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 the determination of the quality and quantity properties of fixed-dose combinations. In particular, this invention relates to the determination of fixed-dose combinations of two anti-HER2 antibodies, and to the determination of subcutaneous formulations comprising pertuzumab and trastuzumab. Background Technology

[0002] To ensure the safety and efficacy of biopharmaceutical products, continuous monitoring of product quality is essential. Certain specific standards, 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] Potency testing, along with numerous other tests, is 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, potency measurements are used throughout all phases of clinical studies and after market approval to confirm that only product batches meeting specified 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 inhomogeneities. IEX is typically a release method where specifications are specifically 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, because it does not involve the addition of denaturing agents, it is one of the few methods that can characterize proteins in their native conformation. IEX is also used as an identification method for certain biologics and is a routine test for stability and shelf-life validation.

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

[0006] Fixed-dose combination (FDC) formulations combine 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 co-formulation of two highly similar monoclonal antibodies. The mechanisms of action of pertuzumab and trastuzumab are believed to be 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 HER signaling pathway. The standard IV regimen (perjeta-based) of perjeta in combination with 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 perjeta-based regimen has shown almost twice the pCR rate of Herceptin and chemotherapy. Furthermore, this combination has been shown to significantly reduce the risk of recurrence, invasive 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 facilitate the dispersion and absorption of 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 co-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:

[0010] a. Contact the FDC with a capture reagent containing the modified HER2 ECD subdomain;

[0011] b. Contact the sample with the detectable antibody;

[0012] c. Use a detection method that can detect antibodies to quantify the amount of antibody bound to the capture reagent.

[0013] 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.

[0014] 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 quantified.

[0015] 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.

[0016] In one embodiment, a binding assay is provided for a fixed-dose combination (FDC) of two anti-HER2 antibodies, wherein binding to the antibody bound to HER2 subdomain II is quantified. In one embodiment, the capture reagent comprises recombinant HER2 extracellular domain IV.

[0017] 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.

[0018] 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 such anti-HER2 antibodies. In one embodiment, bioactivity is quantified by correlating the level of antibody bound to a capture reagent with the bioactivity of the isolated antibody measured in a cell-based assay.

[0019] In one embodiment, a capture reagent is coated onto a microtiter plate. In another embodiment, the antibody targets the F(ab')2 moiety of an anti-HER2 antibody.

[0020] In one embodiment, the fixed-dose combination to be analyzed in the binding assay further comprises hyaluronidase.

[0021] In one embodiment, an isolated protein comprising SEQ ID NO:24 is provided. In another embodiment, an isolated protein comprising SEQ ID NO:29 is provided.

[0022] Further provided is a kit for specifically quantifying the binding of antibodies to HER2 extracellular subdomain II in a fixed-dose combination (FDC), the FDC having a first antibody and a second anti-HER2 antibody binding to HER2 extracellular subdomain II, the kit comprising:

[0023] 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,

[0024] b. Explanation of the binding of antibodies that quantify the binding to HER2 extracellular subdomain II.

[0025] Further provided is a kit for specifically quantifying the binding of an antibody to HER2 extracellular subdomain IV in a fixed-dose combination (FDC), the FDC having an antibody binding to HER2 extracellular subdomain IV and a second anti-HER2 antibody, the kit comprising:

[0026] 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.

[0027] b. Explanation of the binding of antibodies that quantify the binding to the extracellular subdomain IV of HER2.

[0028] In another aspect of the invention, a method for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab is provided, the method comprising:

[0029] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0030] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0031] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0032] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0033] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further includes the following steps:

[0034] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0035] 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.

[0036] In one embodiment, a method of preparing a composition is provided, comprising: (1) producing a fixed-dose composition comprising pertuzumab, trastuzumab, and one or more variants thereof; and (2) analyzing the composition thus produced to assess the amount of the variant, wherein the variant comprises: (i) pertuzumab deamided at HC-Asn-391, a pertuzumab FC sialic acid variant, and a pertuzumab lysine glycosylated 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.

[0037] In one embodiment, a method for preparing a composition is provided, wherein the analytical determination in step (2) comprises:

[0038] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0039] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0040] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0041] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0042] In one embodiment, the analytical determination in step (2) further includes the following steps:

[0043] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0044] In one embodiment, the method is performed at a temperature of 32-40°C.

[0045] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab in step (1) further comprises hyaluronidase.

[0046] 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.

[0047] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated 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.

[0048] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated at HC-Asn-55, at least 38% of a natural pertuzumab antibody, at least 16% of a natural trastuzumab antibody, and less than 9% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain via HC-Asp-102.

[0049] 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 deamidated variants, Fc sialic acid variants and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30 and a trastuzumab variant deamidated at HC-Asn-55, at least 28% of a natural pertuzumab antibody, at least 23% of a natural trastuzumab antibody, and less than 12% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0050] 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:

[0051] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0052] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0053] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0054] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0055] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0056] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0057] 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.

[0058] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0059] 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:

[0060] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0061] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0062] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0063] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0064] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0065] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0066] 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.

[0067] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0068] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises a peak area of ​​less than 21% of the sum of peaks 1 to 3, a peak area of ​​at least 28% of peak 4 (pertuzumab natural antibody), a peak area of ​​at least 23% of peak 7 (trastuzumab natural antibody), and a peak area of ​​less than 12% of peak 8, as determined in a method comprising the following steps:

[0069] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0070] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0071] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0072] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0073] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0074] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0075] 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.

[0076] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0077] In another aspect of the invention, the compositions provided herein can be obtained by a method comprising the following steps:

[0078] a. Add the pre-quantified pertuzumab to the mixing dish.

[0079] b. Add trastuzumab at a 1:1 ratio to pertuzumab or a 1:2 ratio to pertuzumab.

[0080] c. Add rHuPH20.

[0081] In another aspect, a method is provided for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, comprising:

[0082] a. Provide RP-HPLC phenyl column;

[0083] b. Load a fixed-dose combination (FDC) of two anti-HER2 antibodies onto an RP-HPLC column;

[0084] c. The two anti-HER2 antibodies were separated at a flow rate of 0.2-0.4 mL / min, with the column temperature ranging from 64°C to 76°C.

[0085] In one embodiment, the fixed-dose combination comprises pertuzumab and trastuzumab. In another embodiment, the fixed-dose combination of pertuzumab and trastuzumab further comprises hyaluronidase.

[0086] 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).

[0087] 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 approximately 0.3 mL / min.

[0088] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the antibodies are separated over 10 to 20 minutes. In another embodiment, the antibodies are separated over 15 minutes. In one embodiment, the antibodies are separated over 15 minutes at a flow rate of 0.3 mL / min.

[0089] 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.

[0090] 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), XRs Diphenyl (diphenyl) Biphenyl Plus Hexyl Phenyl (phenyl), Ascentis Phenyl (phenyl) and Agilent AdvanceBio RP mAb Diphenyl (diphenyl). Attached Figure Description

[0091] Figure 1 A schematic diagram of the HER2 protein structure and the amino acid sequences of domains I-IV of its extracellular domains are provided (SEQ ID No. 1-4, respectively).

[0092] Figure 2A and 2B The following amino acid sequence alignment is shown: Variable light chain (V) of mouse monoclonal antibody 2C4. L ()( Figure 2A ) and variable heavy chain (V H ()( Figure 2B ) fields (SEQ ID No. 5 and 6, respectively); V of variant 574 / pertuzumab L and V HStructural domains (SEQ ID NO.7 and 8, respectively); and human V L and V H Common frameworks (humκ1, light chain κ subgroup I; humIII, heavy chain subgroup III) (SEQ ID No. 9 and 10, respectively). An asterisk indicates a difference between the variable domains of pertuzumab and mouse monoclonal antibody 2C4, or between pertuzumab and the variable domains of the human framework. Complementarity-determining regions (CDRs) are enclosed in parentheses.

[0093] Figure 3A and 3B The light chain of pertuzumab is shown. Figure 3A ; SEQ ID NO.11) and heavy chain ( Figure 3B The amino acid sequence of SEQ ID No. 12 is shown. 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.

[0094] Figure 4A and 4B The light chain of trastuzumab is shown separately. Figure 4A ; SEQ ID NO.13) and heavy chain ( Figure 4B The amino acid sequence of SEQ ID NO. 14. The boundaries between the variable light chain and variable heavy chain domains are indicated by arrows.

[0095] Figure 5A and 5B The modified pertuzumab light chain sequence is shown below. Figure 5A ; SEQ ID NO.15) and the variant pertuzumab heavy chain sequence ( Figure 5B (SEQ ID NO.16).

[0096] Figure 6 This diagram illustrates the extracellular domains of HER2 and the capture reagents that can be used in the ELISA assays described herein. P-HER2 variant: A modified HER2 ECD used to analyze pertuzumab potency. T-HER2 variant: A modified HER2 ECD used to analyze trastuzumab potency.

[0097] Figure 7A and 7B The selective sensitivity of cell-based assays is shown. Figure 7A : Pertuzumab antiproliferative assay in MDA-MB-175VII cells. Figure 7B Trastuzumab antiproliferative assay was performed on BT-474 cells.

[0098] Figure 8A and8B This demonstrates the complementary mechanism of pertuzumab and trastuzumab in a cell-based antiproliferative assay. Figure 8A Pertuzumab antiproliferative assay: After adding trastuzumab at a 1:1 ratio, the dose-response curve shifted to lower concentrations. Figure 8B Trastuzumab antiproliferative assay: After adding pertuzumab at a 1:1 ratio, the dose-response curve shifted slightly to lower concentrations.

[0099] Figure 9A and 9B The masking effect is shown in the cell-based antiproliferation assay. Figure 9A Pertuzumab antiproliferative assay: The affinity of the pertuzumab mutant (HC S55A) for HER2 was greatly 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 greatly reduced (solid symbol); the loss of affinity of the trastuzumab mutant was masked after the addition of pertuzumab (hollow symbol).

[0100] Figure 10 The representative dose-response curve of pertuzumab ELISA is shown.

[0101] Figure 11 The representative dose-response curves for trastuzumab ELISA are shown.

[0102] Figure 12 A representative chromatogram of the provided IEC method is shown, which is used to analyze the pertuzumab-trastuzumab FDC charge variant.

[0103] Figure 13 The image shows the IE-HPLC chromatograms of the pertuzumab-trastuzumab FDC drug, pertuzumab, and trastuzumab.

[0104] Figure 14A and Figure 14B The HER2 affinity mutant in ELISA is shown. 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) for HER2 (hollow symbol) is much lower than that of trastuzumab (solid symbol).

[0105] Figure 15 An example RP-UHPLC chromatogram used to analyze the protein content of FDC LD reference standards is shown.

[0106] Figure 16 An example RP-UHPLC chromatogram is shown for analyzing the protein content of FDC MD reference standards. Detailed Implementation

[0107] I. Definition

[0108] The term “about” as used in this patent specification is intended to indicate that the 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, a variation within a range of + / -5% in a given value is included.

[0109] The "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family and includes EGFR, HER2, HER3, and HER4 receptors. HER receptors typically contain: 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 having several phosphorylated tyrosine residues. HER receptors can be the "natural sequence" HER receptor or its "amino acid sequence variants." Preferably, the HER receptor is the natural sequence human HER receptor.

[0110] The terms “ErbB2” and “HER2” are used interchangeably in this paper 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) (Genebank Accession Number: X03363). The term “erbB2” refers to the gene encoding human ErbB2, and “neu” refers to the gene encoding rat p185. neu The preferred HER2 gene is the natural human HER2 sequence.

[0111] In this article, "HER2 extracellular domain" or "HER2 ECD" refers to the extracellular HER2 domain anchored to the cell membrane or circulating in the cell, including its segments. The amino acid sequence of HER2 is shown in [image / image / etc.]. Figure 1In one embodiment, the extracellular domain of HER2 may comprise the following four subdomains: “Subdomain I” (approximately amino acid residues 1-195; SEQ ID NO: 1), “Subdomain II” (approximately amino acid residues 196-319; SEQ ID NO: 2), “Subdomain III” (approximately amino acid residues 320-488; SEQ ID NO: 3), and “Subdomain IV” (approximately amino acid residues 489-630; SEQ ID NO: 4) (the residue numbers do not include the signal peptide). 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 the present invention. Figure 1 The “recombinant HER2 extracellular subdomain” or “recombinant HER2 ECD subdomain” contains the corresponding native HER2 ECD subdomain in its 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 may preferably be truncated by up to 6 amino acids at its C-terminus.

[0112] "Anti-HER2 antibodies" or "HER2 antibodies" are antibodies that bind to the HER2 receptor. Optionally, HER2 antibodies further interfere with the activation or function of HER2. The anti-HER2 antibodies of interest in this article are pertuzumab and trastuzumab.

[0113] An antibody “binding to extracellular subdomain II of HER2” binds to residues in domain II (SEQ ID NO:2) of HER2 and optionally to 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.

[0114] For the purposes of this document, “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, respectively, as in SEQ ID NO:7 and 8. Where pertuzumab is a complete 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 optionally generated by recombinant Chinese hamster ovary (CHO) cells. The terms “pertuzumab” and “rhuMAb2C4” herein cover biosimilar forms having a United States Adopted Name (USAN) or International Nonproprietary Name (INN): pertuzumab.

[0115] An antibody that “binds to the extracellular subdomain IV of HER2” binds to residues in domain IV of HER2 (SEQ ID NO:4) and optionally residues in other subdomains. In one embodiment, the antibody that binds to the extracellular subdomain IV is trastuzumab or a variant thereof.

[0116] For the purposes of this document, “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, respectively, in SEQ ID No:13 and 14. Where the trastuzumab is a complete 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 optionally generated via Chinese hamster ovary (CHO) cells. The terms “trastuzumab” and “rhuMAb4D5” herein cover biosimilar forms having a US Adopted Name (USAN) or International Nonproprietary Name (INN): trastuzumab.

[0117] The term “co-preparation” is used herein to refer 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.

[0118] "Fixed-dose combination" or "FDC" as used herein refers to a single alternative pharmaceutical formulation containing two or more active ingredients, including, for example, a single alternative pharmaceutical formulation containing pertuzumab and trastuzumab formulated together for subcutaneous (SC) administration. "Pertuzumab-trastuzumab FDC" contains pertuzumab, trastuzumab, and optionally hyaluronidase.

[0119] The term "hyaluronidase" (or hyaluronidase enzyme) refers to a group of enzymes found throughout the animal kingdom that are typically neutral or acidic in activity. Hyaluronidases vary depending on their substrate 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-acetylhexosaminease that produces tetrasaccharides and hexasaccharides as major end products. It possesses hydrolytic and transglycosidic activities and degrades 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-acetylhexosaminease that functions through a β-elimination reaction that primarily produces disaccharide end 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 acidic-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 plasmahyaluronidase”, Biochem. Biophys. Res. Commun. 1997; 236(1):10-15]. Bovine PH20 attaches very loosely to the plasma membrane and is not anchored via phospholipase-sensitive anchors [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). TM Hyalase TMOther PH20 substances are lipid-anchored enzymes that are usually insoluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane via GPI anchors. Natural macaque sperm hyaluronidase is 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 administration of therapeutic drugs to the hypothalamus. By rapidly depolymerizing HA in the extracellular space, sHASEGP reduces interstitial viscosity, thereby increasing hydraulic conductivity and allowing for the safe and comfortable administration of larger volumes of drugs to SC tissues. The preferred hyaluronidase is human hyaluronidase, with recombinant human hyaluronidase known as rHuPH20 (rHuPH20 α-hyaluronidase) being the most preferred. rHuPH20 is a member of a family of neutral and acid-active β-1,4-glycosyl hydrolases that 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 EU countries include... “Dessau” and Animal-derived hyaluronidase products approved in the United States include Vitrase. TM Hydase TM and Amphadase TM .

[0120] rHuPH20 is currently the first and only recombinant human hyaluronidase available for therapeutic use. TM The amino acid sequence of ) is well known and available at CAS Registry No.: 75971-58-7. The molecular weight is approximately 61 kDa. In one embodiment, pertuzumab-trastuzumab FDC comprises hyaluronidase at an optional concentration of 2000 U / mL.

[0121] The “loading” dose in this article 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 the pertuzumab-trastuzumab FDC consists of 40 mg / mL trastuzumab, 80 mg / mL pertuzumab, and 2000 U / mL rHuPH20.

[0122] The term "maintenance" dose in this document refers to one or more therapeutic doses administered to a 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.

[0123] As used herein, a “capture reagent” refers to any reagent capable of binding to an analyte (e.g., an anti-HER2 antibody). Preferably, a “capture reagent” refers to any reagent specifically bound to an anti-HER2 antibody in a fixed-dose combination of two anti-HER2 antibodies. For specific analysis of binding to 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.

[0124] "Modified HER2 ECD" is a genetically engineered protein or peptide containing one or more recombinant HER2 ECD subdomains. The HER2 ECD is modified so that an anti-HER2 antibody to be evaluated in the FDC (Follicular Unit Discharge) can bind to it, while a second anti-HER2 antibody in the FDC cannot. 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 similar subdomain that does not bind to any anti-HER2 antibody. Preferably, the modified HER2 ECD is constructed to mimic the natural HER2 ECD as closely as possible. The subdomains 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 is preserved or improved when using one or more recombinant HER2 ECD subdomains shortened by about 4 to 5 amino acids at the C-terminus.

[0125] The term “Fc domain” in this article 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 C-terminus. Unless otherwise indicated, the residue numbers in the immunoglobulin heavy chain in this article are 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.

[0126] As used herein, the term "detectable antibody" refers to an antibody linked to a reagent or detectable label that can generate a detectable signal that can be used to assess the presence and / or quantity of a analyte to be detected (i.e., an anti-HER2 antibody).

[0127] The term "label" or "detectable label" refers to any chemical group or portion that can be linked 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 can indicate its presence by participating in a specific binding reaction. Examples of such labels include haptens, antibodies, biotin, streptavidin, his tags, N-triacetic acid, glutathione S-transferase, glutathione, and the like.

