0548] Moving Phase B

[0549] 70% (v / v) 2-propanol, 20% (v / v) acetonitrile, 10% (v / v) mobile phase A

[0550] Preparation of Reference Standard Solution

[0551] Note: To measure the LD and MD of FDC drug samples, separate FDC LD and FDC MD reference standards must be prepared. Two copies of each reference solution (Reference A and Reference B) must be prepared. Dilute each reference standard to a total protein concentration of 1 mg / mL using drug dilution buffer.

[0552] Preparation of Sample Solution

[0553] Dilute the FDC drug with drug dilution buffer to prepare a sample solution containing a total protein concentration of 1 mg / mL.

[0554] Program

[0555] Before injecting the first sample, flush the column with 70% mobile phase A / 30% mobile phase B until a stable baseline is obtained. If necessary, inject a reference solution for column conditioning purposes until a visual assessment of the chromatography indicates that at least two consecutive injections have a continuous pattern.

[0556] Operating Parameters: Detection Wavelength: 280 nm; Injection Volume: See Injection Protocol below; Flow Rate: 0.3 mL / min; Column Temperature: 70℃ ±2℃; Automatic Sampler Temperature: 10°C ±4°C; Run Time: 30 min

[0557] Gradient

[0558] Table 15: Binary Gradient Time (min) Shifting phase A (%) Shifting phase B (%) 0 70 30 15 55 45 20 10 90 twenty one 70 30 30 70 30

[0559] Injection Plan

[0560] For each dosage form, a separate sequence must be performed using the corresponding reference standard. Inject the samples in the order shown in Table 16.

[0561] Table 16: Injection Scheme Sample type illustrate Injection volume (μL) blank sample Drug dilution buffer 10 Standard curve Reference solution A 6 Reference solution A 8 Reference solution A 10 Reference solution A 12 Reference solution A 14 Test control Reference solution B 10 sample Sample solutions (1 to n) 10 Test control Reference solution B 10 blank sample Drug dilution buffer 10 Note: For more than 10 samples, place every 10 sample injection solutions together with the control solution (see B).

[0562] Result

[0563] Typical chromatography patterns are shown in Figure 15 (FDC drug LD) and Figure 16 (FDC drug MD).

[0564] The final method (Example 18) substantially improves the initial protein content method, including improved overall separation / peak separation and elimination of sample residue, i.e., residue does not exceed 0.2% in subsequent analyses. Furthermore, the final method allows for quantitative protein content determination of pertuzumab and trastuzumab at maintenance and loading doses. Different phenyl-based RP columns demonstrate improved specificity for both antibodies, detecting only small amounts of sample residue and allowing for accurate protein content determination. The final reversed-phase U-HPLC method for determining protein content in pertuzumab / trastuzumab FDC separates the two molecules at 70°C on a phenyl-based reversed-phase column (Agilent Zorbax RRHD 300-Diphenyl) using a water-2-propanol / acetonitrile gradient and 0.1% TFA.

[0565] Figure 15 shows an example RP-UHPLC chromatogram used to analyze the protein content of FDC LD reference standard, and Figure 16 shows an example RP-UHPLC chromatogram used to analyze the protein content of FDC MD reference standard.

[0566] Data Analysis

[0567] Integrate the pertuzumab and trastuzumab peaks in the chromatograms of reference solutions A and B and the sample solution. Define the integration using representative chromatograms in Figure 15 (FDC drug LD) and Figure 16 (FDC drug MD).

[0568] A standard curve for each antibody was generated by plotting the peak area against the injection dose (µg) for each standard grade. The standard curve data were fitted using linear regression. The curve was not forced to cross zero.

[0569] The amounts of pertuzumab and trastuzumab were calculated using the standard curve equation and the individual peak areas of each sample solution and reference B injection.

[0570] Slope calibration curve

[0571] To calculate the content of pertuzumab and trastuzumab, divide the amount by the respective injection volume and multiply by the dilution factor.

[0572] Example 19: HI-HPLC for determining the content in FDC

[0573] Evaluation of hydrophobic interaction chromatography (HI-HPLC). HI-HPLC is a commonly used method for analyzing antibodies, especially for identifying their molecular variants (e.g., post-translational modifications or antibody-drug conjugates). Additionally, it can identify misfolded proteins or conformational changes because HI-HPLC is a non-denaturing chromatography method.

