Assay of a fixed-dose formulation

Abstract

This invention provides a binding assay, kit, and evaluation method for analyzing the quality and quantity attributes of a fixed-dose formulation. [Solution] Assays are provided for analyzing the quality and quantity attributes of fixed-dose combination formulations. In particular, assays for fixed-dose combination formulations of two anti-HER2 antibodies, as well as for subcutaneous formulations containing pertuzumab and trastuzumab, are described herein.

Description

[Technical Field]

[0001] This invention relates to an assay for analyzing the quality and quantity attributes of a fixed-dose combination formulation. In particular, this invention relates to an assay for a fixed-dose combination formulation of two anti-HER2 antibodies, and for a subcutaneous formulation containing pertuzumab and trastuzumab. [Background technology]

[0002] To ensure the safety and efficacy of biopharmaceuticals, product quality must be continuously monitored. Before a product batch is released, certain specific criteria, including critical quality attributes (CQAs), must be met. Critical quality attributes (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 conducted as part of product conformity testing, comparability studies, and safety testing. These tests are used to measure product attributes related to product quality and manufacturing control, and are performed to ensure the character, purity, strength (potency), and safety of the product used throughout all stages of clinical trials. Similarly, potency testing is used to demonstrate that only product lots meeting defined specificity or acceptance criteria are administered throughout all stages of clinical investigations and for subsequent marketing authorization.

[0004] Ion exchange chromatography (IEX) is widely used for the detailed characterization of therapeutic proteins and can be considered a reference and powerful technique for the qualitative and quantitative assessment of charge heterogeneity. IEX is typically an extraction method in which specificity is set by the distribution of acidic, major, and basic species of monoclonal antibodies (mAbs). These charged species are considered product-related impurities that can affect titer. Furthermore, it is one of the few methods that can characterize proteins by confirming their natural, undenatured state. IEX can also be used as a characterization method for certain biological products and is a standard test for justifying stability and shelf life.

[0005] The quantity is a CQA, typically measured as protein content. It is important for biotechnological and biological products and should usually be determined using appropriate assays of physicochemical nature. For most biopharmaceuticals, protein content is measured by UV absorption.

[0006] A fixed-dose combination drug (FDC) combines two different active ingredients into a single-dose formulation. The combination of two anti-HER2 antibodies, trastuzumab and pertuzumab, with hyaluronidase enzyme is the first-ever clinical development of a co-formulation of two highly similar monoclonal antibodies. The mechanisms of action of pertuzumab and trastuzumab are thought to be complementary, as both bind to the HER2 receptor at different sites. The pertuzumab and trastuzumab combination is thought to provide a more comprehensive double blockade of the HER signaling pathway. The standard IV formulation of Perjeta in combination with IV Herceptin and chemotherapy (Perjeta-based regimen) is approved in over 100 countries for the treatment of both early and metastatic HER2-positive breast cancer. In the neoadjuvant early breast cancer (eBC) setting, the Perjeta-based regimen has shown nearly twice the pCR rate compared to Herceptin and chemotherapy. In addition, the combination drug has shown a significant reduction in the risk of recurrence or death of invasive disease in the adjuvant eBC setting. In the metastatic setting, the combination drug has demonstrated unprecedented survival benefits in previously untreated (first-line) patients with HER2-positive metastatic breast cancer.

[0007] The enzyme hyaluronidase in FDC enables and optimizes SC drug delivery of appropriate co-administered therapeutics. Recombinant human hyaluronidase PH20 (rHuPH20) is an enzyme that transiently breaks down hyaluronans, which are glycosaminoglycans or natural sugar chains in the body, to aid in the dispersion and absorption of other injectable therapeutics.

[0008] Trastuzumab and pertuzumab share over 93% sequence identity, differing 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 similarities, most conventional analytical methods cannot be applied to this combination. [Overview of the project]

[0009] In one embodiment, a binding assay for a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Contacting FDC with a capture reagent containing a modified HER2 ECD subdomain; b. Contacting the sample with a detectable antibody; c. Quantifying the level of antibody bound to the capture reagent using a detection method for detectable antibodies, A binding assay is provided, including the following.