[0128] The term "detection means" refers to a component or technique used to detect the presence of a detectable antibody via a signal read out in the assay described herein. "Photoluminescence" is the process by which a material emits light after absorbing light (also known as 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.

[0129] 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.

[0130] In this article, "potency" refers to the therapeutic activity or expected biological effect of a biotherapeutic agent. The potency of a biotherapeutic agent can be determined by measuring or quantifying the bioactivity of its active ingredient.

[0131] In this document, the “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 an anti-HER2 antibody in the FDC is related to the ability of the anti-HER2 antibody in the monoclonal antibody formulation to inhibit proliferation in human breast cancer cell lines. A suitable human breast cancer cell line for testing pertuzumab is MDA-MB-175-VII. A suitable human breast cancer cell line for testing trastuzumab is BT-474.

[0132] The term “antibody” is used in the broadest sense in this article and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0133] 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) with the desired specificity, affinity, and capability. In some cases, framework 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 intended 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 hypervariable loops correspond to hypervariable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. Humanized antibodies optionally also contain 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. As described in Table 3 of U.S. Patent 5,821,337 (which is expressly incorporated herein by reference) and as defined herein; and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

[0134] The “intact antibody” described herein comprises two antigen-binding regions and an Fc region. Preferably, the intact antibody has a functional Fc region.

[0135] 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; dimers; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0136] "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 linked to the heavy chain by a covalent disulfide bond, the number of which varies in the heavy chain of different immunoglobulin isotypes. The heavy and light chains also have regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H ), followed by multiple constant structural domains. Each light chain has a variable structural domain (V) at one end. LIt also has a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains.

[0137] The term "hypervariate region" as used in this article 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 CDR (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 and Lesk). J.Mol.Biol.196:901-917(1987)). “Frame region” or “FR” residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0138] Based on the amino acid sequence of their 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. Several 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 antibody types are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known.

[0139] "Naked antibody" is an antibody that has not bound to a foreign molecule (such as a cytotoxic moiety or a radiolabeled molecule).

[0140] Affinity-matured antibodies are antibodies that have one or more alterations in one or more hypervariable regions compared to their unaltered parental antibodies, these alterations improving the antibody's affinity for the antigen. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using methods known in the art. 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., ProcNat.Acad.Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (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).

[0141] "Value" is a container suitable for containing liquid or lyophilized formulations. In one embodiment, the vial is a disposable vial, such as a 10mL or 20mL disposable vial with a stopper, for example, a 10mL disposable glass vial with a 20mm stopper.

[0142] As used in this article, “elution” 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.

[0143] 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 the mixture with an adsorbent that more or less strongly adsorbs or retains the solutes under specific process buffer conditions (due to solute properties such as pi, hydrophobicity, size, and structure).

[0144] The terms “ion exchange” and “ion exchange chromatography” refer to chromatographic processes in which an ionizable solute of interest (e.g., an antibody against FDC and its acidic and basic variants) interacts with oppositely charged ligands linked (e.g., via covalent linkage) 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.

[0145] "Ion exchange chromatography" specifically includes cation exchange (CEX) chromatography, anion exchange chromatography, and mixed-mode chromatography.

[0146] "Cation exchange material" or "CEX material" refers to a negatively charged solid phase having free cations for exchange with cations in an aqueous solution passing over or through the solid phase. Any negatively charged ligands attached to the solid phase suitable for forming a cation exchange material can be used, such as carboxylic acid esters, sulfonates, and other ligands described below. Commercially available cation exchange materials include, but are not limited to, those having, for example, sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S from GE Healthcare, SP Sepharose Fast Flow). TMSP Sepharose High Performance, Toyopearl SP-650S and SP-650M from Tosoh, Macro-PrepHigh 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 SP5PW and SP-5PW-HR from Tosoh, Poros HS-20 and HS 50 from Applied Biosystems); 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 from Biochrom Labs) Hydrocell CM from Inc., Macro-Prep CM from BioRad, Ceramic HyperD CM from Pall Technologies, Trisacryl M CM, Trisacryl LSCM, Matrx Cellufine C500 and C200 from Millipore, 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 J.TBaker, DOWEX MAC-3 from Dow Liquid Separations, Amberlite weak cation exchanger, DOWEX weak cation exchanger and Diaion weak cation exchanger from Sigma-Aldrich, and Fractogel EMD from EMD). COO-); sulfonic acid-based groups (e.g., HydrocellSP from Biochrom Labs Inc., DOWEX fine-mesh strong acid cationic resin from Dow Liquid Separations, and JT...Baker's UNOsphere S, WP Sulfonic, Sartobind S membranes from Sartorius, Amberlite strong cation exchangers from Sigma-Aldrich, DOWEX strong cation exchangers, and Diaion strong cation exchangers; and phosphate-based groups (e.g., PI 1 from Whatman).

[0147] Based 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-acryloylamino-2-methyl-1-propanesulfonic acid)).

[0148] The "isoelectric point" or "pI" of a protein or antibody corresponds to the pH value at which the protein or antibody's net charge is neutral. pI can be determined using standard experimental methods, such as isoelectric focusing, or by calculation ("theoretical pI"). An example of a calculation method is the free online standard tool "ExPASy" (…). http: / / web.expasy.org / compute_pi / The pI is calculated 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.

[0149] A "mobile phase" is a liquid or gas that flows through a chromatographic system to cause the materials to be separated to move at different rates through the stationary phase. Preferably, the mobile phase is a liquid. In one example, the mobile phase may be a loading buffer ("mobile phase A") or an elution buffer (mobile phase B).

[0150] The "loading buffer" provides conditions to ensure that the target molecules interact effectively with the ligands of the ion-exchange chromatographic material, and are retained by the affinity medium as all other molecules flow through the column.

[0151] "Eluting buffer" is used to first wash away unbound proteins and then release charged variants and natural antibodies at a higher concentration of self-ligands.

[0152] The terms "major type antibody" or "natural antibody" herein refer to the antibody amino acid sequence structure that is 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 is an antibody that binds to extracellular subdomain II of HER2 and an antibody that binds to extracellular subdomain IV. In one embodiment, the major type antibody in the FDC is pertuzumab and trastuzumab.

[0153] A "charge variant" is a variant of a major antibody species that has a different overall charge than the major antibody species. Examples of charge variants are acidic variants and basic variants.

[0154] "Acidic variants" are variants of the major antibody class that are more acidic than the major antibody class. Compared to the major antibody class, acidic variants have acquired a negative charge or lost a positive charge. These acidic variants can be resolved using separation methods that separate proteins based on charge (e.g., ion exchange chromatography). After separation by cation exchange chromatography, acidic variants of the major antibody class elute before 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 deamidated at the heavy chain asparaginic acid at position 391 (HC-Asn-391), pertuzumab Fc sialic acid variant, and pertuzumab lysine glycation variant. Examples of acidic trastuzumab variants include trastuzumab deamidated at LC-Asn-30 and trastuzumab deamidated at HC-Asn-55.

[0155] "Basic variants" are variants of the major antibody class that are more basic than the major antibody class. Compared to the major antibody class, basic variants have acquired 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 the major antibody class elute later than the main peak. Basic variants of pertuzumab and trastuzumab can be separated and quantified using the ion-exchange chromatography method described herein.

[0156] As used herein, the term "gradient" refers to a change in the properties of the mobile phase during chromatographic sample run. 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 chromatographic flow) can be changed incrementally, i.e., gradually, compared to a linear change.

[0157] 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 an elution procedure used in liquid chromatography where the mobile phase is significantly more polar than the stationary phase.

[0158] 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 the silica surface. Some phenyl columns with phenyl groups have alkyl spacers between the phenyl group and the silica surface. Increasing the length of the alkyl spacers enhances steric and aromatic selectivity. RP-HPLC phenyl columns are distinguished by the number of aromatic groups (monophenyl-p-biphenyl), the length of the alkyl spacers between the silica surface and the phenyl group, the nature of the substituents on the bonded ligands (typically methyl or spatially larger isobutyl), whether oxygen atoms are incorporated into the linker to activate the π-electron system in the aromatic ring, and whether the silica stationary surface is capped. For example, RP-HPLC phenyl columns may have the following groups: ethylphenyl with methyl side groups and end-capped silica surfaces; 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). XRsDiphenyl, Biphenyl Plus Hexyl Phenyl, AscentisPhenyl, Agilent Zorbax RRHD 300-Diphenyl and Agilent AdvanceBio RP mAb Diphenyl.

[0159] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth.

[0160] "Advanced" cancer is cancer that has spread beyond the source site or organ through local invasion ("locally advanced") or metastasis ("metastatic"). Accordingly, the term "advanced" cancer includes both locally advanced disease and metastatic disease.

[0161] Metastatic cancer refers to cancer that has spread from one part of the body (such as the breast) to another part of the body.

[0162] "Refractory" cancer is cancer that progresses even when the patient is given antitumor agents (such as chemotherapy) or biological therapies (such as immunotherapy). An example of refractory cancer is platinum-resistant cancer.

[0163] “Recurrent” cancer is cancer that has regrowth at the initial site or at a distant site after a response to an initial treatment (such as surgery).

[0164] "Locally recurrent" cancer is cancer that recurs in the same place as the previously treated cancer after treatment.

[0165] "Non-resectable" or "unresectable" cancers cannot be removed (excised) through surgery.

[0166] 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.

[0167] "Neoadjuvant therapy" or "neoadjuvant treatment" or "neoadjuvant administration" refers to systemic therapy given prior to surgery.

[0168] "Adjunctive therapy" or "adjunctive treatment" or "adjunctive application" refers to systemic therapies administered after surgery.

[0169] In this article, "patient" or "individual" refers to a human patient. A patient may be a "cancer patient," that is, a patient who has one or more symptoms of cancer, especially breast cancer, or who is at risk of having these symptoms.

[0170] A “patient population” refers to a group of cancer patients. These populations can be used to demonstrate statistically significant efficacy and / or safety of drugs such as pertuzumab and / or trastuzumab.

[0171] A "relapsed" patient is one who has developed signs or symptoms of cancer after a period of remission. Optionally, the patient relapses after adjuvant or neoadjuvant therapy.

[0172] Cancer or biological samples that “show HER expression, amplification, or activation” are those that express (including overexpress) the HER receptor in diagnostic tests, have an amplified HER gene, and / or additionally show HER receptor activation or phosphorylation.

[0173] Cancer or biological samples that “show HER activation” are those that demonstrate activation or phosphorylation of the HER receptor in diagnostic tests. This activation can be determined directly (e.g., by measuring HER phosphorylation using an ELISA) or indirectly (e.g., by gene expression profiling or by detecting HER heterodimers, as described herein).

[0174] 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 by increased transcription or translation. HER receptor overexpression or amplification can be assessed in diagnostic or prognostic assays by evaluating increased levels of HER proteins present on the cell surface (e.g., via immunohistochemical IHC assay). Alternatively or additionally, the level 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., HER extracellular domains) 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 the art can utilize various in vivo assays. For example, cells in a patient's body can be exposed to antibodies optionally labeled with detectable markers (e.g., radiolabels) for in situ analysis of radioactivity or analysis of biopsies obtained from patients pre-exposed to antibodies.

[0175] "HER2-positive" cancers include cancer cells with HER2 levels higher than normal. Optionally, 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, having an ISH / FISH / CISH amplification ratio of ≥2.0.

[0176] "HER2-mutant" cancers encompass 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). Specifically, "HER2-mutant" cancers 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 previously reported identical non-synonymous putative activating mutations (or insertions / deletions) 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 Sci USA 2012; 109:14476-81; and Herter-Sprie et al., Front Oncol 2013; 3:1-10.

[0177] In this article, "antitumor agents" refers to drugs used to treat cancer. Non-limiting examples of antitumor agents in this article include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, antihormonal 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.

[0178] "Treatment" refers to both therapeutic treatment and preventative or preventative measures. Those who require treatment include those who already have cancer and those who need cancer prevention. Therefore, the patients in this article who require treatment may have been diagnosed with cancer or are susceptible to or prone to cancer.

[0179] The term "effective dose" refers to the amount of drug that is effective in treating a patient's cancer. An effective dose of drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down or preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down or preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more cancer-related symptoms to some extent. To some extent, the drug can prevent the growth of existing cancer cells and / or kill them; it can be for the purpose of inhibiting cell growth and / or cytotoxicity. An effective dose can prolong progression-free survival (e.g., as measured by 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 assessed by FOSI).

[0180] 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., At). 211 ,I 131 ,I 125 ,Y 90 Re 186 Re 188 ,Sm 153 ,Bi 212 ,P 32 Radioactive isotopes of 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).

[0181] Chemotherapy uses chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents used in chemotherapy include alkylating agents, such as thiotepa and... Cyclophosphamide; 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; TLK286 (TELCYTA) TM) ; polyacetogenin (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapacol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan) CPT-11 (irinotecan) Acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid. Illinicacid); Teniposide; Cryptophycin (especially Cryptophycin 1 and Cryptophycin 8); Dolastatin; Duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); Elutherobin; Pancratistatin; Sarcodictyin; Spongistatin; Nitrogen mustards Mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamineoxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil Mustard; nitrosoureas, 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, alcarubicin, daunorubicin, doxorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, valrubicin, KRN5500, menogaril, dynemicin (including dynemicin A), esperamicin, neocarzinostatin, chromophores and related chromoproteins. Antiobiotic chromophores, aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-leucine. (Doxorubicin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esoubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid Folic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogs, such as denopterin, pteropterin, and trimetrexat. e) Purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergic agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as leucovorin.Acetylglucuronolactone; antifolate and antitumor agents, for example; 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), as well as thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors (e.g., raltitrexed (TOMUDEX)). RMTDX); inhibitors of dihydropyrimidine dehydrogenase, such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK7 polysaccharide complex (JHSNatural) 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 Dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine 6-Thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine. Etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Vinorelbine (Catharanthus alkaloids) Novotrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutical 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 oxaliplatin. TM () is an abbreviation for a combination therapy regimen with 5-FU and formyltetrahydrofolate.

[0182] This definition also includes antihormonal 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...). Tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Toremifene; aromatase inhibitors; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane cyclopentane nucleoside cytosine analog); 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, for example... vaccine, Vaccines and Vaccine; Purulent rIL-2; Topoisomerase 1 inhibitor; Abaric rmRH; and any of the medicinal salts, acids or derivatives thereof.

[0183] Taxanes are chemotherapeutic agents that inhibit mitosis and interfere with microtubules. Examples of taxanes include paclitaxel. Bristol-Myers Squibb Oncology, Princeton, NJ; paclitaxel or nab-paclitaxel cremophor-free, albumin-engineered nanoparticle formulations (ABRAXANE) TM American Pharmaceutical Partners, Schaumberg, Illinois); and docetaxel ( -Poulenc Rorer, Antony, France).

[0184] "Anthracycline drugs" are a class of antibiotics derived from the fungus *Streptococcus peucetius*, including daunorubicin, doxorubicin, epirubicin, and any other anthracycline chemotherapeutic agents (including those listed above).

[0185] "Allium ring-based chemotherapy" refers to chemotherapy regimens consisting of or including one or more anthracene rings. 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.

[0186] For the purposes of this article, "carboplatin-based chemotherapy" refers to a chemotherapy regimen consisting of or including one or more carboplatins. An example is TCH (docetaxel / Carboplatin and trastuzumab ).

[0187] "Aromatase inhibitors" inhibit aromatase, an enzyme that regulates the production of estrogen in the adrenal glands. Examples of aromatase inhibitors include: 4(5)-imidazole, aminoglutethimide, megestrol acetate Exemestane, Formestan, Fadrozole vorozole Letrozole and Anastrozole. In one embodiment, the aromatase inhibitor described herein is letrozole or anastrozole.

[0188] "Antimetabolite chemotherapy" uses agents that are structurally similar to metabolites but cannot be effectively utilized by the body. Many antimetabolite chemotherapy therapies interfere with the production of nucleic acids, RNA, and DNA. Examples of antimetabolite chemotherapy agents include gemcitabine. 5-Fluorouracil (5-FU), Capecitabine (XELODA) TM ), 6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosine (ARA-C), and cytarabine. Dacarbazine azocytosine, deoxycytosine, pyrimidine, and fludarabine. Cladribine, 2-deoxy-D-glucose, etc.

[0189] "Chemotherapy-resistant" cancer refers to cancer that progresses while the patient is receiving chemotherapy (i.e., the patient is "chemotherapy-refractory"), or cancer that progresses within 12 months (e.g., within 6 months) after the patient completes chemotherapy.

[0190] The term “platin” is used in this document to refer to platinum-based chemotherapy, including but not limited to cisplatin, carboplatin, and oxaliplatin.

[0191] The term "fluoropyrimidine" is used in this document to refer to antimetabolite chemotherapy, including but not limited to capecitabine, fluorouridine, and fluorouracil (5-FU).

[0192] The term "fixed" or "uniform" dose for therapeutic agents in this article refers to the dose administered to a human patient without regard to the patient's weight (WT) or body surface area (BSA). Therefore, fixed or uniform doses are not expressed in mg / kg or mg / m². 2 It is not provided in dose, but in absolute quantities as a therapeutic agent.

[0193] II. Measurement

[0194] Co-formulation of therapeutic monoclonal antibodies (mAbs) into fixed-dose combinations (FDCs) increases drug complexity and poses challenges to the characterization and control of product quality. These challenges are 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 used to determine the critical quality attributes (CQAs) of a fixed-dose combination of two anti-HER2 antibodies.

[0195] On one hand, these assays are suitable for analyzing a fixed-dose combination of two anti-HER2 antibodies, trastuzumab and pertuzumab. Trastuzumab and pertuzumab share more 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 co-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: a loading dose and a maintenance dose, differing in the ratio of pertuzumab SC drug substance to trastuzumab SC drug substance.