[0574] The main differences between HI-HPLC and RP-UHPLC are as follows: HI chromatography is non-destructive and proteins retain their folds. Due to the natural folding of proteins, protein-column interactions originate only from amino acids located on the protein surface. Escape cannot be promoted by increasing the concentration of organic solvents, but it can be promoted by reducing, for example, the amount of ammonium sulfate to weaken the hydrophobic-hydrophobic interactions between the protein and the stationary phase. Therefore, substances with lower hydrophobicity are escaped earlier.

[0575] Two types of columns were tested for use in HIC-HPLC: - TSKgel Ether column, 75 mm × 7.5 mm, 10 μm particle size - TSKgel Butyl column, 35 mm × 4.6 mm, 2.5 μm particle size

[0576] Mobile Phase Test: - Extraction Agent A: 50 mM sodium phosphate, pH 7.0 ± 0.05, 5% (v / v) ethanol - Extraction Agent B: 50 mM sodium phosphate, 2 M ammonium sulfate, pH 7.0 ± 0.05

[0577] Result:

[0578] HI-HPLC can separate pertuzumab / trastuzumab FDC molecules using any column type. For co-blended samples, Butyl columns offer significantly better resolution than Ether columns (data not shown). In comparison of RP-UHPLC and HI-HPLC (especially for protein content analysis), RPUHPLC is superior to HI-HPLC. HI chromatography can separate both antibodies, but lacks overall resolution and exhibits a noticeable tailing effect.

[0579] Reversed-phase chromatography (RPC) improves the resolution of pertuzumab and trastuzumab compared to high-performance liquid chromatography (HI-HPLC). Specifically, the resolution of the shoulder peaks of pertuzumab and trastuzumab on RPC is superior to that on HI. Furthermore, the horizontal baseline generated in RPC is superior to the sloping baseline in HI. Additionally, the higher-low salt gradient makes it easier to use a water-organic solvent gradient on the HPLC system.

[0580] Table 18: Operating conditions and HIC gradient for HI-HPLC assays Flow rate 0.5 mL / min Maximum pressure 20 bar / 290 psl wavelength 214min Runtime 55min Column temperature 25℃ ±3℃ Sample temperature 5℃ ±3℃ Sample concentration na Injection volume 10μL gradient minute % Extraction Agent B 0 70 5 70 35 0 40 0 45 70 55 70

[0581] Although certain embodiments of the invention have been shown and described herein, those skilled in the art will understand that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will be conceived by those skilled in the art without departing from the invention. It should be understood that various alternative forms of the embodiments of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and are intended to cover methods and structures within the scope of the claims and their equivalents. [Simplified Explanation of the Diagram]