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

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

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

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

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

[0015] In one embodiment, a binding assay for a fixed-dose combination (FDC) of two anti-HER2 antibodies is provided, where the binding assay is for analyzing the biological activity of one of the anti-HER2 antibodies. In one embodiment, the biological activity is quantified by measuring in a cell-based assay and correlating the level of the antibody bound to the capture reagent with the biological activity of the isolated antibody.

[0016] In one embodiment, the capture reagent is coated on a microtiter plate. In one embodiment, the detectable antibody targets the F(ab’)2 portion of the anti-HER2 antibody.

[0017] In one embodiment, the fixed-dose combination analyzed by the binding assay additionally contains hyaluronidase.

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

[0019] Further provided is a kit for specifically quantifying the binding of an antibody that binds to the HER2 extracellular subdomain II in a fixed-dose combination (FDC) of a first antibody that binds to the HER2 extracellular subdomain II and a second anti-HER2 antibody, a. a container containing as a capture reagent a protein comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 34, b. instructions for quantifying the binding of an antibody that binds to the HER2 extracellular subdomain II, and comprising the kit.

[0020] Furthermore, the kit provided is for specifically quantifying the binding of an antibody to the HER2 extracellular subdomain IV in a fixed-dose combination (FDC) of an antibody that binds to the HER2 extracellular subdomain IV and a second anti-HER2 antibody. a. A container containing proteins including SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 4 as a capture reagent, b. Instructions for quantifying antibody binding to HER2 extracellular subdomain IV, A kit that includes this.

[0021] In another embodiment of the present invention, a method for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, a. Using a loading buffer, the antibody is bound to the ion exchange material, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. Elute the antibody with elution buffer, ensuring the pH of the elution buffer is approximately 7.5 to 7.7. A method is provided that includes this.

[0022] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0023] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0024] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

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

[0026] In one embodiment, a method is provided for preparing a composition, comprising (1) producing a fixed-dose combination containing pertuzumab, trastuzumab, and one or more variants thereof, and (2) subjecting the composition thus produced to an analytical assay to evaluate the amount of variants, wherein the variants include (i) pertuzumab deamidated with HC-Asn-391, pertuzumab FC sialic acid variant, and pertuzumabridin glycated variant, (ii) a natural antibody of pertuzumab, (iii) a natural antibody of trastuzumab, and (vi) trastuzumab having a sole isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0027] In one embodiment, a method for preparing a composition, wherein the analytical assay in step (2) is a. Using a loading buffer, the antibody is bound to the ion exchange material, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. Elute the antibody with elution buffer, ensuring the pH of the elution buffer is approximately 7.5 to 7.7. A method including this is provided.

[0028] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0029] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0030] In one embodiment, the analytical assay in step (2) is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

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

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

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

[0034] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 23% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 28% of pertuzumab native antibody, at least 16% of trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0035] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 23% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 38% of pertuzumab native antibody, at least 16% of trastuzumab native antibody, and less than 9% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0036] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 21% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 28% of pertuzumab native antibody, at least 23% of trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0037] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, the composition comprising less than 23% of the peak region of peaks 1-3 combined, at least 28% of the peak region of peak 4 (pertuzumab natural antibody), at least 16% of the peak region of peak 7 (trastuzumab natural antibody), and less than 12% of the peak region of peak 8. a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. A step in which the antibody is eluted with elution buffer, and the pH of the elution buffer is approximately 7.5 to approximately pH 7.7, It is determined by a method that includes the following.

[0038] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0039] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0040] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

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

[0042] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0043] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, the composition comprising less than 23% of the peak region of peaks 1-3 combined, at least 38% of the peak region of peak 4 (pertuzumab natural antibody), at least 16% of the peak region of peak 7 (trastuzumab natural antibody), and less than 9% of the peak region of peak 8. a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. A step in which the antibody is eluted with elution buffer, and the pH of the elution buffer is approximately 7.5 to approximately pH 7.7, It is determined by a method that includes the following.

[0044] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0045] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0046] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

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

[0048] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0049] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, the composition comprising less than 21% of the peak region of peaks 1-3 combined, at least 28% of the peak region of peak 4 (pertuzumab natural antibody), at least 233% of the peak region of peak 7 (trastuzumab natural antibody), and less than 12% of the peak region of peak 8. a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. A step in which the antibody is eluted with elution buffer, and the pH of the elution buffer is approximately 7.5 to approximately pH 7.7, It is determined by a method that includes the following.