[0196] (i) efficacy determination

[0197] Potency is a controllable quality assessment (CQA) component of the control system for the release and stability testing of biological therapeutics, including therapeutic monoclonal antibodies. Potency monitors the cumulative effect of product quality properties on biological activity, which can potentially influence safety and efficacy; that is, higher potency may raise safety concerns, 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) document "Guidance for Industry on Potency Tests for Cellular and Gene Therapy Products," the traditional approach to assessing the potency of biological products is to develop quantitative bioassays (bioassays) that measure the product activity associated with a specific ability to achieve a given result. Bioassays can measure potency by evaluating the active ingredient of the product within a living biological system. Bioassays can include in vivo animal studies, in vitro organ, tissue, or cell culture systems, or any combination of these assays. A widely used example of a bioassay for determining or quantifying potency is a cell-based assay. This study evaluated the suitability of two different cell-based assays (designed specifically to measure cell growth inhibition and antiproliferative activity of pertuzumab or trastuzumab) for controlling the bioactivity of a fixed-dose pertuzumab-trastuzumab combination. The evaluation confirmed that these assays are not suitable for the fixed-dose combination due to limitations that prevent control over variations in the quality of the individual antibodies combined into the co-formulation. Because of the nature of co-formulations of two antibodies that bind to the same receptor and inhibit similar signaling pathways, no alternative HER2-expressing cell lines can overcome these limitations.

[0198] 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 mediated through their binding activity to the corresponding epitopes on HER2. Therefore, potential molecular changes in antibodies affecting their potency 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, as well as CDR affinity mutants), as illustrated in the examples presented herein. This study confirmed that binding differences (as detected by the binding assays provided herein) 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 herein to detect single antibody quality changes affecting potency, the novel binding assays provided herein are considered the best possible assay for controlling for relevant changes in product quality affecting target binding and HER2 signaling.

[0199] In one embodiment, pertuzumab-trastuzumab FDC drugs are 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 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 OpinDrug Metab Toxicol 2015; 11:1647-63.). Trastuzumab's binding to the HER2 subdomain IV inhibits ligand-independent HER2 signaling by blocking its homodimerization (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 HER2ectodomain 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 subdomain II of HER2 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 arecombinant 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 signal transduction, thereby inducing cell growth arrest and apoptosis in human tumor cells that overexpress HER2.

[0200] Pertuzumab and trastuzumab bind to these distinct and non-overlapping epitopes on the HER2 ECD without competing with each other, and they possess complementary mechanisms that disrupt HER2 signaling. When pertuzumab and trastuzumab are administered in combination, this enhances their antiproliferative activity in vitro and in vivo (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, ensuring control over the quality of each of the two antibodies in the pertuzumab-trastuzumab FDC drug.

[0201] In one embodiment, a binding assay for a fixed-dose combination (FDC) of two anti-HER2 antibodies is provided, comprising:

[0202] a. Contact the FDC with the capture reagent, wherein the capture reagent is a modified HER2 ECD.

[0203] b. Contact the sample with the detectable antibody.

[0204] c. Use the detection method that can detect antibodies to quantify the amount of antibody bound to the capture reagent.

[0205] A fixed-dose combination of two anti-HER2 antibodies is contacted with and incubated together with a capture reagent, thereby capturing or binding the target anti-HER2 antibody to make it detectable in an assay step. The capture reagent is a modified HER2 ECD containing one or more recombinant HER2 ECD subdomains. In one embodiment, the modified HER2 ECD is a genetically engineered protein or peptide containing one or more recombinant HER2 ECD subdomains. In one embodiment, the HER2 ECD is modified so that one anti-HER2 antibody to be evaluated in the FDC binds 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 bound by the second anti-HER2 antibody or by replacing that subdomain with a structurally similar subdomain that does not bind to either anti-HER2 antibody. A structurally similar subdomain can be any subdomain that, when included in the modified HER2 ECD, does not disrupt the three-dimensional conformation of 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 conformation that closely mimics the native HER2 ECD. The subdomains 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 is preserved or improved when using one or more recombinant HER2 ECD subdomains shortened by approximately 4 to 5 amino acids at the C-terminus.

[0206] 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 include biotin, bovine serum albumin (BSA), and Fc domains. In a modified HER2, an extracellular domain is fused to an Fc domain. In one embodiment, the Fc domain is from a species different from the Fc domain of the anti-HER2 antibody to be analyzed. For example, if the anti-HER2 antibody to be analyzed contains a human Fc domain, the capture reagent should contain a non-human Fc domain, such as an Fc domain from a mouse, porcupine, rat, rabbit, etc. In one embodiment, the Fc domain of the recombinant HER2 ECD subdomain is a mouse Fc domain. In one embodiment, the Fc domain comprises SEQ ID NO. 35.

[0207] In the next step, the sample containing the capture reagent and the captured anti-HER2 antibody is incubated 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, thereby detecting the presence or amount of the anti-HER2 antibody of interest in the FDC.

[0208] 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. 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 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 a 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 mean maxima (MD) of the pertuzumab-trastuzumab FDC. In one embodiment, a binding assay is used to analyze a 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.

[0209] In one embodiment, the binding of pertuzumab and trastuzumab is determined in two separate binding assays.

[0210] The pertuzumab binding assay determines the bioactivity as the ability of pertuzumab to specifically bind to its epitopes on 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.

[0211] In one embodiment, the modified HER2 ECD comprises HER2 ECD subdomains I, II, and III, or portions thereof. In one embodiment, the modified HER2 ECD comprises human HER2 ECD subdomains I, II, and III, or portions thereof. In one embodiment, the modified HER2 ECD does not comprise subdomain IV. The inventors have discovered that by including a recombinant subdomain III truncated at the C-terminus, a modified HER2 ECD having a three-dimensional conformation similar to that of the natural HER2 ECD can be produced. 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, the modified HER2 ECD comprises 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.

[0212] 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 embodiments described above, 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.

[0213] 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.

[0214] 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 comprises SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:3, and SEQ ID NO:28.

[0215] In one embodiment, the modified HER2 ECD includes SEQ ID NO.29. In another embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO.29.

[0216] 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 embodiments described above, the capture agent used to evaluate trastuzumab binding does not contain HER2 subdomain II. In one embodiment, the capture agent comprises 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.

[0217] In any of the embodiments described above, the detectable antibody comprises a label that allows it to be detected by various means. These 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 these labels include radioisotopes 32P, 14C, 125I, 3H, and 131I; fluorophores, such as rare earth chelates or luciferin and its derivatives; rhodamine and its derivatives; ruthenium; dansyl; umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; 2,3-dihydrodiazanaphthyldione; HRP; alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; and sugar oxidases, such as glucose. Oxidases, 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 (detectable by, for example, avidin, streptavidin, streptavidin-HRP, and streptavidin-β-galactosidase with MUG); spinning labels; phage labels; stable free radicals; and the like.

[0218] The preferred label for detectable antibodies is horseradish peroxidase (HRP). Substrates used for HRP are typically categorized into different classes, 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'-azido-di-[3-ethylbenzothiazoline-6-sulfonate] (ABTS)), fluorescent (e.g., ADHP), and chemiluminescent (e.g., enhanced chemiluminescence (ECL)) substrates, depending on whether they produce color, fluorescence, or luminescence, respectively. ABTS is the preferred substrate.

[0219] In one embodiment, the antibody targeting the F(ab')2 portion of human IgG can be detected. In another embodiment, the antibody targeting the F(ab')2 portion of an anti-HER2 antibody can be detected.

[0220] In one embodiment, the binding assay is an enzyme-linked immunosorbent assay (ELISA). In ELISA, a capture reagent attaches 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 suitable for immunoassay, including carriers in the form of surfaces, particles, porous matrices, etc. Examples of commonly used carriers include flakes, Gels, polyvinyl chloride, plastic beads, and assay plates or tubes (including 96-well microtiter plates) 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, it is suitable to immobilize the capture reagent using a reactive, water-insoluble matrix (e.g., cyanogen bromide-activated carbohydrates and reactive substrates) 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 capture reagent is coated onto the microtiter plate, and the preferred solid phase used is a porous microtiter plate suitable for the simultaneous analysis of several samples. The preferred microtiter plate 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 best option is or 96-hole ELISA plates, such as those from NUNC or For sale.

[0221] 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. 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 prior to 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).

[0222] The amount of capture reagent used is large enough to obtain a good signal, but should not be molar excess 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.

[0223] The coated plate is then typically treated with a blocking agent that nonspecifically binds to and saturates the binding sites to prevent unnecessarily 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.

[0224] After coating and blocking, standards or FDC samples to be analyzed are added to the coated plate in the form of standard dilutions. In one embodiment, incremental concentrations of pertuzumab-trastuzumab FDC (standards, product controls, and samples to be analyzed) are added to the coated plate.

[0225] The incubation conditions for the FDC sample and the immobilized capture reagent are selected to maximize assay sensitivity and minimize dissociation, ensuring that the anti-HER2 antibody to be evaluated in the FDC sample binds to the immobilized capture reagent. Preferably, incubation is achieved at a relatively constant temperature in the range of about 0°C to about 40°C, preferably at room temperature or about room temperature. The incubation time typically does not exceed about 10 hours. Preferably, the incubation time is about 0.5 to 3 hours at room temperature or about room temperature, 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.

[0226] The immobilized capture reagent, which is conjugated with any anti-HER2 antibody, is preferably brought into 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 employed.

[0227] In another embodiment, the binding assay is electrochemiluminescence (ECL).

[0228] 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 following steps:

[0229] d. Establish a correlation between the level of antibody binding to the capture reagent and the biological activity of that antibody.

[0230] In one embodiment, the dose-response curve generated for the sample is compared with the dose-response curve for a standard. In another 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.

[0231] In one embodiment, nonlinear 4-parameter dose-response curves generated for samples and standards are compared. After evaluating similarities between the standard dose-response curve and the sample dose-response curve, the relative potency of the samples 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.

[0232] In one embodiment, the binding assay is used for the fractional release of a fixed-dose combination of pertuzumab and trastuzumab. In another 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.

[0233] (ii) Analysis of charge variants

[0234] 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 is considered a reference and powerful 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 immobilized in a column packing material. The adsorption of molecules to the solid support is driven by ionic interactions between the two moieties. The strength of the interaction depends on the number and location of charges on the molecules and on the stationary phase. IEX is typically a flare method, where specifications are specifically set for the distribution of each acidic, major, and basic substance for each mAb. These charged substances can be considered product-related impurities that can affect potency. Furthermore, because it does not involve the addition of denaturing agents, this is one of the few methods that can characterize proteins in their native conformation. IEX can also be used as an identification method for certain biological agents and is a routine test for stability and shelf-life verification.

[0235] 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.

[0236] 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 elute with a shorter residence time than basic substances.

[0237] After column equilibration and sample application, FDC's anti-HER2 antibodies, pertuzumab and trastuzumab, adsorb onto the column ligands. The column is then washed to remove unadsorbed proteins, and elution is performed by varying the ionic strength of the mobile phase while maintaining the pH within a predefined range. In one embodiment, the pH is maintained at a constant value.

[0238] The ionic strength is altered by applying a gradient of increasing salt concentration, which can be either a stepwise or continuous gradient. The inventors have found that the pH range of the loading buffer (mobile phase A) and elution buffer (mobile phase B) is critical when analyzing charge variants of the free-current concentrations (FDCs) of two anti-HER2 antibodies, pertuzumab and trastuzumab. Using a predefined pH range of pH 7.5–7.65 for the loading buffer (mobile phase A) and a predefined pH range of pH 7.5–7.7 for the elution buffer (mobile phase B) optimizes the separation of charge variants. In one embodiment, the pH is maintained at a constant value. In one embodiment, this constant pH value for the loading buffer is 7.5, 7.55, 7.6, or 7.65. In one embodiment, this constant pH value for the elution buffer is 7.5, 7.55, 7.6, 7.65, or 7.7.

[0239] After elution, the column is then reequilibrated using loading buffer (mobile phase A).

[0240] In one embodiment, the pertuzumab-trastuzumab fixed-dose combination is contacted with a cation exchange material, and the charged variant and the native antibody are eluted using a salt gradient while maintaining the pH of the mobile phase within a predefined range. In one embodiment, the salt gradient is a continuous salt gradient. In one embodiment, the pH of the loading buffer (mobile phase A) is between pH 7.5 and pH 7.65. In one embodiment, the pH of the elution buffer (mobile phase B) is between pH 7.5 and pH 7.7.

[0241] 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 elution buffer (mobile phase B) is between pH 7.5 and pH 7.7.

[0242] In one embodiment, a method for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab is provided, the method comprising:

[0243] a. Use a loading buffer to bind the antibody to the ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65.

[0244] b. Elute the antibody using an elution buffer with a pH between approximately pH 7.5 and approximately pH 7.7.

[0245] In one embodiment, elution in step b is performed using a salt gradient. 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 elution buffer contains sodium. In one embodiment, the elution buffer contains sodium ions (Na+). In one embodiment, the sodium gradient is a sodium chloride (NaCl) gradient. In one embodiment, the elution buffer contains NaCl. Suitable buffers for loading and elution are MES (2-ethanesulfonic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazine 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 (piperazine-N,N′-bis(2-ethanesulfonic acid)), and TPP (Tris, phosphate, piperazine). The preferred buffer solutions are ACES and HEPES.

[0246] In one embodiment, the sodium chloride concentration of the elution 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.

[0247] In one embodiment, the ion exchange material is a cation exchange material. In further optimizing the method of the invention, the inventors found that the separation of charge variants was 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 elution relative ion. 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 elution buffer contains sodium. In such an embodiment, the elution buffer contains sodium ions. In one embodiment, the SCX column is non-porous. Preferred cation exchange columns that can be used in this embodiment include: YMCBio Pro SP-F column, MabPac SCX-10, Waters BioResolve SCX mAb, Sepax Proteomix SCX-NP1.7, or non-porous Agilent Bio SCX.

[0248] 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.

[0249] In one embodiment, ion exchange chromatography is performed using a loading of about 50 μg to 149 μg or about 51 μg to 153 μg of total protein. In one embodiment, ion exchange chromatography is performed using a loading of a pertuzumab-trastuzumab FDC with a total protein content of about 50 μg to 149 μg. In one embodiment, ion exchange chromatography is performed using a loading of a maintenance dose of pertuzumab-trastuzumab FDC with a total protein content of about 51 μg to 153 μg. In one embodiment, the total protein loaded onto the ion exchange chromatograph is about 100 μg.

[0250] In one embodiment, a method for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab is provided, the method comprising:

[0251] a. Use a loading buffer to bind the antibody to the ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65.

[0252] b. Elute the antibody using an elution buffer with a pH between approximately pH 7.5 and approximately pH 7.7.

[0253] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0254] In one embodiment, acidic, natural, and basic variants of trastuzumab and pertuzumab in a fixed-dose combination are selectively detected.

[0255] 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 column.

[0256] 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 comprises 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 the pertuzumab and trastuzumab FDC. In one embodiment, the charge variant of pertuzumab and trastuzumab is 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 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, 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 comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further comprises 2000 U / mL rHuPH20.

[0257] In one embodiment, the natural antibody and its acidic and basic variants are eluted in a 1–100% (solvent B) salt gradient for at least 44 minutes. In one embodiment, the salt gradient increases from 1% solvent B to 47% solvent B over 43 minutes. In one embodiment, the salt gradient increases from about 1.8 mM NaCl to 103.4 mM NaCl. In another embodiment, the salt gradient increases from about 2 mM NaCl to about 94 mM NaCl.

[0258] 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.

[0259] (ii) Quantity / protein content determination

[0260] UV spectrophotometry is a typical method for determining the total protein content of pharmaceutical preparations. However, a different approach is required for a fixed-dose combination (FDC) of two anti-HER2 antibodies because conventional methods cannot individually and quantitatively analyze the protein content of each anti-HER2 antibody in the FDC. Different chromatographic methods are tested, such as hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC). For the individual and quantitative analysis of the protein content of pertuzumab-trastuzumab FDCs, reversed-phase chromatography has proven to be the most suitable method.

[0261] 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 the column. The adsorption of molecules to the solid support is driven by hydrophobic / nonpolar interactions 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 elute from the stationary phase with a longer residence time than polar molecules. Because the two anti-HER2 antibodies, trastuzumab and pertuzumab, share more than 93% sequence identity and differ by only 30 Da in total, a stable method was developed to provide reliable overall resolution and peak separation without significant sample residue (i.e., 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-U HPLC columns are known in the art and can have the following groups: ethylphenyl with methyl side groups and a capped silica surface; phenylhexyl phase with extended (hexyl) ligand spacer methyl side groups; ethylphenyl ligand 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 phenyl (e.g., monophenyl, biphenyl, diphenyl, phenylhexyl, phenylpropyl) modified stationary phases are readily available from most major column suppliers. One example of a phenyl column that can be used in this document is the Agilent ZorbaxRRHD 300-Diphenly column. In one embodiment, the column is a 2.1 × 100 mm column.

[0262] This article provides a method for analyzing the protein content of two fixed-dose combinations (FDC) of anti-HER2 antibodies, which includes...

[0263] a. Provide RP-HPLC phenyl column

[0264] b. The fixed-dose combination (FDC) of the two anti-HER2 antibodies was loaded onto an RP-HPLC column.

[0265] c. The two anti-HER2 antibodies were separated at a flow rate of 0.2-0.4 mL / min, with the column temperature ranging from 64°C to 76°C.

[0266] The principle of RP-HPLC separation is based on the hydrophobic association between peptide solutes and hydrophobic ligands on the surface of chromatographic resins. RP-HPLC columns are typically part of a UHPLC system equipped with an online vacuum degasser, an automated sampler with a sample cooler, a column heater, and a UV / VIS detector. Examples of suitable UHPLC systems include the Waters Aquity and the Thermo Ultimate 3000 RS.