[0063] Figure 1 provides a schematic diagram of the HER2 protein structure and the amino acid sequences of its extracellular domains I-IV (SEQ ID Nos. 1-4, respectively). Figures 2A and 2B illustrate the alignment of the following amino acid sequences: the variable light chain (VL) (Figure 2A) and variable heavy chain (VH) (Figure 2B) domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6, respectively); the VL and VH domains of variant 574 / pertuzumab (SEQ ID Nos. 7 and 8, respectively); and the human VL and VH common framework (hum κ1, light chain κ subgroup I; humIII, heavy chain subgroup III) (SEQ ID Nos. 9 and 10, respectively). An asterisk indicates the difference between the variable domains of pertuzumab and murine monoclonal antibody 2C4 or between the variable domains of pertuzumab and the human framework. Complementarity-determining regions (CDRs) are enclosed in square brackets. Figures 3A and 3B show the amino acid sequences of the pertuzumab light chain (Figure 3A; SEQ ID NO. 11) and heavy chain (Figure 3B; SEQ ID NO. 12). CDRs are shown in bold. The calculated molecular weights of the light and heavy chains are 23,526.22 Da and 49,216.56 Da (cysteine ​​in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain. Figures 4A and 4B show the amino acid sequences of the trastuzumab light chain (Figure 4A; SEQ ID NO. 13) and heavy chain (Figure 4B; SEQ ID NO. 14), respectively. The boundaries of the variable light and variable heavy chain domains are indicated by arrows. Figures 5A and 5B show the sequences of the variant pertuzumab light chain (Figure 5A; SEQ ID NO. 15) and the variant pertuzumab heavy chain (Figure 5B; SEQ ID NO. 16), respectively. Figure 6 illustrates the extracellular domain of HER2 and the capture reagents used in the ELISA assays described herein. P-HER2 variant: Modified HER2 ECD used to analyze pertuzumab potency. T-HER2 variant: Modified HER2 ECD used to analyze trastuzumab potency. Figures 7A and 7B illustrate the selectivity sensitivity of cell-based assays. Figure 7A: Pertuzumab antiproliferative assay using MDA-MB-175 VII cells. Figure 7B: Trastuzumab antiproliferative assay using BT-474 cells. Figures 8A and 8B illustrate the complementary mechanism of pertuzumab and trastuzumab in cell-based antiproliferative assays. Figure 8A: Pertuzumab antiproliferative assay: The dose-response curve shifts to lower concentrations after adding trastuzumab at a 1:1 ratio.Figure 8B: Trastuzumab antiproliferative assay: After adding pertuzumab at a 1:1 ratio, the dose-response curve shifted slightly towards lower concentrations. Figures 9A and 9B illustrate the masking effect of cell-based antiproliferative assays. Figure 9A: Pertuzumab antiproliferative assay: The affinity of the pertuzumab mutant (HC S55A) for HER2 was significantly reduced (solid symbol); the loss of affinity of the pertuzumab mutant was masked after the addition of trastuzumab (hollow symbol). Figure 9B: Trastuzumab antiproliferative assay: The affinity of the trastuzumab mutant (LC H91A) for HER2 was significantly reduced (solid symbol); the loss of affinity of the trastuzumab mutant was masked after the addition of pertuzumab (hollow symbol). Figure 10 shows a representative dose-response curve for pertuzumab ELISA. Figure 11 shows a representative dose-response curve for trastuzumab ELISA. Figure 12 shows a representative chromatogram of the provided IEC method for analyzing the charge variant of pertuzumab-trastuzumab FDC. Figure 13 shows the IE-HPLC chromatograms of pertuzumab-trastuzumab FDC, pertuzumab, and trastuzumab. Figures 14A and 14B show the HER2 affinity mutant in ELISA. Figure 14A: Pertuzumab ELISA: The binding activity of the pertuzumab mutant (HC S55A) to HER2 (hollow symbol) is much smaller than that of pertuzumab (solid symbol). Figure 14B: Trastuzumab ELISA: The affinity of the trastuzumab mutant (LC H91A) to HER2 (hollow symbol) is much smaller than that of trastuzumab (solid symbol). Figure 15 shows an example RP-UHPLC chromatogram used to analyze the protein content of the FDC LD reference standard. Figure 16 shows an example RP-UHPLC chromatogram used to analyze the protein content of FDC MD reference standards. [Sequence List]