[0050] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0051] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0052] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

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

[0054] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0055] In a further embodiment of the present invention, the compositions provided herein are comprised of the following steps: a. A step of adding a predetermined amount of pertuzumab to a compounding container, b. A step of adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or a 1:2 ratio of trastuzumab to pertuzumab, The process involves adding c.rHuPH20, It is obtained by a method that includes the following.

[0056] In a further embodiment, a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Prepare the RP-HPLC phenyl column; b. Loading a fixed-dose combination (FCD) of two anti-HER2 antibodies into an RP-HPLC column; c. Separate two anti-HER2 antibodies at a flow rate of 0.2-0.4 mL / min, with a column temperature of 64°C-76°C. A method including this is provided.

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

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

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

[0060] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, the antibodies are separated over a period of 10-20 minutes. In one such embodiment, the antibodies are separated over a period of 15 minutes. In one embodiment, the antibodies are separated over a period of 15 minutes at a flow rate of approximately 0.3 mL / min.

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

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

[0063] [Figure 1] This provides schematic diagrams of the HER2 protein structure and the amino acid sequences of its extracellular domains I-IV (SEQ ID NOs: 1-4, respectively). [Figure 2A-B] The diagram illustrates the amino acid sequence alignments of the variable light chain (VL) (Figure 2A) and variable heavy chain (VH) (Figure 2B) domains of the mouse monoclonal antibody 2C4 (SEQ ID NOs. 5 and 6, respectively); the VL and VH domains of the mutant 574 / pertuzumab (SEQ ID NOs. 7 and 8, respectively); and the human VL and VH consensus framework (hum κ1, light chain kappa subgroup I; hum III, heavy chain subgroup III) (SEQ ID NOs. 9 and 10, respectively). Asterisks highlight differences between the variable domain of pertuzumab and the mouse monoclonal antibody 2C4, or between the variable domain of pertuzumab and the human framework. Complementarity-determining regions (CDRs) are enclosed in square brackets. [Figure 3A-B] The amino acid sequences of the pertuzumab light chain (Figure 3A: SEQ ID NO: 11) and heavy chain (Figure 3B: SEQ ID NO: 12) are shown. The CDR is shown in bold. The calculated molecular weights of the light and heavy chains are 23,526.22 Da and 49,216.56 Da (cysteines in the reduced form). The hydrocarbon moiety is attached to Asn299 of the heavy chain. [Figure 4A] The amino acid sequence of the trastuzumab light chain (SEQ ID NO: 13) is shown. The boundary of the variable light chain domain is indicated by an arrow. [Figure 4B] The amino acid sequence of the trastuzumab heavy chain (SEQ ID NO: 14) is shown. The boundary of the variable heavy chain domain is indicated by an arrow. [Figure 5A] The light chain sequence of the pertuzumab variant (SEQ ID NO: 15) is depicted. [Figure 5B] The heavy chain sequence of the pertuzumab variant (SEQ ID NO: 16) is depicted. [Figure 6]A schematic diagram of the HER2 extracellular domain and the capture reagents useful for the ELISA assays described herein is shown. P-HER2 variant: A modified HER2 ECD for analyzing pertuzumab titer. T-HER2 variant: A modified HER2 ECD for analyzing trastuzumab titer. [Figure 7A-B] This describes selective sensitivity in cell-based assays. Figure 7A: Pertuzumab antiproliferative assay using MDA-MB-175 VII cells. Figure 7B: Trastuzumab antiproliferative assay using BT-474 cells. [Figure 8A-B] The complementary mechanisms of pertuzumab and trastuzumab in cell-based antiproliferative assays are described. Figure 8A: Pertuzumab antiproliferative assay: When trastuzumab is added in a 1:1 ratio, the dose-response curve shifts toward lower concentrations. Figure 8B: Trastuzumab antiproliferative assay: When pertuzumab is added in a 1:1 ratio, the dose-response curve shifts toward lower concentrations. [Figure 9A-B] This illustrates the shielding effect in cell-based antiproliferative assays. Figure 9A: Pertuzumab antiproliferative assay: The affinity of the pertuzumab mutant (HC S55A) to HER2 is significantly reduced (solid symbol), and the addition of trastuzumab shields the affinity loss of the pertuzumab mutant (hollow symbol). Figure 9B: Trastuzumab antiproliferative assay: The affinity of the trastuzumab mutant (LC H91A) to HER2 is significantly reduced (solid symbol), and the addition of pertuzumab shields the affinity loss of the trastuzumab mutant (hollow symbol). [Figure 10] This diagram illustrates a typical dose-response curve for pertuzumab ELISA. [Figure 11] This diagram illustrates a typical dose-response curve for trastuzumab ELISA. [Figure 12] A representative chromatogram of the IEC method provided herein for analyzing pertuzumab-trastuzumab FDC charge variants is shown. [Figure 13]The IE-HPLC chromatograms of pertuzumab and trastuzumab FDC drugs are depicted. [Figure 14A-B] ELISA shows the HERT2 affinity mutant. Figure 14A: Pertuzumab ELISA: Significantly reduced binding activity of the pertuzumab mutant (HC S55A) to HER2 (hollow symbol) compared with pertuzumab (solid symbol). Figure 14B: Trastuzumab ELISA: Significantly reduced affinity of the trastuzumab mutant (LC H91A) to HER2 (hollow symbol) compared with trastuzumab (solid symbol). [Figure 15] This diagram illustrates an example of an RP-UHPLC chromatogram analyzing the protein content of an FDC LD reference standard. [Figure 16] This diagram illustrates an example of an RP-UHPLC chromatogram analyzing the protein content of an FDC MD reference standard. [Modes for carrying out the invention]