[0267] The free concentrations (FDCs) of two anti-HER2 antibodies are loaded onto the column by injecting the sample into an RP-HPLC system. Typically, the sample is diluted to a concentration of, for example, 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 hydrochloride monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20. By using the formulation buffer as a diluent, the risk of altering the sample and reference solutions due to the use of a different diluent than previously observed 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 one embodiment, the total protein load on the column is 10 μg.

[0268] Proteins bind to an RP-HPLC column in an aqueous mobile phase and elute from the column by increasing the hydrophobicity of the mobile phase. The proteins are then separated based on 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 (eluent A) of water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and is then eluted with an increasing concentration of an organic phase containing acetonitrile. In one such embodiment, the organic phase (eluent B) contains 2-propanol:acetonitrile:eluent A (70:20:10). Due to the phenyl-based column type, improved specificity is obtained, and novel substances are detected only using 2-propanol, but not using pure acetonitrile. The difference in specificity is due to the interaction of the analyte with the phenyl-based column via classical hydrophobic interactions and other π-π interactions. As demonstrated in the literature, pure acetonitrile inhibits 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 areversed-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 is added to reduce the back pressure in the system.

[0269] In one embodiment, the aqueous mobile phase comprises 70% eluent A and 30% eluent B, wherein eluent A comprises water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and eluent B comprises 2-propanol:acetonitrile:eluent A (70:20:10). In one such embodiment, the organic phase (eluent B) is increased to 55% eluent A and 45% eluent B. In one embodiment, the gradient is increased to 45% eluent B over 15 minutes.

[0270] In one embodiment, the organic phase (eluent B) is increased to 10% eluent A and 90% eluent B. In another embodiment, the gradient is increased to 90% eluent B over 20 minutes.

[0271] 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.

[0272] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FDC) of two anti-HER2 antibodies, the antibodies are separated over 10 to 20 minutes. In another such embodiment, the antibody system is separated over 15 minutes. In one embodiment, the antibodies are separated over 15 minutes at a flow rate of 0.3 mL / min.

[0273] In addition to loading and elution (separation) steps, RP-HPLC purification may also include other steps such as equilibration, washing, and regeneration. In one embodiment, an RP-HPLC phenyl column is equilibrated using 70% eluent A and 30% eluent B, wherein eluent A comprises water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and eluent B comprises 2-propanol:acetonitrile:eluent A (70:20:10). In another embodiment, an RP-HPLC phenyl column is washed using 10% eluent A and 90% mobile phase B, wherein eluent A comprises water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and eluent B comprises 2-propanol:acetonitrile:eluent A (70:20:10).

[0274] 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 preferred resolution and separation. Several column temperatures were tested, and 70°C showed 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.

[0275] 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) column, etc. XRs Diphenyl, Biphenyl PlusHexyl Phenyl, Ascentis Phenyl, BioResolve RP mAb Polyphenyl, and Agilent AdvanceBioRP mAb Diphenyl. In one embodiment, the phenyl column is an Agilent Zorbax RRHD 300-Diphenyl column. In another embodiment, the column is a BioResolve RP mAb Polyphenyl column.

[0276] In one embodiment, the protein is detected by UV light. In another embodiment, the detection wavelength is 280 nm.

[0277] 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 comprises 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 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., in the same method. In one embodiment, this method is used to analyze the protein content of a 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, 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 comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC further comprises 2000 U / mL rHuPH20.

[0278] III. Anti-HER2 antibodies and compositions

[0279] (i) Anti-HER2 antibody

[0280] The HER2 antigen intended for antibody production may be, for example, a soluble form containing, a portion of, the extracellular domain 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 can be used for antibody production will be apparent to those skilled in the art.

[0281] Various methods for preparing the monoclonal antibodies described herein are available in the art. For example, the monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (US Patent No. 4,816,567).

[0282] The anti-HER2 antibodies pertuzumab and trastuzumab used in this invention are commercially available.

[0283] 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.

[0284] 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 defined herein; and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

[0285] The humanized antibody described herein may, for example, contain non-human hypervariable residues incorporated into the human variable heavy chain domain, and may further contain 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 contains FR substitutions at two or all of positions 69H, 71H, and 73H.

[0286] The exemplary humanized antibodies of interest herein comprise the 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), optionally comprising amino acid modifications of such CDR residues, for example wherein these modifications substantially maintain or improve the antibody's affinity. For example, antibody variants used in the methods of the present invention may have about one to about seven or about five amino acid substitutions in the aforementioned variable heavy chain CDR sequence. Such antibody variants can be prepared by affinity maturation, as described below, for example.

[0287] For example, in addition to the variable heavy chain domain CDR residues mentioned in the previous paragraph, humanized antibodies may also contain the variable light chain domain complementarity-determining residues KASQDVSIGVA (SEQ ID NO:20); SASYX 1 X 2 X 3 , where X 1 R or L, X are preferred. 2 The preferred choice is Y or E, and X 3 Preferably, it is T or S (SEQ ID NO:21); and / or QQYYIYPYT (SEQ ID NO:22). This humanized antibody optionally includes amino acid modifications of the aforementioned CDR residues, such that these 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. Such antibody variants can be prepared by affinity maturation, as described below, for example.

[0288] 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 antibodies comprising the variable light chain sequence and / or variable heavy chain sequence (i.e., VL and / or VH containing 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 mouse 2C4 or pertuzumab, for example, about two or four times to about 100 or about 1000 times the modified affinity, as assessed by ELISA, for example. Exemplary variable heavy chain CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two, three, four, five, six, or seven of these residues. The CDR residues used for modification include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 or two or more (e.g., combinations of two to three, four, five or up to about ten of these residues).

[0289] Humanization of mouse 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 mouse 4D5 antibodies 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 components integrated into V L Variable light chains (V) in the common framework of κ subgroup I L CDR residues and integration into V H Variable heavy chains (V) in the common frame of subgroup III H CDR residues. The antibody is further applied to V... H Positions 71, 73, 78, and 93 (Kabat numbers of FR residues) contain framework region (FR) substitutions; and at V L The FR substitution is located at position 66 (Kabat number of the FR residue). Trastuzumab contains a non-A allogeneic human γ1Fc region.

[0290] Consider various forms of humanized antibodies or affinity-matured antibodies. For example, humanized antibodies or affinity-matured antibodies can be antibody fragments. Alternatively, humanized antibodies or affinity-matured antibodies can be complete antibodies, such as complete IgG1 antibodies.

[0291] (ii) Pertuzumab composition

[0292] In one embodiment of the HER2 antibody composition, the composition comprises a natural pertuzumab antibody and a mixture of one or more variants thereof. A preferred embodiment of the natural pertuzumab antibody described 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 most 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 natural pertuzumab antibody and a mixture of its amino acid sequence variants 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 dominant 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 include 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 comprises or consists of VHS-. The presence of an amino-terminal leader extension in a composition can be detected using 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 typically between the amount constituting the detection limit for any assay used to detect the variant (preferably N-terminal sequence analysis) and less than the amount of the dominant antibody class. Typically, about 20% or less (e.g., about 1% to about 15%, e.g., 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 analysis is preferably used (preferably using a high-resolution, weak cation exchange column, such as PROPAC WCX-10). TM The percentage amount was determined using a cation exchange column. In addition to amino-terminal leader extension variants, other amino acid sequence changes of the major class of antibodies and / or variants were also considered, including but not limited to antibodies containing C-terminal lysine residues on one or both heavy chains, deamidated antibody variants, etc.

[0293] Furthermore, major types of antibodies or variants may further include glycosylation changes, with non-limiting examples including antibodies containing G1 or G2 oligosaccharide structures attached to the Fc region, antibodies containing carbohydrate moieties attached to light chains (e.g., attached to one or two light chains of the antibody, such as 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.

[0294] The composition can be recovered from genetically modified cell lines (e.g., Chinese hamster ovary (CHO) cell lines expressing HER2 antibodies) or obtained through peptide synthesis.

[0295] For more information on exemplary pertuzumab compositions, see U.S. Patent Nos. 7,560,111 and 7,879,325 and US 2009 / 0202546A1.

[0296] (iii) Trastuzumab composition

[0297] Trastuzumab compositions typically comprise a mixture of a major class of antibodies (containing the light chain and heavy chain sequences of SEQ ID NO: 13 and 14, respectively) and their variant forms, particularly acidic variants (including deamidated 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, B 752:233-245 (2001). These forms include peak A (Asn30 deamidated to Asp in two light chains); peak B (Asn55 deamidated to isoAsp in one heavy chain); peak 1 (Asn30 deamidated to Asp in one light chain); peak 2 (Asn30 deamidated 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 converted to succinimide (Asu) in one heavy chain).

[0298] (iv) Trastuzumab-pertuzumab combination in fixed-dose combination

[0299] This invention discloses extensive research on various charge variants found in the trastuzumab-pertuzumab fixed-dose combination. Acceptance criteria were established based on clinical experience and hypothetical impacts on bioactivity / PK and safety / immunogenicity profiles. The compositions presented herein are considered to have the bioactivity and PK required for safe biologics without increasing the risk of immunogenicity or safety.

[0300] 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 deamidated variants, Fc sialic acid variants and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30 and a trastuzumab variant deamidated at HC-Asn-55, at least 28% of a natural pertuzumab antibody, at least 16% of a natural trastuzumab antibody, and less than 12% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0301] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated at HC-Asn-55, at least 38% of a natural pertuzumab antibody, at least 16% of a natural trastuzumab antibody, and less than 9% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain via HC-Asp-102.

[0302] 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 deamidated variants, Fc sialic acid variants and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30 and a trastuzumab variant deamidated at HC-Asn-55, at least 28% of a natural pertuzumab antibody, at least 23% of a natural trastuzumab antibody, and less than 12% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0303] In one embodiment, a composition comprising pertuzumab, trastuzumab, and their charge variants is analyzed by ion exchange chromatography. 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 antibody and charge variant is equal to the peak area determined by ion exchange chromatography according to any of the foregoing embodiments, wherein (i) the pertuzumab variant deamided at HC-Asn-391, the pertuzumab FC sialic acid variant, the pertuzumab lysine glycated variant, the trastuzumab deamided at LC-Asn-30, and the trastuzumab deamided at HC-Asn-55 elute in peaks 1 to 3, and thus the percentage of these variants in the composition is equal to the peak area. The sum of 1 to 3, (ii) pertuzumab natural antibody eluted in peak 4 and the percentage of pertuzumab natural antibody in the composition is equal to the peak area of ​​peak 4, (iii) trastuzumab natural antibody eluted in peak 7 and the percentage of trastuzumab natural antibody in the composition is equal to the peak area of ​​peak 7, (iv) trastuzumab monoisomerized to isoflavone at one heavy chain eluted in peak 8 and the percentage of this variant in the composition is equal to the peak area of ​​peak 8.

[0304] 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 following steps:

[0305] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0306] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0307] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0308] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0309] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0310] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0311] 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.

[0312] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0313] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises a peak area of ​​less than 23% of the sum of peaks 1 to 3, a peak area of ​​at least 38% of peak 4 (pertuzumab natural antibody), a peak area of ​​at least 16% of peak 7 (trastuzumab natural antibody), and a peak area of ​​less than 9% of peak 8, as determined by the method described in any of the embodiments above. In one aspect, the method comprises the following steps:

[0314] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0315] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0316] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0317] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0318] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0319] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0320] 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.

[0321] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0322] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, wherein the composition comprises a peak area of ​​less than 21% of the sum of peaks 1 to 3, a peak area of ​​at least 28% of peak 4 (pertuzumab natural antibody), a peak area of ​​at least 23% of peak 7 (trastuzumab natural antibody), and a peak area of ​​less than 12% of peak 8, as determined by the method described in any of the embodiments above. In one aspect, the method comprises the following steps:

[0323] a. Use a loading buffer to bind such antibodies to ion exchange material, wherein the pH of the loading buffer is between approximately pH 7.5 and approximately pH 7.65;

[0324] b. Elute the antibody with an elution buffer having a pH between approximately pH 7.5 and approximately pH 7.7.

[0325] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatographic material is a strong cation exchange material. In one embodiment, the cation exchange material contains sulfonic acid groups.

[0326] In one embodiment, step b is performed using a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0327] In one embodiment, the method for evaluating the above-described fixed-dose composition comprising pertuzumab and trastuzumab further comprises the following steps:

[0328] c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition.

[0329] 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.

[0330] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0331] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated at HC-Asn-55, at least 29.2% of a natural pertuzumab antibody, at least 21.8% of a natural trastuzumab antibody, and less than 5% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain via HC-Asp-102.

[0332] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated at HC-Asn-55, at least 39.4% of a natural pertuzumab antibody, at least 21.8% of a natural trastuzumab antibody, and less than 4.1% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0333] 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 deamidated variants, Fc sialic acid variants, and lysine glycosylated variants, a trastuzumab variant deamidated at LC-Asn-30, and a trastuzumab variant deamidated at HC-Asn-55, at least 29.2% of a natural pertuzumab antibody, at least 31% of a natural trastuzumab antibody, and less than 5% of a trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0334] In one embodiment, the composition comprising pertuzumab and trastuzumab comprises 40 to 60 mg / mL trastuzumab and 60–80 mg / mL pertuzumab.

[0335] In another aspect of the invention, the composition provided herein can be obtained by a method comprising the following steps:

[0336] a. Add the predetermined amount of pertuzumab to the mixing dish.

[0337] b. Add trastuzumab at a 1:1 ratio to pertuzumab or a 1:2 ratio to pertuzumab.

[0338] c. Add rHuPH20.

[0339] In one embodiment, a 1:1 trastuzumab / pertuzumab ratio produces a composition comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, a 1:2 trastuzumab / pertuzumab ratio produces 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.

[0340] IV. Recombinant HER2 extracellular domain

[0341] The inventors have discovered that by including a recombinant subdomain III truncated at the C-terminus, a modified HER2 ECD lacking subdomain IV can be produced, exhibiting a three-dimensional conformation 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 another embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO.24 is provided.

[0342] 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.

[0343] In one embodiment, a modified ECD comprising SEQ ID NO:33, SEQ ID NO:3, and SEQ ID NO:4 is provided. In another embodiment, the modified ECD comprises SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:3, and SEQ ID NO:4.

[0344] The inventors have discovered that by including a recombinant subdomain I 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, exhibiting a three-dimensional conformation similar to that of the natural HER2 ECD. In one embodiment, a modified HER2 ECD comprising SEQ ID NO. 29 is provided. In another embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 29 is provided.

[0345] 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 embodiments described above, 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 HER2ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO. 32 is provided.

[0346] Recombinant HER2 extracellular domains can be generated and purified using methods known in the art. In one embodiment, a method for preparing a recombinant HER2 extracellular domain is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the recombinant HER2 extracellular domain under conditions suitable for expression of the recombinant HER2 extracellular domain, and optionally recovering the recombinant HER2 extracellular domain from the host cell (or host cell culture medium). In 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 cloning and / or expression in host cells. Such nucleic acid can be isolated and sequenced using conventional procedures, generated by recombinant methods, or obtained through chemical synthesis.

[0347] Host cells used for cloning or expressing 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. Following expression, the recombinant HER2 extracellular domain can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0348] Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning 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 conjunction 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 adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines include monkey kidney CV1 cell lines transformed with 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); young 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); and mouse mammary tumors (MMT). 060562); TRI cells (as described in the following literature: e.g., Mather, JP et al., Annals N.Y. Acad. 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 respect, the host cell is, for example, Chinese hamster ovary (CHO) cells.

[0349] V. Reagent Kit

[0350] This invention also provides a kit for specifically quantifying the binding of antibodies to HER2 extracellular subdomain II in a fixed-dose combination (FDC), the fixed-dose combination containing a first antibody and a second anti-HER2 antibody binding to HER2 extracellular subdomain II, the kit comprising:

[0351] (a) A container containing proteins comprising SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:34 as a capture agent.

[0352] (b) Explanation of the binding of antibodies that quantify binding to HER2 extracellular subdomain II.

[0353] 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.

[0354] 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.

[0355] In one embodiment, these descriptions further include instructions on establishing a relationship between the binding of a first antibody to the extracellular subdomain II of HER2 and its potency.

[0356] In one embodiment, the second antibody binds to an epitope different from that of the first antibody. In one embodiment, the second antibody is an antibody that binds to the HER2 extracellular subdomain IV.

[0357] In one embodiment, the first antibody is pertuzumab. In another embodiment, the second antibody is trastuzumab.

[0358] 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.

[0359] This invention also provides a kit for binding of an antibody to the HER2 extracellular subdomain IV in a specific quantification fixed-dose combination (FDC), the fixed-dose combination containing an antibody to the HER2 extracellular subdomain IV and a second anti-HER2 antibody, the kit comprising:

[0360] (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 agent.

[0361] (b) Explanation of the binding of antibodies that quantify binding to the extracellular subdomain IV of HER2.

[0362] 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.

[0363] 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.

[0364] In one embodiment, these descriptions further include instructions on establishing a correlation between the binding of an antibody to the HER2 extracellular subdomain IV and its potency.

[0365] In one embodiment, the second antibody binds to an epitope different from that of the first antibody. In one embodiment, the second antibody is an antibody that binds to the HER2 extracellular subdomain II.

[0366] In one embodiment, the first antibody is trastuzumab. In another embodiment, the second antibody is pertuzumab.

[0367] 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.

[0368] VI. Manufacturing Method

[0369] In one embodiment, a method of 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 deamided at HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumab lysine glycation variant, trastuzumab deamided at LC-Asn-30, trastuzumab deamided at HC-Asn-55, (ii) a natural pertuzumab antibody, (iii) a natural trastuzumab antibody, and (vi) trastuzumab monoisomeric to isaspartic acid at one heavy chain at HC-Asp-102.