[0582] <![CDATA[ <110> Genentech, Inc. (USA) and F. Hoffmann-La Roche AG (Switzerland) <![CDATA[ <120> For determination of fixed-dose combinations]]> <![CDATA[ <130> P36263-WO]]> <![CDATA[ <160> 35 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 195]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 1]]> Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp IlePhe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg 195 <![CDATA[<210> 2]]> <![CDATA[<211> 124]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 2]]> Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40 45 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 50 55 60 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 65 70 75 80 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 85 90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val 115 120 <![CDATA[<210> 3]]> <![CDATA[<211> 169]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]><![CDATA[<400> 3]]> Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr 1 5 10 15 Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 20 25 30 Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 35 40 45 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu 50 55 60 Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp 65 70 75 80 Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His 85 90 95 Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu 100 105 110 Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His 115 120 125 His Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 130 135 140 Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu 145 150 155 160 Asp Glu Cys Val Gly Glu Gly Leu Ala 165 <![CDATA[<210> 4]]> <![CDATA[<211> 142]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 4]]> Cys His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr 1 5 10 15 Gln Cys ValAsn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 20 25 30 Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 35 40 45 His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val 50 55 60 Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr 65 70 75 80 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro 85 90 95 Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala 100 105 110 Cys Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp 115 120 125 Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr 130 135 140 <![CDATA[<210> 5]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 5]]> Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr AspPhe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 6]]> <![CDATA[ <211> 119]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Little house mouse]]> <![CDATA[ <400> 6]]> Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <![CDATA[ <210> 7]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide"<![CDATA[<400> 7]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 8]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<221> Source]]> <![CDATA[<223> Note= "Artificial Sequence Description: Synthetic polypeptide"]]> <![CDATA[<400> 8]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly ArgPhe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[ <210> 9]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 9]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[ <210> 10]]> <![CDATA[ <211> 119]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213>Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 10]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[ <210> 11]]> <![CDATA[ <211> 214]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 11]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 12]]> <![CDATA[<211> 448]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<221> Source]]> <![CDATA[<223> Note= "Artificial Sequence Description: Synthetic polypeptide"]]><![CDATA[<400> 12]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <![CDATA[ <210> 13]]> <![CDATA[ <211> 214]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 13]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 14]]> <![CDATA[<211> 449]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]><![CDATA[<221> Source]]> <![CDATA[<223> Note= "Artificial Sequence Description: Synthetic polypeptide"]]> <![CDATA[<400> 14]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe TyrAla Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <![CDATA[ <210> 15]]> <![CDATA[ <211> 217]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 15]]> Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 15 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val 20 25 30 Ser Ile Gly ValAla Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile 85 90 95 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 16]]> <![CDATA[<211> 449]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<221> Source]]> <![CDATA[<223>Note = "Artificial Sequence Description: Synthetic Polypeptide"]]> <![CDATA[<400> 16]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <![CDATA[ <210> 17]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <220> ]]> <![CDATA[ <221> Mutants <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Replace = "Ser"]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Note: "The residues given in the sequence have no preference for the residues in the annotation at that position." <![CDATA[ <400> 17]]> Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp 1 5 10 <![CDATA[ <210> 18]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 18]]> Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5 10 15 Gly <![CDATA[ <210> 19]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]><![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 19]]> Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10 <![CDATA[ <210> 20]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 20]]> Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10 <![CDATA[ <210> 21]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <220> ]]> <![CDATA[ <221> Mutants <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> Replace = "Leu"]]> <![CDATA[ <220> ]]> <![CDATA[ <221> Mutants <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Replace = "Glu"]]> <![CDATA[ <220> ]]> <![CDATA[ <221> Mutants <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> Replace = "Ser"]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(7)]]> <![CDATA[ <223> Note: "The residues given in the sequence have no preference for the residues in the annotation at that position." <![CDATA[ <400> 21]]> Ser Ala Ser Tyr Arg Tyr Thr 1 5 <![CDATA[ <210> twenty two]] ><![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> Note = "Artificial Sequence Description: Synthetic Peptide" <![CDATA[ <400> 22]]> Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5 <![CDATA[ <210> 23]]> <![CDATA[ <211> 119]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 23]]> Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40 45 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 50 55 60 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 65 70 75 80 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 85 90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys 115 <![CDATA[ <210> 24]]> <![CDATA[ <211> 483]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 24]]> Thr Gln Val Cys Thr Gly Thr Asp MetLys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly CysThr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285 Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser TrpLeu Gly 420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val <![CDATA[<210> 25]]> <![CDATA[<211> 725]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Capture Reagent for Anti-HER2 Antibody]]> <![CDATA[<400> 25]]> Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu IleLeu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285 Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala ValThr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val Arg Arg Ala Gln Val Thr Asp Lys Lys Ile Glu Pro Arg 485 490 495 Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn 500 505 510 Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp 515 520 525 Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val ValAsp 530 535 540 Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn 545 550 555 560 Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn 565 570 575 Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp 580 585 590 Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro 595 600 605 Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala 610 615 620 Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys 625 630 635 640 Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile 645 650 655 Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn 660 665 670 Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys 675 680 685 Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys 690 695 700 Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe 705 710 715 720 Ser Arg Thr Pro Gly 725 <![CDATA[<210> 26]]> <![CDATA[<211> 756]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]><![CDATA[<223> Capture reagent for anti-HER2 antibody]]> <![CDATA[<400> 26]]> Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Leu Glu Ser Thr 20 25 30 Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro 35 40 45 Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val 50 55 60 Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser Leu 65 70 75 80 Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile Ala 85 90 95 His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg 100 10Cys Ser Pro Met Cys 195 200 205 Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser Leu 210 215 220 Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu 225 230 235 240 Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro 245 250 255 Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile 260 265 270 Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe 275 280 285 Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys 290 295 300 Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys 305 310 315 320 Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly 325 330 335 Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys Tyr 340 345 350 Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala 355 360 365 Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu Ala 370 375 380 Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro 385 390 395 400 Leu Gln Pro Glu Gln Leu Gln Val Phe Glu ThrLeu Glu Glu Ile Thr 405 410 415 Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu Ser 420 425 430 Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn Gly 435 440 445 Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly Leu 450 455 460 Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His Asn 465 470 475 480 Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe Arg 485 490 495 Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu 500 505 510 Cys Val Arg Arg Ala Gln Val Thr Asp Lys Lys Ile Glu Pro Arg Gly 515 520 525 Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu 530 535 540 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val 545 550 555 560 Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val 565 570 575 Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 580 585 590 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser 595 600 605 Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln HisGln Asp Trp Met 610 615 620 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala 625 630 635 640 Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro 645 650 655 Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln 660 665 670 Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr 675 680 685 Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 690 695 700 Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu 705 710 715 720 Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser 725 7Met Lys Leu Arg Leu Pro Ala Ser Pro 35 40 45 Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val 50 55 60 Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser Leu 65 70 75 80 Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile Ala 85 90 95 His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg 100 105 110 Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp Asn 115 120 125 Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro Gly 130 135 140 Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys Gly 145 150 155 160 Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile 165 170 175 Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu 180 185 190 Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met Cys 195 200 205 Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser Leu 210 215 220 Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu 225 230 235 240 Pro Thr Asp Cys Cys His Glu Gln Cys AlaAla Gly Cys Thr Gly Pro 245 250 255 Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile 260 265 270 Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe 275 280 285 Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys 290 295 300 Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys 305 310 315 320 Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly 325 330 335 Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys Tyr 340 345 350 Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala 355 360 365 Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu Ala 370 375 380 Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro 385 390 395 400 Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile Thr 405 410 415 Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu Ser 420 425 430 Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn Gly 435 440 445 Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly IleSer Trp Leu Gly Leu 450 455 460 Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His Asn 465 470 475 480 Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe Arg 485 490 495 Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu 500 505 510 Cys Val Arg Arg Ala Gln Val Thr Asp Lys Lys Ile Glu Pro Arg Gly 515 520 525 Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu 530 535 540 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val 545 550 555 560 Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val 565 570 575 Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 580 585 590 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser 595 600 605 Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met 610 615 620 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala 625 630 635 640 Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro 645 650 655 Gln Val Tyr Val Leu Pro Pro Pro Glu Glu GluMet Thr Lys Lys Gln 660 665 670 Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr 675 680 685 Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 690 695 700 Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu 705 710 715 720 Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser 725 730 735 Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser 740 745 750 Arg Thr Pro Gly Lys 755 <![CDATA[<210> 28]]> <![CDATA[<211> 136]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 28]]> Cys His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr 1 5 10 15 Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 20 25 30 Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 35 40 45 His Cys Leu Pro Cys His Pro Glu C...