[0370] In one embodiment, the variant comprises (i) less than 23% of the following variants: pertuzumab deamidated at HC-Asn-391, pertuzumab FC sialic acid variant and pertuzumab lysine glycation variant, trastuzumab deamidated at LC-Asn-30, and trastuzumab deamidated at HC-Asn-55; (ii) at least 28% of natural pertuzumab antibody; (iii) at least 16% of natural trastuzumab antibody; and (iv) less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain.

[0371] In one embodiment, the variant comprises (i) less than 23% of the following variants: pertuzumab deamidated at HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumab lysine glycation variant, trastuzumab deamidated at LC-Asn-30, or trastuzumab deamidated 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.

[0372] In one embodiment, the variant comprises (i) less than 21% of the following variants: pertuzumab deamidated at HC-Asn-391, pertuzumab FC sialic acid variant and pertuzumab lysine glycation variant, trastuzumab deamidated at LC-Asn-30, and trastuzumab deamidated at HC-Asn-55; (ii) at least 28% of natural pertuzumab antibody; (iii) at least 23% of natural trastuzumab antibody; and (iv) less than 12% of trastuzumab monoisomerized to isaspartic acid at one heavy chain at HC-Asp-102.

[0373] In one embodiment, the analytical assay is ion exchange chromatography. In another embodiment, the analytical assay is ion exchange chromatography according to any of the embodiments described above. In one embodiment, these percentages are equal to the peak areas measured by ion exchange chromatography according to any of the embodiments described above, wherein (i) the pertuzumab variant deamided at HC-Asn-391, the pertuzumab FC sialic acid variant, the pertuzumab lysine glycated variant, the trastuzumab deamided at LC-Asn-30, and the trastuzumab deamided at HC-Asn-55 elute in peaks 1 to 3 and thus the percentage of such variants in the composition is equal to the total peak areas 1 to 3. (ii) Pertuzumab natural antibody elutes out in peak 4 and the percentage of pertuzumab natural antibody in the composition is equal to the peak area of ​​peak 4; (iii) Trastuzumab natural antibody elutes out in peak 7 and the percentage of trastuzumab natural antibody in the composition is equal to the peak area of ​​peak 7; (iv) Trastuzumab monoisomerized to isoflavone at one heavy chain elutes out in peak 8 and the percentage of this variant in the composition is equal to the peak area of ​​peak 8.

[0374] In one embodiment, the amounts of the following other variants are analyzed in an 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 deamidation, trastuzumab with lysine glycosylation, and trastuzumab with increased Fc sialic acid content.

[0375] In one embodiment, the analytical assay is ion exchange chromatography. In another embodiment, the analytical assay is ion exchange chromatography of any of the embodiments described above. In one embodiment, these percentages are equal to the peak areas measured by ion exchange chromatography of any of the embodiments described above, wherein (vii) pertuzumab having N-terminal VHS on both the heavy and light chains, pertuzumab having C-terminal lysine on the heavy chain, trastuzumab having HC-Asn-392 deamidation, trastuzumab having lysine glycosylation, and trastuzumab with increased Fc sialic acid content elute in peaks 5-6, thereby the percentage of these variants in the composition is equal to the peak areas of peaks 5-6.

[0376] In one embodiment, the amounts of the following other variants are analyzed in an analytical assay: (vi) trastuzumab monoisomeric to succinimide at one heavy chain and trastuzumab with Fc oxidation.

[0377] 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.

[0378] They elute in peaks 9-10. Therefore, the percentage of these variants in the composition is equal to the peak area of ​​peaks 9-10.

[0379] 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.

[0380] In one embodiment, step (1) of the preparation method described above includes the following steps:

[0381] a. Add the predetermined amount of pertuzumab to the mixing dish.

[0382] b. Add trastuzumab at a 1:1 ratio to pertuzumab or a 1:2 ratio to pertuzumab.

[0383] c. Add rHuPH20.

[0384] In one embodiment, a 1:1 trastuzumab / pertuzumab ratio produces a composition comprising 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, a 1:2 trastuzumab / pertuzumab ratio produces a composition comprising 40 mg / mL trastuzumab and 80 mg / mL pertuzumab.

[0385] In one embodiment, rHuPH20 is added to the composition to achieve a final concentration of 2000 U / ml rHuPH20.

[0386] VII. Selecting patients for treatment

[0387] 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... (Dako) and HER2 (immunohistochemical (IHC) assay) and and HER2 FISH pharmDx TM (FISH assay). For information on validation and performance of each assay, users should refer to the packaging insert of the specific assay kit.

[0388] For example, through IHC, for example using (Dako) was used to analyze HER2 expression or overexpression. Paraffin-embedded tissue sections from tumor biopsy can undergo IHC assay and meet the following HER2 protein staining intensity criteria:

[0389] Score 0: No staining was observed or membrane staining was observed in less than 10% of tumor cells.

[0390] Score 1+: Weak / virtually imperceptible membrane staining was detected in more than 10% of tumor cells. Staining occurred only in the membrane portion of the cells.

[0391] Score 2+: Weak to moderate complete membrane staining was observed in more than 10% of tumor cells.

[0392] Score 3+: Moderate to strong complete membrane staining was observed in more than 10% of tumor cells.

[0393] Tumors with a HER2 overexpression score of 0 or 1+ can be characterized as HER2 negative, while those with a score of 2+ or 3+ can be characterized as HER2 positive.

[0394] Tumors that overexpress HER2 can be graded based on immunohistochemical scores corresponding to the number of HER2 molecules expressed per cell, and this can be determined biochemically.

[0395] 0 = 0-10,000 copies / cell

[0396] 1+ = at least approximately 200,000 copies / cell.

[0397] 2+ = at least approximately 500,000 copies / cell,

[0398] 3+ = at least approximately 2,000,000 copies / cell.

[0399] HER2 overexpression at grade 3+ can lead to ligand-independent activation of tyrosine kinases (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 impaired (Slamon et al., Science, 244:707-712 (1989); Slamon et al., Science, 235:177-182 (1987)).

[0400] 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) can be performed on formalin-fixed, paraffin-embedded tumor tissue. TM (Sold by Ventana, Arizona) or (Vysis, Illinois) to determine the extent of HER2 amplification in the tumor (if present).

[0401] Most typically, archived paraffin-embedded tumor tissue is used and any of the aforementioned methods are used to confirm HER2 positivity.

[0402] 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.

[0403] 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 mutations G309A, G309E, S310F, D769H, D769Y, V777L, P780-Y781insGSP, V842I, R896C (Bose et al., CancerDiscov 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.

[0404] For alternative assays and examples for screening patients for pertuzumab therapy, see also U.S. Patent No. 7,981,418.

[0405] Table 1: Sequences

[0406]

[0407]

[0408] Table 2 - List of abbreviations and definitions for terms

[0409]

[0410]

[0411] Example 1: Pertuzumab-Trastuzumab FDC

[0412] 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.

[0413] FDC Pharmaceuticals provides sterile, colorless to light brownish solutions for subcutaneous injection. They are preservative-free. Two formulations are available as described below:

[0414] Loading dose: FDC drug LD

[0415] 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 at 80 mg / mL pertuzumab and 40 mg / mL trastuzumab. The excipients used in the formulation are L-histidine, L-histidine hydrochloride monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0416] Maintenance dose: FDC drug MD

[0417] 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 at 60 mg / mL pertuzumab and 60 mg / mL trastuzumab. The excipients used in the formulation are L-histidine, L-histidine hydrochloride monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0418] Example 2: Potency of pertuzumab-trastuzumab FDC as determined by cell-based assay

[0419] This method determined 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 microtiter plates 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.

[0420] Fluorescence was measured using excitation at 530 nm and emission at 590 nm.

[0421] AlamarBlue dye is blue and non-fluorescent in its oxidized state, but it is reduced by the intracellular environment of the cell to a highly fluorescent pink form. 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 procedure was used to estimate the antiproliferative activity of the FDC samples relative to the reference standard.

[0422] Cell-based assays are selectively sensitive to one or more antibodies in FDC drugs, but not to both. Figure 7AAs shown in Figure B. When analyzed individually, trastuzumab exhibited antiproliferative activity against BT-474 cells but not against MDA-MD-175VII cells; while pertuzumab showed antiproliferative activity against MDA-MB-175VII 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-175VII), rather than differences in HER2 affinity. Additionally, HER3-expression levels and other potential parameters involving overall antiproliferative activity (e.g., the presence or absence of HER3 endogenous ligand regulatory proteins) can contribute to differences in sensitivity. Furthermore, in cell-based assays of drug substances, the presence of one antibody can affect the response of the other, thus 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. Figure 8A And B). Although trastuzumab alone could not inhibit the proliferation of MDA-MB-175VII cells in the pertuzumab anti-proliferation assay ( Figure 7A (and B), but adding it to pertuzumab shifts the trastuzumab dose-response curve to a lower EC50 value, reflecting the higher potency when combined with pertuzumab. Figure 8A Therefore, no slight quality change of pertuzumab in the FDC drug was detected in the MDA-MB-175VII antiproliferative assay. Similar observations were obtained for pertuzumab in the BT-474 antiproliferative assay, but less pronounced. Figure 8B In addition, slight changes in antibody mass along opposite directions can produce 100% potency.

[0423] To confirm that no substantial quality change of any antibody in the FDC drug was detected in the antiproliferative assay, HER2 affinity mutants of pertuzumab and trastuzumab with directed changes in CDR (HC S55A and L C H91A mutations, respectively) were tested in the pertuzumab and trastuzumab antiproliferative assays. Figure 9A (and B). The significantly reduced affinity of the mutant for HER2 was associated with its decreased antiproliferative activity in certain cell-based assays. Adding pertuzumab to the trastuzumab mutant (or vice versa) partially restored the dose-response profile and thus the antiproliferative activity.

[0424] 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 co-prepared formulation. These limitations prevent antiproliferative assays from being used to determine and control the biological activity 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.

[0425] Example 3: Potency of pertuzumab in FDC as measured by ELISA

[0426] 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.

[0427] Pertuzumab is a monoclonal IgG1 antibody targeting HER2, specifically HER2's extracellular subdomain II. Upon binding, pertuzumab blocks HER2 activation by preventing heterodimerization of HER2 with ligand-activating members of the HER receptor family. This inhibits downstream signaling pathways in HER2-overexpressing cells.

[0428] Pertuzumab ELISA will determine the biological activity of pertuzumab by its ability to specifically bind to the epitope (i.e., subdomain II) of recombinant HER2. Figure 6 A schematic diagram of the capture reagents used in pertuzumab ELISA and trastuzumab ELISA is shown (see Example 6 for details).

[0429] Binding is measured using a secondary antibody that binds to peroxidase. Dose-response curves generated for 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 dilution preparation. Pertuzumab ELISA is used for both the LD and MD of the FDC drug.

[0430] Equipment and materials

[0431] 96-well immunoassay plate (e.g., Maxisorp ELISA)

[0432] Absorption brightness reading

[0433] Computers equipped with 4-parameter data reduction software and parallelism analysis software (such as SoftMaxPro)

[0434] Microplate scrubber

[0435] reagents

[0436] • Pertuzumab coating reagent: recombinant HER2 extracellular domains I, II, and III fused to mouse Fc; without domain IV (containing trastuzumab epitope) (SEQ ID NO:27).

[0437] • Antibody detection: HRP-bound goat anti-human antibodies (specific to the F(ab')2 moiety of human IgG) (e.g., Jackson ImmunoResearch)

[0438] • 1X DPBS, calcium and magnesium-free

[0439] • Purified water, such as Milli-Q.

[0440] ·BSA Component 5

[0441] Tween 20

[0442] ABTS substrate solution

[0443] Concentrated phosphoric acid (85%)

[0444] solution

[0445] Note: The formula is for the nominal amount of reagent and can be adjusted proportionally according to the measurement requirements.

[0446] Washing buffer: 1X DPBS, 0.05% Tween 20

[0447] Diluents for assay: 1X DPBS, 0.05% Tween 20, 0.5% BSA (fraction 5)

[0448] Coating solution: Pertuzumab coating reagent (1 μg / mL) in 1X DPBS

[0449] Detection antibody: 0.8 mg / mL HRP-bound goat anti-human antibody

[0450] Detection solution: The detection solution is prepared by diluting the detection antibody (0.8 mg / mL) to a concentration of 16 ng / mL in the assay diluent. Prepare fresh before use.

[0451] Termination solution: 1M phosphoric acid

[0452] Reference Standard FDC MD Reference Standard

[0453] Coated plate

[0454] Transfer 100 μL of the coating solution into each well of the microtiter plate.

[0455] – Incubate the coated plate at 2℃-8℃ for 30-60 minutes.

[0456] Blocking plate

[0457] Excess coating solution was removed by washing all coated plates three times with 300 μL / well washing buffer.

[0458] – Block all plates by adding 100 μL of assay diluent to each well.

[0459] Incubate the plate at ambient temperature with gentle shaking for 60-90 minutes.

[0460] – Wash the plate three times with 300 μL / well of washing buffer.

[0461] Sample transfer

[0462] – Transfer 100 μL / well of FDC reference standard, product control and sample dilution to the wells of the immunoassay plate.

[0463] Incubate the plate at ambient temperature with gentle shaking for 60-90 minutes.

[0464] Detection

[0465] – Transfer 100 μL of the detection solution (16 ng / mL) into each well of the plate.

[0466] Incubate the plate at ambient temperature with gentle shaking for 30-90 minutes.

[0467] Wash the plate three times with 300 μL / well of washing buffer.

[0468] Substrate transfer and measurement

[0469] – Transfer 100 μL / well of ABTS substrate solution into each well of the plate.

[0470] Incubate the plate at ambient temperature with gentle shaking for 20-35 minutes.

[0471] – To terminate the reaction, transfer 100 μL / well of the termination solution into each well of the plate.

[0472] – Mix the plate by gently stirring for at least 1 minute.

[0473] – OD values ​​were measured over 30 minutes using an absorbance reading instrument at a wavelength of 405 nm (reference wavelength 490 nm).

[0474] Evaluate

[0475] –Calculate the OD value for each pore as follows: OD(405nm) - OD(490nm)

[0476] Where: OD(405nm): Detected absorbance at 405nm, OD(490nm): Reference absorbance at 490nm.

[0477] – The average OD value of parallel measurements is taken to determine the mean OD.

[0478] – Dose-response curves for standards, product controls, and samples are generated by plotting the average OD(y) against pertuzumab antibody concentration (in ng / mL) (x).

[0479] – Apply nonlinear regression using the following 4-parameter equation:

[0480]

[0481] in:

[0482] A: Lower asymptote

[0483] B: Hill slope

[0484] C:EC 50 value

[0485] D: Lower asymptote

[0486] – Calculate the R² of the standard curve, product control curve, and sample curve.

[0487] –Calculate the standard ΔOD as follows:

[0488] Standard ΔOD = (Average maximum OD of standard products) - (Average minimum OD of standard products)

[0489] –The maximum OD is determined as follows:

[0490] The maximum OD value is the maximum OD value obtained at 405 nm across all parallel measurements of the dose-response curve.

[0491] Calculation of effectiveness

[0492] – Use 4-parameter parallel line analysis to calculate a common Hill slope, lower asymptote, and lower asymptote for the standard curve and the sample (or product control) curve.

[0493] –The equations for the curves obtained from standards and samples (or product controls) are as follows:

[0494]

[0495] in:

[0496] A = Common Lower Asymptote

[0497] B = Common Hill Slope

[0498] C 标准 =Standard EC 50 value

[0499] D = Common Lower Asymptote

[0500] ρ = the effectiveness of the sample and product control relative to the reference standard.

[0501] – The relative effectiveness is calculated as follows:

[0502] Relative efficacy = ρ × Activity of reference standard

[0503] Reference Standard Validity Assignment

[0504] 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 both MD and LD samples of the FDC drug. The FDC MD reference standard is selected as the potency reference standard.

[0505] Details regarding the efficacy assignment of the FDC MD reference standard are provided below:

[0506] - Set the potency to 1.00×10 4 U / mg.

[0507] - The potency of pertuzumab was determined by ELISA relative to the commercial pertuzumab IV reference standard anti2C4907-2.

[0508] - Trastuzumab potency was determined by ELISA relative to the commercial trastuzumab SC reference standard G005.03EP1.

[0509] 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 middle.

[0510] Example 4: Specificity of pertuzumab ELISA

[0511] To assess the specificity of the pertuzumab ELISA in Example 3, formulation buffers and structure-related molecules were tested in duplicate on a single plate at the highest assay concentration. 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).

[0512] These results confirm that the pertuzumab ELISA is specific for pertuzumab:

[0513] • 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.

[0514] • Structure-related molecules (including trastuzumab, except pertuzumab, see below) do not interfere with pertuzumab ELISA. This is demonstrated by the following results: the mean OD value of parallel assays is less than three times the mean OD value of the lower asymptote of the dose-response curve of the reference standard.

[0515] As expected, pertuzumab SC drug substance and IV pertuzumab in FDC drug formulation exhibited pertuzumab ELISA interference because they bind to the same HER2 domain II.

[0516] The results are shown in Table 3.

[0517] Table 3: ELISA Specificity of Pertuzumab

[0518]

[0519]

[0520] Example 5: Robustness of pertuzumab ELISA

[0521] The robustness of pertuzumab ELISA was assessed by deliberately varying the assay parameters, which are potential sources of variation in practice. Robustness results were evaluated by comparing the obtained dose-response curve parameters, system suitability, and similarity standards with the methodological procedures. Overall robustness results are summarized in Table 4.

[0522] Table 4: Robustness results of pertuzumab ELISA.

[0523]

[0524] Example 6: Potency of trastuzumab in FDC as measured by ELISA

[0525] Two ELISA-like assays 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.

[0526] 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.

[0527] This inhibits downstream signaling pathways in HER2-overexpressing cells.

[0528] Trastuzumab ELISA will determine the biological activity of trastuzumab by its ability to specifically bind to its epitope (i.e., subdomain IV) of recombinant HER2. Figure 6 A schematic diagram of the capture reagent used in trastuzumab ELISA is shown.

[0529] Binding is measured using a secondary antibody that binds to peroxidase. Dose-response curves generated for 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 dilution preparation. Trastuzumab ELISA is used for both LD and MD of the FDC drug.

[0530] Reagents, buffer solutions, and procedures are as outlined in Example 3, except for the coating reagent and coating solution:

[0531] • Trastuzumab coating reagent: recombinant HER2 extracellular domains I, III, and IV fused to mouse Fc; domain II is replaced by the structure-associated domain II (SEQ ID NO:32) in EGFR that cannot bind pertuzumab.

[0532] • Coating solution: Trastuzumab coating reagent (1 μg / mL) in 1X DPBS

[0533] Evaluate

[0534] –Calculate the OD value for each pore as follows: OD(405nm) - OD(490nm)

[0535] Where: OD(405nm): Detected absorbance at 405nm, OD(490nm): Reference absorbance at 490nm.

[0536] – The average OD value of parallel measurements is taken to determine the mean OD.

[0537] – Dose-response curves for standards, product controls, and samples are generated by plotting the average OD(y) against trastuzumab antibody concentration (in ng / mL) (x).

[0538] – Apply nonlinear regression using the following 4-parameter equation:

[0539]

[0540] in:

[0541] A: Lower asymptote

[0542] B: Hill slope

[0543] C:EC50 value

[0544] D: Lower asymptote

[0545] – Calculate the R-values ​​for the standard curve, product control curve, and sample curve. 2 .

[0546] –Calculate the standard ΔOD as follows:

[0547] Standard ΔOD = (Average maximum OD of standard products) - (Average minimum OD of standard products)

[0548] –The maximum OD is determined as follows:

[0549] The maximum OD value is the maximum OD value obtained at 405 nm across all parallel measurements of the dose-response curve.

[0550] Calculation of effectiveness

[0551] – Use 4-parameter parallel line analysis to calculate a common Hill slope, lower asymptote, and lower asymptote for the standard curve and the sample (or product control) curve.

[0552] –The equations for the curves obtained from standards and samples (or product controls) are as follows:

[0553]

[0554] in:

[0555] A = Common Lower Asymptote

[0556] B = Common Hill Slope

[0557] C 标准 =Standard EC 50 value

[0558] D = Common Lower Asymptote

[0559] ρ = the effectiveness of the sample and product control relative to the reference standard.

[0560] – The relative effectiveness is calculated as follows:

[0561] Relative efficacy = ρ × Activity of reference standard

[0562] Reference Standard Validity Assignment

[0563] 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 both MD and LD samples of the FDC drug. The FDC MD reference standard is selected as the potency reference standard.

[0564] Details regarding the efficacy assignment of the FDC MD reference standard are provided below:

[0565] - Set the potency to 1.00×10 4 U / mg.

[0566] - The potency of pertuzumab was determined by ELISA relative to the commercial pertuzumab IV reference standard anti2C4907-2.

[0567] - Trastuzumab potency was determined by ELISA relative to the commercial trastuzumab SC reference standard G005.03EP1.

[0568] Results: Trastuzumab binding in a pertuzumab-trastuzumab fixed-dose combination was analyzed in a trastuzumab ELISA assay. Representative dose-response curves are shown in [image / data missing]. Figure 11 middle.

[0569] Example 7: Specificity of trastuzumab ELISA

[0570] To assess the specificity of trastuzumab ELISA, formulation buffer and structure-related molecules were tested in duplicate on a single plate at the highest assay concentration. 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).

[0571] The results confirmed that trastuzumab ELISA is specific for trastuzumab:

[0572] • 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.

[0573] • Results-related molecules (including pertuzumab, excluding trastuzumab, see below) do not interfere with trastuzumab ELISA. This is demonstrated by the following results: the mean OD value of parallel assays is less than three times the mean OD value of the lower asymptote of the dose-response curve of the reference standard.

[0574] As expected, trastuzumab (IV and SC) and trastuzumab-etansine exhibited trastuzumab ELISA interference because they bind to the same HER2 domain IV.

[0575] The results are shown in Table 5.

[0576] Table 5: ELISA Specificity of Trastuzumab

[0577]

[0578]

[0579] Example 8: Robustness of trastuzumab ELISA

[0580] The robustness of trastuzumab ELISA was assessed by intentionally varying the assay parameters, which are potential sources of variation in practice. Robustness results were evaluated by comparing the obtained dose-response curve parameters, system suitability, and similarity criteria with the methodological procedures. Overall robustness results are summarized in Table 6.

[0581] Table 6: Robustness results of trastuzumab ELISA.

[0582]

[0583] Example 9: Development of IEC for analyzing FDC charge variants

[0584] Various ion exchange chromatography protocols have been tested to resolve 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.

[0585] The research and development testing methods are used to separate the following peaks / peak groups and determine their relative abundance (expressed as % of total peak area):

[0586] -Sum of peaks 1-3

[0587] - Peak 4 (pertuzumab main peak)

[0588] -Sum of peaks 5-6

[0589] - Peak 7 (trastuzumab main peak)

[0590] -Peak 8

[0591] -Sum of peaks 9-10

[0592] A FDC IE-HPLC method has been developed and optimized to achieve optimal separation between 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).

[0593] In the first step, registration IE-HPLC methods for individual molecules of IV Pertuzumab and IV / SC Herceptin were 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) was used (see Methods 1 and 2 in Table 7). In the next step, the ProPac WCX-10 column was tested using operating conditions successfully applied to 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 buffers and pH values ​​were tested. The parameters used and the results are summarized in Table 7 below.

[0594] In the next step, different columns were screened. Optimal separation was achieved using a strong cation exchange column (Mab PAC SCX-10). Different buffers 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 optimal results (see Table 8).

[0595] result

[0596] Method 1: When using the conditions of Method 1 to analyze pertuzumab-trastuzumab FDC, the peak resolution was not satisfactory for the requirements of product release assay: the resolution between peak 7 (trastuzumab main peak) and peak 8 (trastuzumab IsoAsp102) was poor and the basic pertuzumab region overlapped with the trastuzumab main peak.

[0597] Method 2: When using the conditions of Method 2 to analyze pertuzumab-trastuzumab FDC, the peak separation was not satisfactory for the requirements of product release assay: the basic pertuzumab region completely overlapped with the main trastuzumab peaks (peak 7) and 8, which was therefore unacceptable.

[0598] 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.

[0599] Method 4: The basic pertuzumab region overlaps with the main peak of trastuzumab and the IsoAsp102 peak (peak 8) of trastuzumab.

[0600] Method 5: 1: pH 7.5: Thoroughly separate the two main peaks and peak 8. The alkaline pertuzumab region has only a small overlap with the trastuzumab main peak.

[0601] 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.

[0602] Method 6: Thoroughly separate all substances of interest.

[0603] Table 7: Development of IEC schemes for analyzing FDC charge variants.

[0604]

[0605] Table 8: Development of the IEC scheme used to analyze FDC charge variants. The test parameters of Method 6 above are investigated.

[0606]

[0607] Based on the HPLC parameters described in Table 8 above, several experimental designs (Design of Experiments, DoE) were implemented. The following parameters were tested:

[0608] Gradient type

[0609] • Flow rate (0.5–1.0 mL / min)

[0610] • pH values ​​of mobile phase A and mobile phase B (7.4-7.6 and 6.8, respectively)

[0611] • NaCl concentration in mobile phase B (100-300 mM)

[0612] Column temperature (25-40℃)

[0613] By analyzing the data obtained within these experimental designs, the following test parameters demonstrate optimal results:

[0614] • Eluent A: 20mM ACES, pH 7.5

[0615] • Eluent B: 20mM ACES, 200mM NaCl, pH 7.5

[0616] ·Column MabPac SCX-10,BioLC,4×250mm

[0617] Column temperature: 40℃

[0618] • Flow rate: 0.8 mL / min

[0619] • Injection volume: 10 μL (100 μg protein)

[0620] • Gradient: 1% to 47% B over 40 minutes

[0621] To analyze the robustness of this research method, a DoE-based experimental factorial design was performed. Therefore, the following parameters varied in the matrix:

[0622] • ACES concentration: 18–22 mM

[0623] • NaCl concentration: 180–220 mM

[0624] Column temperature: 36-44℃

[0625] • Flow rate: 0.7-0.9 mL / min

[0626] pH: 7.4-7.6

[0627] Injection volume: 8-12 μL (80–120 μg)

[0628] The results of these experiments show that the testing method is relatively stable within the testing range for the trastuzumab main peak (peak 7) and peak 8. However, highly variable purity values ​​were observed for the pertuzumab main peak (peak 4) and the intermediate region (the region between the pertuzumab main peak and the trastuzumab main peak). This variability is primarily dependent on pH and column temperature. Statistical evaluation of these experiments yields the following setup for the final method:

[0629] • Eluent A: 20mM ACES, pH 7.6

[0630] • Eluent B: 20mM ACES, 200mM NaCl, pH 7.6

[0631] Column temperature: 36℃

[0632] • Flow rate: 0.8 mL / min

[0633] • Injection volume: 10 μL (100 μg protein)

[0634] • Gradient: 1% to 47% B over 40 minutes

[0635] Evaluate possible alternatives for determining charge inhomogeneity in pertuzumab / trastuzumab FDC variants. Among these alternatives, assess the suitability of different column types and pH gradient methods.

[0636] Several separation attempts were also carried out using a weak anion exchange column, ProPac WAX-10bio LC (4×250mm), under the following chromatographic conditions:

[0637] • Prepare the following eluents A and B and perform tests:

[0638] 1.A=20mM CAPSO(pH 10.0),B=20mM CAPSO+250mM NaCl(pH 10.0)

[0639] 2. A = 20 mM hexahydropyrazine (pH 10.0), B = 20 mM hexahydropyrazine + 250 mM NaCl (pH 10.0)

[0640] 3.A=20mM trisma(pH 10.5),B=20mM trisma+250mM NaCl(pH10.5)

[0641] 4.A=20mM trisma(pH 8.0),B=20mM trisma+250mM NaCl(pH 8.0)

[0642] 5. A = 20 mM phosphate (pH 11.0), B = 20 mM phosphate + 250 mM NaCl (pH 11.0)

[0643] Column temperature: 30℃

[0644] • Flow rate: 0.8 mL / min; 1.0 mL / min

[0645] • Injection volume: 5 μL (50 μg protein)

[0646] Gradient 1: 0 to 100% B over 60 minutes

[0647] Gradient 2: 0 to 100% B over 40 minutes

[0648] For all the conditions tested on the weak anion exchange column, the substance of interest was not retained on the column and eluted along with the injection peak, thus demonstrating that these experimental conditions are simply not suitable for separating the charge variants of pertuzumab / trastuzumab FDC.

[0649] Experiments in pH-gradient separation mode

[0650] The suitability of the pH gradient-based IEC method was assessed as a possible alternative to the salt gradient method. A strong cation exchange column (MabPac SCX-10 column) and the following HPLC test parameters were used:

[0651] • Eluent A: 10mM Tris, 10mM phosphate, 10mM hexahydropyrazine, pH 6.0

[0652] • Eluent B: 10mM Tris, 10mM phosphate, 10mM hexahydropyrazine, pH 11.0

[0653] • Eluent C: 100mM NaCl

[0654] • Eluent D: Pure water

[0655] ·Column MabPac SCX-10,BioLC,4×250mm

[0656] Column temperature: 35℃

[0657] • Flow rate: 0.5 mL / min

[0658] • Injection volume: 10 μL (10 μg protein)

[0659] • Gradient: 10% to 50% B over 45 minutes (see details below)

[0660] Equipment: Waters Alliance

[0661] Eluents 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 eluent C / eluent D varied from 0% eluent C / 50% eluent D (0 mM NaCl) to 50% eluent C / 0% eluent 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% eluent A / 10% eluent B.

[0662] Optimal separation was achieved using 40 mM NaCl; however, under these conditions, the peaks eluted very early, and the two main peaks exhibited broad shapes and relatively short heights.

[0663] The DoE design was implemented using a salt concentration of 40 mM NaCl, while simultaneously varying the gradient pattern and flow rate. However, no statistical model could be established, demonstrating a lack of robustness in the investigated method, even with minor modifications to the test conditions.

[0664] 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.

[0665] Example 10: IEC used to analyze FDC charge variants

[0666] Purpose and Principle

[0667] 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 elute first in a salt gradient, followed by more basic substances. The same method was applied to the LD and MD of FDC pharmaceuticals. FDC MD reference standards were used to test the LD and MD of FDC pharmaceuticals.

[0668] Equipment and materials

[0669] • HPLC system equipped with a UV detector (WatersAlliance 2695 / e2695 with 2487 / 2489 detector or equivalent)

[0670] • HPLC column (Thermo Scientific MAbPac SCX-10, 4mm-250mm, particle size: 10μm or equivalent particle size)

[0671] solution

[0672] • Drug product dilution buffer: 20 mM L-histidine / L-histidine monohydrochloride, 105

[0673] mM trehalose, 100mM sucrose, 10mM methionine, 0.04% [w / v] polysorbate 20, pH 5.5±0.2

[0674] • Mobile phase A: 20 mM ACES, pH 7.60 ± 0.05

[0675] • Mobile phase B: 20 ​​mM ACES, 200 mM sodium chloride, pH 7.60 ± 0.05

[0676] • CpB solution: 1 mg / mL CpB (in mobile phase A)

[0677] Preparation of sample solution:

[0678] The FDC drug was diluted with mobile phase A to prepare a sample solution with a total protein concentration of approximately 10 mg / mL and a CpB concentration of approximately 0.08 mg / mL.

[0679] Preparation of blank solution:

[0680] Dilute the drug dilution buffer in the same way as the sample.

[0681] CpB digestion

[0682] The reference standard solution, sample solution, and blank solution were incubated at 37℃±2℃ for 20±5 minutes.

[0683] Store the sample at 10℃±4℃ until analysis, and HPLC analysis must be completed within 24 hours.

[0684] program

[0685] Before injecting the first sample, flush the column with 99% mobile phase A until a stable baseline is obtained. Optionally, inject a reference solution for column conditioning purposes until a visual evaluation of the chromatogram shows that at least two consecutive injections have a continuous pattern.

[0686] Operating parameters

[0687] • Detection wavelength: 280nm

[0688] Injection volume: 10 μL

[0689] • Flow rate: 0.8 mL / min

[0690] Column temperature: 36℃±2℃

[0691] • Automatic sampler temperature: 10℃±4℃

[0692] Runtime: 60 minutes

[0693] gradient

[0694]

[0695] Injection scheme

[0696] The injection will be performed in the following order:

[0697] 1. Mobile phase A

[0698] 2. Blank solution

[0699] 3. Reference Standard

[0700] 4. Samples (maximum 10 samples)

[0701] 5. Reference Standard

[0702] 6. Blank solution

[0703] Note: If more than 10 samples are to be analyzed, place every 10 samples together with the reference standard injection solution.

[0704] result:

[0705] 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 reference). Figure 13 It can control all important charge variants of individual molecules in FDC drugs because all relevant peaks can be separated. The reported assay 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 middle.

[0706] Example 11: HPLC Robustness and Reproducibility Study

[0707] Different experiments were performed to evaluate the robustness of the analytical procedure in Example 10 for different input variables. These input variables are, in particular:

[0708] • Column temperatures (32℃, 36℃, 40℃)

[0709] ·Flow rate (0.7mL / min, 0.8mL / min, 0.9mL / min)

[0710] • pH of mobile phases A and B (pH 7.5-7.7)

[0711] • Sodium chloride concentration in mobile phase B (180 mM to 220 mM)

[0712] The analytical patterns and results obtained after changing the parameters of interest were compared with those obtained based on the target parameters. The relative differences between the results were calculated using the relative peak areas (expressed as area %) of peaks 4, 7, the sum of peaks 1-3, and peak 8. These results met the acceptance criteria, thus confirming that the procedure is appropriately stable for its intended purpose.

[0713] The repeatability of the analysis procedure is shown within the following ranges for peaks 4, 7, the sum of peaks 1-3, and peak 8:

[0714] – Injected protein of FDC drug LD ranging from 50 μg to 149 μg, covering 50% to 149%.

[0715] Nominal workload (100μg protein)

[0716] –51 μg to 153 μg of FDC drug MD injected protein, covering 51% to 153 nominal working amount (100 μg protein).

[0717] Example 12: Stability Indicator Property

[0718] 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 photostress. Impurities and related substances with different charges were separated. (Compared to unstressed samples...) Figure 12 and Figure 13 In contrast, the chromatogram of the stressed sample shows the sum of peaks 1–3 and peak 8 (data not shown). In conclusion, this procedure can indicate stability.

[0719] Example 13: ELISA was used to detect trastuzumab and pertuzumab charge variants and CDR affinity mutations in FDC. The efficacy of variants

[0720] This demonstrates the ability of ELISA to reflect the antiproliferative activity of charge variants and CDR affinity mutants:

[0721] The 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) for the HER2 affinity mutants confirmed a significant reduction in similar potency.

[0722] To evaluate charge variants, supportive technology batches of FDC drugs containing MD formulation buffers of trastuzumab or pertuzumab were used to exclude the aforementioned cross-interference of FDC drugs in cell-based assays.

[0723] 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 oxidation at Met261). Although this Fc oxidation at Met261 should not affect the target binding activity of CDRs, this variant showed reduced potency in trastuzumab ELISAs (73% vs. 91% in cell-based assays).

[0724] It cannot be definitively determined whether the fractionation process of this isoform, present in only trace 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 respect because its indication of reduced power was not reflected by cell-based antiproliferative assays.

[0725] ELISA's ability to control the bioactivity of known bioactive product variants is equivalent to that of antiproliferative assays, as detailed below:

[0726] • In both assays, the trastuzumab deamidation product variants HC Asn55 / isoAsp55 and LCAsn30 / Asp30 in peak 1 both showed reduced activity.