Claims

1. A method for analyzing a fixed-dose combination (FDC) of antibodies binding to HER2 extracellular subdomain II and antibodies binding to HER2 extracellular subdomain IV, comprising: a. contacting the FDC with a capture reagent comprising a modified HER2 ECD subdomain containing the amino acid sequence shown in SEQ ID NO: 24 to quantify the binding of the antibody binding to HER2 extracellular subdomain II; b. contacting the FDC with a capture reagent comprising a modified HER2 ECD subdomain containing the amino acid sequence shown in SEQ ID NO: 29 to quantify the binding of the antibody binding to HER2 extracellular subdomain IV; c. contacting the samples obtained from steps a. and b. with detectable antibodies, respectively; and d. quantifying the amount of antibody bound to the capture reagent using a detection method for the detectable antibody.

2. The method of claim 1, which is used to analyze the potency of one of the anti-HER2 antibodies.

3. The method of claim 2, wherein efficacy is quantified by correlating the amount of antibody bound to the capture reagent with the bioactivity of the isolated antibody as measured in a cell-based assay.

4. The method of any one of claims 1 to 3, wherein the capture reagent is coated on a microplate.

5. The method of any one of claims 1 to 3, wherein the detectable antibody targets the F(ab')2 portion of the anti-HER2 antibody.

6. The method of any one of claims 1 to 3, wherein the fixed dose combination further comprises hyaluronidase.

7. The method of any one of claims 1 to 3, wherein the antibody binding to HER2 extracellular subdomain II is pertuzumab and the antibody binding to HER2 extracellular subdomain IV is trastuzumab.

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