[0727] • In both assays, the trastuzumab product variants in peak 10, which contain succinimide at the Asp102 position in each heavy chain and have increased Fc Met oxidation, both exhibited reduced activity.

[0728] • 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 invariant activity between 80% and 120%.

[0729] Furthermore, although similar peak 8 potency was obtained in cell-based assays and ELISA, it was confirmed that no known negative impact of HC IsoAsp102 on the potency of trastuzumab IV was observed in this study. The HC Asp102-to-IsoAsp isomerization at one heavy chain of trastuzumab eluted 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 have shown that the effect of SC trastuzumab is less significant than that of IV trastuzumab.

[0730] 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.

[0731] Table 9: Association between binding activity and antiproliferative activity of trastuzumab and pertuzumab

[0732]

[0733]

[0734] a For fraction characterization, refer to Example 14.

[0735] b Qualitative estimates relative to reference standards are provided because the dose-response curves of the samples and reference standards are not similar and therefore the relative potency cannot be reported (n≥3 single-plate results).

[0736] c FDC drugs contain only the trastuzumab isoform in IE-HPLC peaks 7 to 10.

[0737] Example 14: Characterization of charge variants

[0738] Characterization of charge variants of FDC drugs separated and isolated by IE-HPLC method. Figure 13 In addition, individual antibody charge variants at release from the FDC formulation were separated and characterized using the same IE-HPLC method. Figure 13 ).

[0739] Perform comprehensive peak characterization studies using the following methods to confirm the charge variants of FDC drugs:

[0740] • LC-MS / MS for evaluating trypsin antibody peptides at chemical degradation sites.

[0741] Boric acid affinity chromatography for assessing the degree of lysine saccharification

[0742] • Combined with HILIC for the analysis of 2-AB markers in Fc glycosylation

[0743] LC-MS peptide localization:

[0744] 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 using 8 mol / L guanidine hydrochloride (pH 6.0) and reduced with dithiothreitol at 50 °C for 1 h.

[0745] Samples were buffer-exchanged (0.02 mol / L histidine hydrochloride, 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 achieved using a Synapt G2HDMS Q-ToF mass spectrometer. Relative quantification of modified peptides was performed using GRAMS AI software.

[0746] Boric acid affinity chromatography:

[0747] Boronate affinity chromatography was performed using a TSKgel Boronate-5PW affinity column. Separation was carried out at 280 nm on an HPLC system equipped with UV detection using an elution 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.).

[0748] Glycan analysis:

[0749] To assess 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. HILIC separation and fluorescence detection were performed on the labeled glycan structures 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 GFc-glycosylation profiles--part 1: separation-based methods. MAbs. 2015; 7:167-79.)

[0750] Results and conclusions:

[0751] All charge variants (relative abundance ≥1%) found in individual pertuzumab and trastuzumab molecules in FDC formulations were also detected in FDC pharmaceutical products. No new charge variants were detected in FDC pharmaceutical products compared to individual antibodies in FDC formulations at release and after storage. Table 10 summarizes these findings.

[0752] Table 10: IE-HPLC peak characterization results of FDC drugs

[0753]

[0754]

[0755] Table 10 (continued): IE-HPLC peak characterization results of FDC drugs

[0756]

[0757]

[0758] The sum of peaks 1-3 contains acidic variants of pertuzumab (HC-Asn-391 deamidation, FC sialic acid and lysine glycation) and trastuzumab (LC-Asn-30 deamidation and HC-Asn-55).

[0759] Peak 4 contains the major charge variant of pertuzumab (i.e., the natural antibody) and a lower amount of the acidic trastuzumab variant (LC-Asn-30 deamidation and HC-Asp-102 isomerization).

[0760] The sum of peaks 5-6 contains pertuzumab basic variant (N-terminal VHS on both heavy and light chains and C-terminal lysine on the heavy chain) and trastuzumab acidic variant (HC-Asn-392 deamidation, lysine glycosylation, and increased Fc sialic acid content).

[0761] Peak 7 contains the major charge variant of trastuzumab (i.e., the natural antibody) and does not show overlap with pertuzumab variants.

[0762] Peak 8 contains trastuzumab monoisomeric to isoflavone (at one heavy chain) and does not show overlap with the charge variant of pertuzumab.

[0763] 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.

[0764] 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 are controllable.

[0765] No additional co-elution or increase in existing peak co-elution was observed or anticipated during stability testing because the stress-induced charge variants of pertuzumab and trastuzumab shifted to earlier and later elution times, respectively: the peak pattern of stressed pertuzumab shifted to the acidic region of the chromatogram, while the peak pattern of stressed trastuzumab shifted to the basic region.

[0766] Example 15: FDC Composition

[0767] The sum of peaks 1-3 of the FDC drug as determined by IE-HPLC

[0768] The sum of peaks 1-3 of FDC drugs is composed of the following variants:

[0769] • Acidic variants of pertuzumab (HC Asn391 deamidation, Fc sialic acid and lysine glycosylation).

[0770] • The deamidation of Asn327 in pertuzumab was only observed in trace amounts in the IE-HPLC peak characterization study of FDC drugs.

[0771] • Acidic variants of trastuzumab (primarily deamidation of LC Asn30 and HC Asn55). Compared to LC Asn30, HC Asn55 exhibits lower degradation susceptibility under biological processes 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).

[0772] This study validates end-of-storage acceptance criteria for FDC pharmaceutical products based on clinical experience and anticipated impacts on PK / biological activity and safety / immunogenicity. The proposed acceptance criteria are suitable for controlling product quality and cover the potential impacts of pharmaceutical substances and handling and storage.

[0773] For FDC drugs, the following end-of-life acceptance criteria are established: the 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 morphology. Extensions beyond current clinical experience are considered reasonable because the impact on bioactivity and PK is low and poses no risk to immunogenicity / safety.

[0774] Safety and immunogenicity considerations: Since the acidic variants found in pertuzumab and trastuzumab materials are modifications commonly found in IgG antibodies, any increase in the content of any acidic variant within the acceptance criteria is not expected to represent a new form and therefore is not expected to increase the risk of toxicity or the incidence of ADA. This is evidenced by the low incidence of ADA and favorable safety profile in clinical studies of FDC drugs. During pivotal studies, expired 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, for trastuzumab, it has been disclosed that degradation of solvent-accessible residues is located in both conserved Fc (deamidation of HC Asn387, Asn392, and Asn393) and CDR (primarily LC Asn30 deamidation 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 deamidation site in endogenous human antibodies was significantly higher than that observed in liquid drug formulations stored at 5°C. This suggests that these degradations do not increase the safety / immunogenic risk in patients (Liu YD, vanEnk JZ, Flynn GC. Human antibody Fc deamidation in vivo. Biologicals 2009; 37:313-22). This conclusion also applies to pertuzumab deamidation, where only one deamidation site was detected in this peak region and located in the Fc region (HC Asn391).

[0775] In summary, the potential 4.3% increase in area of ​​peaks 1-3 exceeding the patient exposure value is not expected to alter the immunogenicity and safety profile of the product.

[0776] Biological activity considerations: Based on the maximum clinical experience at 18.7 area % (LD) and 16.5 area % (MD) for the acidic variants of pertuzumab and trastuzumab (sum of peaks 1-3), the standard 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 potency obtained by the ELISA values ​​described in Table 8). A 4% change in biological activity is not considered impactful. Therefore, the presence of the sum of peaks 1-3 at the standard limits is expected to maintain efficacy.

[0777] 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, deamidation in CDR is not expected to affect PK. Although charge properties are known to affect the PK behavior of antibodies, a single negative charge introduced by deamidation should not affect PK (Khawli et al., 2010). Notably, only a low degree of Fc deamidation 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 standard limits, PK is not expected to be affected.

[0778] Peak 4 of FDC drug as determined by IE-HPLC

[0779] Peak 4 of the FDC drug is part of the reported assay parameters of the IE-HPLC method and constitutes the required major charge isoform of pertuzumab. Its inclusion in the specification ensures consistent product purity.

[0780] Acceptance criteria for drug substance and drug product release and stability testing are set based on other reported assay parameters obtained by IE-HPLC, taking into account manufacturing experience and stability effects. FDC drug product acceptance criteria of ≥38% area (LD) and ≥28% area (MD) at the end of shelf life ensure product purity and proper control of the manufacturing process.

[0781] The sum of peaks 5-6 of the FDC drug as determined by IE-HPLC

[0782] The sum of FDC drug peaks 5-6 consists of the following variants:

[0783] • Basic variants of pertuzumab (N-terminal VHS, N-terminal pyroglutamic acid on the heavy and light chains, and C-terminal lysine and proline amide on the heavy chain)

[0784] • Acidic variants of trastuzumab (HC Asn392 deamidation, lysine glycation, and increased FC sialic acid content)

[0785] The sum of peaks 5 and 6 was 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 considered as stability indicators.

[0786] Peak 7 of FDC drug as determined by IE-HPLC

[0787] Peak 7 of the FDC drug is part of the output of the IE-HPLC method and constitutes the major charge isoform required for trastuzumab. Its specification ensures consistent product purity. Acceptance criteria for drug release and stability testing are set based on other reported assay parameters from the IE-HPLC method, taking into account manufacturing experience and stability effects. Acceptance criteria for the FDC drug at the end of its shelf life of ≥16.0 area % (LD) and ≥23.0 area % (MD) ensure proper control of product quality and the manufacturing process.

[0788] Peak 8 of FDC drug as determined by IE-HPLC

[0789] Peak 8 of the FDC drug is composed of trastuzumab monoisomeric to isoflavone (at one heavy chain) and does not exhibit co-elution with pertuzumab charge variants.

[0790] Peak 8 can be controlled in FDC drug release and stability tests.

[0791] This study validates the end-of-life acceptance criteria for FDC pharmaceutical products with ≤9.0 area % (LD) / ≤12.0 area % (MD) based on clinical experience and anticipated impacts on PK / biological activity and safety / immunogenicity. The proposed acceptance criteria are suitable for controlling product quality and cover the potential impacts of pharmaceutical substances and handling and storage.

[0792] Safety and immunogenicity considerations: Because the acidic variants found in the trastuzumab material are modifications commonly found in IgG antibodies, any increase in the level of any acidic variant within the acceptance criteria is unlikely to represent a new form and is unlikely to increase the risk of toxicity or the incidence of ADAs. 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 low incidence of ADAs (≤5%) and no clinical consequences related to PK, efficacy, or safety.

[0793] During pivotal studies, expired clinical trial material with up to 6.4% area of ​​FDC drug LD peak 8 and 9.4% area of ​​FDC drug MD peak 8 was 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 (deamidation of HC Asn387, Asn392, and Asn393) and CDR sites (primarily LC Asn30 deamidation 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 degree of degradation of the same FcAsn deamidation sites in endogenous human antibodies was significantly higher than that observed in liquid drug formulations stored at 5°C. It is inferred that these degradations do not increase the safety / immunogenic risk in patients (Liu et al., 2009). The potential 2.5% area increase of peak 8 (HCAsp102 isomerization to isoflavone) exceeding patient exposure is not expected to alter the immunogenicity of the product.

[0794] Bioactivity considerations: Compared to the reference standard, enriched peak 8 (92% peak purity, primarily containing a monoisomerization of HC Asp102 to isoflavone at one heavy chain) exhibits similar trastuzumab activity (100% binding activity). Therefore, the presence of peak 8 within the specification limits is expected to maintain the efficacy of the FDC drug.

[0795] PK Considerations: In the CDRs of pertuzumab and trastuzumab, isomerization of aspartate to isoflavone does not change the charge and is not expected to affect PK. Therefore, single aspartate isomerization of trastuzumab HC Asp102 should not affect PK.

[0796] The sum of peak values ​​9-10 of FDC drugs as determined by IE-HPLC

[0797] The sum of peaks 9-10 of the FDC drug consists of trastuzumab monoisomerized to succinimide (at one heavy chain) from HC Asp102, and does not show overlap with the charge variant of pertuzumab. Additionally, low levels of trastuzumab Fc oxidation were detected in these peaks. Due to the low level, no impact is expected. 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.

[0798] Therefore, the sum of peaks 9-10 in the control system does not require acceptance criteria.

[0799] Example 16: Production of FDC Compositions

[0800] The pertuzumab SC drug substance is transferred 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.

[0801] 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 target amount of trastuzumab is defined (e.g., a 1:1 API ratio at maintenance dose). 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.

[0802] Based on the volume (depending on mass and density) of the pertuzumab SC and trastuzumab SC drug substances added to the mixing vessel, the required amount of thawed rHuPH20 is added to the mixing vessel (based on the rHuPH20 solution content and activity). Multiple rHuPH20 batches can be combined for drug manufacturing.

[0803] After all components have been transferred to the mixing vessel, the solution is then homogenized by mixing.

[0804] Example 17: Development of RP-UPHLC for determining FDC content

[0805] equipment

[0806] Equivalent instruments and appropriate operating conditions can be used.

[0807] HPLC system: An HPLC system equipped with data acquisition software (with an online vacuum degasser).

[0808] Detector: UV / visible light absorbance detector or photodiode array detector

[0809] Membrane filter: 0.2μm filter (e.g., Corning catalog number: 430049)

[0810] Column: TSK-Gel G3000SWXL, 7.8×300mm, 5μm (Tosoh Bioscience, Catalog No.: 08541); or BioSuite 250, 7.8×

[0811] 300mm, 5μm (Waters, catalog number: 186002165)

[0812] reagents

[0813] • Purified water (water treated with Milli-Q)

[0814] Trifluoroacetic acid (TFA) (Fluka, Cat. Nr. 40967)

[0815] Acetonitrile (Merck, Cat. Nr. 1.00030.2500)

[0816] • Anhydrous L-histidine (Sigma, Cat.Nr.H8000)

[0817] • Sucrose (Merck, Cat. Nr. 1.07687)

[0818] L-Methionine (Sigma, Cat. Nr. 64319)

[0819] Glacial acetic acid (Merck.Cat.Nr.1.00063.1000)

[0820] Polysorbate 20 (Sigma, Cat. Nr. 93773)

[0821] Solvent A: 0.1% TFA in Milli-Q water

[0822] Solvent B: 0.1% TFA in acetonitrile

[0823] Formulation buffer: 20 mM histidine acetate, 240 mM sucrose, 10 mM methionine, and polysorbate 20, 0.02% [w / v], pH 5.7 ± 0.2

[0824] Dilution buffer: 20 mM acetate histidine, pH 5.5

[0825] Column storage solution: 60% acetonitrile (v / v)

[0826] 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.

[0827] mg / mL.

[0828] Blank: Inject formulation buffer and dilution buffer without dilution.

[0829] Flow rate: 0.4 mL / min

[0830] Maximum pressure: 400 bar / 6000 psi

[0831] Wavelength: 280nm

[0832] Runtime: 29 minutes

[0833] Column temperature setting: 60℃

[0834] Automatic sampler temperature setting: ≤10℃

[0835] Injection volume: Sample and reference standard: 25 μg protein (nominal); Blank and mobile phase: Injection volume same as reference standard.

[0836] gradient

[0837] 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

[0838] Although this method effectively separates the peaks of pertuzumab and trastuzumab, the main legacy issues become apparent. Trace amounts of Herceptin / pertuzumab were still detected after injecting five blank samples (formulation buffer). Therefore, further method development is needed. Different chromatographic 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.

[0839] The effect of column type on separation

[0840] Different types of columns were used for FDC testing of pertuzumab / trastuzumab.

[0841] Table 11: Columns tested for protein content determination using the RP-UHPLC method, and the individual temperatures used for testing.

[0842]

[0843]

[0844] Several potential columns were identified for pertuzumab / trastuzumab FDC. 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 most suitable column was determined to be the Agilent Zorbax RRHD300-Diphenyl (2.1 × 100 mm) column, which exhibited low residuals and improved separation of the two antibodies compared to the initial method.

[0845] DoE (Experimental Design) of Agilent Zorbax RRHD 300-Diphenyl Column

[0846] Mobile phase, flow rate, gradient, and column temperature were tested on an Agilent Zorbax RRHD 300-Diphenyl (2.1 × 100 mm) column. Using... The DoE (DoE) was set for the development of a reverse-phase protein content method. A summary of the factors tested within the DoE range is listed in Table 12.

[0847] Table 12: Factors used for DoE screening as determined by the results.

[0848] name abbreviation unit type set up accuracy flow Flow mL / min Quantification 0.6-0.8 mL / min 0.005 mL / min temperature Temp ℃ Quantification 70-90℃ 0.5℃ Duration Hold Min Quantification 0-3min 0.05min gradient Grad Min Quantification 10-20 min 0.3min start Start ％B Quantification 20-30％B 0.05％B

[0849] Evaluation of "Trastuzumab / Pertuzumab split":

[0850] 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 temperature and starting conditions have a weak but insignificant effect on the method. A relatively high starting condition of 70°C and 30% B has been shown to produce optimal results. Increasing the holding time has no effect on resolution.

[0851] Assessment of "the sum of secondary forms":

[0852] The total height of the secondary forms depends on the initial concentration (high) and column temperature (low). Flow rate and gradient time have only minor effects. Hold time alone is insignificant, but it exhibits some effect when combined with flow rate and column temperature.

[0853] Assessment of "trastuzumab height ratio":

[0854] 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.

[0855] Evaluation of "pertuzumab USP tailing":

[0856] To reduce the tailing of the pertuzumab peak, the flow rate should be increased and the gradient and initiation conditions reduced. Similarly, additional hold time does not demonstrate an effect.

[0857] Based on the DoE results, select the following parameters:

[0858] Flow rate: 0.8 mL / min

[0859] Wavelength: 280nm

[0860] Column temperature: 70℃

[0861] Automatic sampler temperature: 10℃

[0862] Running time: 20 minutes

[0863] Table 13: DoE gradient

[0864]

[0865] Based on these results, the column temperature, gradient, and flow rate were further optimized.

[0866] Effect of column temperature on separation

[0867] High temperatures in reversed-phase chromatography can significantly affect peak separation, tailing, and system pressure. Three different temperatures were selected for testing on an Agilent Zorbax RRHD 300 Diphenyl column. Temperature measurements were performed within the DoE range (results not shown). In summary, as the column temperature increased, the residence time shifted towards earlier elution. This is expected because eluent viscosity and secondary column interactions decrease with increasing temperature. However, the overall resolution decreased somewhat with increasing column temperature. Therefore, the optimal column temperature within the experimental range was 70°C.

[0868] The influence of gradient pattern on separation

[0869] 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 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 gradients for the RP protein determination method are listed in gradient 5 of Table 14. The initial DoE gradient (Table 13) was changed to obtain optimal separation and equilibration time using a new flow rate (0.3 mL / min).

[0870] Table 14: Morphology 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).

[0871]

[0872] observe:

[0873] All five test gradients demonstrated adequate protein retention and optimized starting conditions (between 20-30% B). Any starting condition within this range was suitable for pertuzumab / trastuzumab FDC separation. However, a 30% starting condition was chosen to shorten the gradient and run time. Considering the gradient time, the tested range (10-20 min) was optimized. Because 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.

[0874] Using a 10-minute separation time, and especially with a 30% B gradient steepness, both antibodies eluted within a window of only 1-2 minutes. However, a 20-minute separation time and a 20% B gradient steepness resulted in a wider elution pattern and a weaker detector signal.

[0875] The final 15-minute separation time was combined with a 15% B gradient steepness. Combined with a slow flow rate (0.3 mL / min), it demonstrated good baseline separation of the two antibodies without significant loss of signal intensity.

[0876] Effect of flow rate on separation

[0877] Finally, the optimal flow rate must be determined. A faster flow rate generally means earlier elution, 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 an Agilent Zorbax RRHD 300 Diphenyl column using samples containing a single mAb. For direct comparison, the column temperature was consistently set at 70 °C, and all separations were performed using the smallest gradients listed in Table 13.

[0878] Reducing the flow rate results in a narrower peak shape and higher signal intensity. Retention time aligns with later elution. Separation (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 operating at 0.3 mL / min demonstrates sufficient column reequilibration.

[0879] 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 limits of the Phase III and BLA / MAA method validation experiments, flow rates of 0.4 mL / min and 0.2 mL / min were tested and found to have no significant impact on method performance.

[0880] Example 18: RP-UPHLC determination of FDC content

[0881] Note: Equivalent instruments, appropriate operating conditions, and solvents, chemicals, and reagents of equivalent quality may be used.

[0882] 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 difference in hydrophobicity. An external calibration curve was generated for calculating the individual contents of pertuzumab and trastuzumab in each analytical sequence by injecting different volumes of FDC reference standards. The same method was applied to the LD and MD dosage forms of the FDC drugs. Measurements were taken for each dosage form against the corresponding reference standards.

[0883] Equipment and materials

[0884] • UHPLC system equipped with a UV detector (Thermo Ultimate 3000RS or equivalent)

[0885] • UHPLC column (Agilent Zorbax RRHD 300-Diphenyl, 2.1 mm × 100 mm, particle size: 1.8 μm or equivalent particle size)

[0886] reagents

[0887] ·2-Propanol

[0888] Acetonitrile

[0889] ·TFA

[0890] Anhydrous L-histidine

[0891] L-histidine monohydrochloride monohydrate

[0892] ·sucrose

[0893] Trehalose

[0894] L-methionine

[0895] ·Polysorbide 20

[0896] Sodium hydroxide

[0897] ·hydrochloric acid

[0898] • Purified water (e.g., MilliQ)

[0899] solution

[0900] Drug product dilution buffer

[0901] 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

[0902] Mobile phase A

[0903] 2% (v / v) 2-propanol, 0.1% (v / v) TFA / water

[0904] Mobile phase B

[0905] 70% (v / v) 2-propanol, 20% (v / v) acetonitrile, 10% (v / v) mobile phase A

[0906] Preparation of reference standard solution

[0907] 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 the corresponding reference solutions (Reference Solution A and Reference Solution B) must be prepared. Dilute the corresponding reference standards to a total protein concentration of 1 mg / mL using drug dilution buffer.

[0908] Preparation of sample solution

[0909] The FDC drug was diluted with a drug dilution buffer to prepare a sample solution containing a total protein concentration of 1 mg / mL.

[0910] program

[0911] Before injecting the first sample, flush the column with 70% mobile phase A / 30% mobile phase B until a stable baseline is obtained. Optionally, inject a reference solution for column conditioning purposes until a visual evaluation of the chromatogram shows that at least two consecutive injections have a continuous pattern.

[0912] Operating parameters

[0913] • Detection wavelength: 280nm

[0914] • Injection volume: See the injection protocol below.

[0915] • Flow rate: 0.3 mL / min

[0916] Column temperature: 70℃±2℃

[0917] • Automatic sampler temperature: 10℃±4℃

[0918] • Running time: 30 minutes

[0919] gradient

[0920] Table 15: Binary Gradients

[0921]

[0922] Injection scheme

[0923] 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.

[0924] Table 16: Injection Scheme

[0925]

[0926] Note: For more than 10 samples, place every 10 sample injection solutions and the control solution in a separate container.

[0927] Together (see B).

[0928] result

[0929] Typical chromatographic patterns are shown in Figure 15 (FDC Drug LD) and Figure 16 (FDC Drug MD)

[0930] The final method (Example 18) substantially improves the initial protein content method, including improved overall resolution / peak separation and elimination of sample residue, i.e., no more than 0.2% residue in subsequent analyses. Additionally, the final method allows for the quantification of protein content in 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.

[0931] Figure 15 An example RP-UHPLC chromatogram used to analyze the protein content of FDC LD reference standards is shown. Figure 16 An example RP-UHPLC chromatogram is shown for analyzing the protein content of FDC MD reference standards.

[0932] Data Analysis

[0933] The pertuzumab and trastuzumab peaks in the chromatograms of reference solutions A and B and the sample solution were integrated. Figure 15 (FDC Drug LD) and Figure 16 The integral is defined with the help of a representative chromatogram in (FDC Drug MD).

[0934] A standard curve for each antibody is generated by plotting the peak area against the injection dose (μg) for each standard grade. The standard curve data are fitted using linear regression. Do not force the curve to cross zero.

[0935] 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.

[0936]

[0937] Slope calibration curve

[0938] To calculate the content of pertuzumab and trastuzumab, divide the amount by the respective injection volume and multiply by the dilution factor.

[0939]

[0940] Example 19: HI-HPLC for determining the content in FDC

[0941] 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 chromatographic method.

[0942] The main differences between HI-HPLC and RP-UHPLC are as follows:

[0943] • HI chromatography is non-destructive and preserves protein folding.

[0944] Due to the natural folding of proteins, protein-column interactions originate solely from amino acids located on the protein surface.

[0945] Elution 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 proteins and the stationary phase. Therefore, less hydrophobic substances elute earlier.

[0946] Two types of columns were tested for use in HIC-HPLC:

[0947] -TSKgel Ether column, 75mm × 7.5mm, 10μm particle size

[0948] -TSKgel Butyl column, 35mm × 4.6mm, 2.5μm particle size

[0949] Mobile phase testing:

[0950] - Eluent A: 50mM sodium phosphate, pH 7.0±0.05, 5% (v / v) ethanol

[0951] - Eluent B: 50mM sodium phosphate, 2M ammonium sulfate, pH 7.0±0.05

[0952] result:

[0953] 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 a 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 separation and exhibits a noticeable tailing effect.

[0954] 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 produced 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.

[0955] Table 18: Operating conditions and HIC gradient for HI-HPLC assays

[0956]

[0957]

[0958] While certain embodiments of the invention have been shown and described herein, those skilled in the art will understand that these embodiments are provided merely by way of example. 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 thereby intended to cover the methods and structures within the scope of these claims and their equivalents. sequence list <110> Hofmeister, Roche, Inc.; Genentech, Inc. <120> Determination of fixed-dose combinations <130> P36263-WO <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 195 <212> PRT <213> Homo sapiens <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 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 195 <210> 2 <211> 124 <212> PRT <213> Homo sapiens <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 <210> 3 <211> 169 <212> PRT <213> Homo sapiens <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 <210> 4 <211> 142 <212> PRT <213> Homo sapiens <400> 4 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 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 <210> 5 <211> 107 <212> PRT <213> Mus musculus <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 Asp Phe 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 <210> 6 <211> 119 <212> PRT <213> Mus musculus <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 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 <210> 8 <211> 119 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 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 115 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 <210> 10 <211> 119 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 <210> 11 <211> 214 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 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 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 <210> 12 <211> 448 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 195 200 205 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 Val Asp 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 <210> 13 <211> 214 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 130 135 140 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 <210> 14 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / Remarks = "Description of artificial sequence: synthetic polypeptide" <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 Tyr Ala 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 Gln Asp 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 <210> 15 <211> 217 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 Val Ala 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 <210> 16 <211> 449 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" "Polypeptide" <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 His Lys 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 Val Asp 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 <210> 17 <211> 10 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <220> <221> variants <222> (10)..(10) <223> / replaces "Ser" <220> <221> MISC_FEATURE <222> (10)..(10) <223> / Note="The residues given in the sequence for the position" The residues in the annotations have no preference. <400> 17 Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp 1 5 10 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <400> 18 Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5 10 15 Gly <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <400> 19 Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <400> 20 Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <220> <221> variants <222> (5)..(5) <223> / replaces "Leu" <220> <221> variants <222> (6)..(6) <223> / replaces "Glu" <220> <221> variants <222> (7)..(7) <223> / replaces "Ser" <220> <221> MISC_FEATURE <222> (5)..(7) <223> / Note="The residues given in the sequence for the position" The residues in the annotations have no preference. <400> twenty one Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthesis" peptide <400> twenty two Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5 <210> twenty three <211> 119 <212> PRT <213> Homo sapiens <400> twenty three 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 <210> 24 <211> 483 <212> PRT <213> Homo sapiens <400> 24 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 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 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 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 <210> 25 <211> 725 <212> PRT <213> artificial sequence <220> <223> Anti-HER2 antibody capture reagent <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 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 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 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 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 Val Asp 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 <210> 26 <211> 756 <212> PRT <213> Artificial sequence <220> <223> Capture reagent for anti-HER2 antibody <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 100 105 110 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 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 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 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 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 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 725 730 735 Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser 740 745 750 Arg Thr Pro Gly 755 <210> 27 <211> 757 <212> PRT <213> Artificial Sequence <220> <223> Capture Agent for Anti-HER2 Antibody <400> 27 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 100 105 110 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 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 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 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 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 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 725 730 735 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 <210> 28 <211> 136 <212> PRT <213> Homo sapiens <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 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 130 135 <210> 29 <211> 624 <212> PRT <213> Synthetic Sequence <220> <223> Capture Reagent for Anti-HER2 Antibody <400> 29 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 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 Lys 180 185 190 Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly 195 200 205 Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr 210 215 220 Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu 225 230 235 240 Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr 245 250 255 Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala 260 265 270 Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly 275 280 285 Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp 290 295 300 Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys 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 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 Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 <210> 30 <211> 866 <212> PRT <213> Artificial sequence <220> <223> Capture reagent for anti-HER2 antibody <400> 30 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 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 Lys 180 185 190 Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly 195 200 205 Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr 210 215 220 Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu 225 230 235 240 Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr 245 250 255 Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala 260 265 270 Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly 275 280 285 Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp 290 295 300 Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys 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 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 Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 Arg Arg Ala Gln Val Thr Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr 625 630 635 640 Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly 645 650 655 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met 660 665 670 Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu 675 680 685 Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val 690 695 700 His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu 705 710 715 720 Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 725 730 735 Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile 740 745 750 Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val 755 760 765 Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr 770 775 780 Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu 785 790 795 800 Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro 805 810 815 Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val 820 825 830 Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val 835 840 845 His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr 850 855 860 Pro Gly 865 <210> 31 <211> 899 <212> PRT <213> Artificial Sequence <220> <223> Capture Reagent for Anti-HER2 Antibody <400> 31 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 Ala Ala 20 25 30 Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala 35 40 45 Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys 50 55 60 Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala 65 70 75 80 Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu 85 90 95 Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile 100 105 110 Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu 115 120 125 Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser 130 135 140 Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu 145 150 155 160 Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp 165 170 175 Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu 180 185 190 Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro 195 200 205 Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln 210 215 220 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 225 230 235 240 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 245 250 255 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 260 265 270 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 275 280 285 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 290 295 300 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 305 310 315 320 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 325 330 335 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 340 345 350 Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr 355 360 365 Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 370 375 380 Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 385 390 395 400 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu 405 410 415 Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp 420 425 430 Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His 435 440 445 Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu 450 455 460 Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His 465 470 475 480 His Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 485 490 495 Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu 500 505 510 Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg 515 520 525 Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln 530 535 540 Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly 545 550 555 560 Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro 565 570 575 Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala 580 585 590 Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val 595 600 605 Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile 610 615 620 Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn 625 630 635 640 Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu 645 650 655 Gln Arg Arg Ala Gln Val Thr Asp Lys Lys Ile Glu Pro Arg Gly Pro 660 665 670 Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu 675 680 685 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 690 695 700 Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser 705 710 715 720 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 725 730 735 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 740 745 750 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 755 760 765 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 770 775 780 Ile Glu Arg Thr Ile Ser...

Claims

1. A method for analyzing a fixed-dose combination (FDC) of anti-HER2 antibodies binding to HER2 extracellular subdomain II and anti-HER2 antibodies binding to HER2 extracellular subdomain IV, comprising: a. Contact the fixed-dose combination (FDC) with a capture agent for quantifying the binding of an anti-HER2 antibody to HER2 extracellular subdomain II, wherein the capture agent does not contain HER2 subdomain IV, wherein (1) the capture agent is a modified HER2 ECD subdomain having the amino acid sequence shown in SEQ ID NO: 24, or (2) the capture agent contains a modified HER2 ECD subdomain having the amino acid sequence shown in SEQ ID NO: 24 and is fused to a peptide or protein that facilitates immobilization of the capture agent to a solid substrate; b. Contact the sample with the detectable antibody; c. Quantifying the level of antibody bound to the capture reagent using a detection method targeting the detectable antibody, wherein the binding of anti-HER2 antibodies to HER2 extracellular subdomain II is quantified, and d. Contact the fixed-dose combination (FDC) with a capture agent for quantifying the binding of an anti-HER2 antibody to the extracellular subdomain IV of HER2, wherein the capture agent does not contain the HER2 subdomain II, wherein (1) the capture agent is a modified HER2 ECD subdomain having the amino acid sequence shown in SEQ ID NO: 29, or (2) the capture agent contains a modified HER2 ECD subdomain having the amino acid sequence shown in SEQ ID NO: 29 and is fused to a peptide or protein that facilitates immobilization of the capture agent to a solid substrate; e. Contact the sample with the detectable antibody; f. The level of antibody bound to the capture reagent is quantified using a detection method for the detectable antibody, wherein the binding of anti-HER2 antibody to the HER2 extracellular subdomain IV is quantified.

2. The method of claim 1, wherein potency is quantified by correlating the level of the antibody bound to the capture reagent with the bioactivity of the isolated antibody as measured in a cell-based assay.

3. The method of claim 1, wherein the capture reagent is coated on a microtiter plate.

4. The method of claim 1, wherein the detectable antibody targets the F(ab')2 portion of the anti-HER2 antibody.

5. The method of claim 1, wherein the fixed-dose combination further comprises hyaluronidase.

6. The method of claim 1, wherein the peptide or protein that facilitates immobilization of the capture reagent onto the solid substrate is biotin, bovine serum albumin (BSA), or an Fc domain.

7. The method of claim 1, wherein the amino acid sequence of the capture reagent for quantifying the binding of anti-HER2 antibody to HER2 extracellular subdomain II is shown in SEQ ID NO:

25.

8. The method of claim 1, wherein the amino acid sequence of the capture reagent for quantifying the binding of anti-HER2 antibody to the extracellular subdomain IV of HER2 is as shown in SEQ ID NO:

30.

9. A kit for specifically quantifying the binding of the first anti-HER2 antibody to HER2 extracellular subdomain II and the binding of the second anti-HER2 antibody to HER2 extracellular subdomain IV in a fixed-dose combination (FDC) of a first anti-HER2 antibody binding to HER2 extracellular subdomain II and a second anti-HER2 antibody binding to HER2 extracellular subdomain IV, said kit comprising: a. A container containing a protein as a capture agent for quantifying the binding of an anti-HER2 antibody to HER2 extracellular subdomain II, wherein (1) the protein comprises SEQ ID NO: 24, or (2) the protein contains SEQ ID NO: 24 and is fused to a peptide or protein that facilitates immobilization of the capture agent to a solid substrate. b. Instructions for use in quantifying the binding of anti-HER2 antibodies to HER2 extracellular subdomain II, and c. A container containing a protein as a capture agent for quantifying the binding of an anti-HER2 antibody to the extracellular subdomain IV of HER2, wherein (1) the protein comprises SEQ ID NO: 29, or (2) the protein comprises SEQ ID NO: 29 and is fused to a peptide or protein that facilitates immobilization of the capture agent to a solid substrate. d. Instructions for use in quantifying the binding of anti-HER2 antibodies to the extracellular subdomain IV of HER2.

10. The kit according to claim 9, wherein the peptide or protein that facilitates immobilization of the capture reagent to the solid substrate is biotin, bovine serum albumin (BSA), or an Fc domain.

11. The kit according to claim 9, wherein the capture reagent for quantifying the binding of anti-HER2 antibody to HER2 extracellular subdomain II is composed of SEQ ID NO:

25.

12. The kit according to claim 9, wherein the capture reagent for quantifying the binding of anti-HER2 antibody to HER2 extracellular subdomain IV is composed of SEQ ID NO: 30.

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