[0064] I. Definition When used in this patent specification, the term "approximately" is intended to indicate that a particular value provided may vary to some extent, meaning, for example, that a variation of +10% is included in the given value. In one embodiment, a variation of + / -5% is included in the given value.

[0065] A "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, including the EGFR, HER2, HER3, and HER4 receptors. A HER receptor generally comprises an extracellular domain to which a HER ligand can bind and / or which can be dimerized by another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain containing several phosphorylated tyrosine residues. A HER receptor may be a "natural sequence" HER receptor or an "amino acid sequence variant" thereof. Preferably, the HER receptor is a natural sequence human HER receptor.

[0066] The terms "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein described, for example, Semba et al., PNAS (USA) Vol. 82: pp. 6497-6501 (1985) and Yamamoto et al., Nature Vol. 319: pp. 230-234 (1986) (Genebank acceptance number X03363). The term "erbB2" refers to the gene encoding human ErbB2, and "neu" refers to rat p185 neu This refers to the gene that codes for HER2. The preferred HER2 is the naturally occurring human HER2 sequence.

[0067] In this specification, “HER2 extracellular domain” or “HER2 ECD” refers to a domain of HER2 located outside the cell, either fixed to the cell membrane or containing fragments of it in circulation. The amino acid sequence of HER2 is shown in Figure 1. In one embodiment, the extracellular domain of HER2 may include four subdomains: “Subdomain I” (approximately 1–195 amino acid residues; SEQ ID NO: 1), “Subdomain II” (approximately 196–319 amino acid residues; SEQ ID NO: 2), “Subdomain III” (approximately 320–488 amino acid residues; SEQ ID NO: 3), and “Subdomain IV” (approximately 489–630 amino acid residues; SEQ ID NO: 4) (residue numbering without signal peptide). See Garrett et al., Mol. Cell. Vol. 11: pp. 495-505 (2003), Cho et al., Nature Vol. 42: pp. 756-760 (2003), Franklin et al., Cancer Cell Vol. 5: pp. 317-328 (2004), and Plowman et al., Proc. Natl. Acad. Sci. Vol. 90: pp. 1746-1750 (1993), as well as Figure 1 of this specification. A "recombinant HER2 extracellular subdomain" or "recombinant HER2 ECD subdomain" includes the corresponding full-length or cleaved native HER2 ECD subdomain. The recombinant HER2 ECD subdomain can be cleaved by up to six amino acids, preferably at the C-terminus, so that the three-dimensional structure of the modified HER2 ECD is as similar as possible to that of the native HER2 ECD.

[0068] An "anti-HER2 antibody" or "HER2 antibody" is an antibody that binds to the HER2 receptor. Optionally, HER2 antibodies further interfere with the activation or function of HER2. The anti-HER2 antibodies of interest as used herein are pertuzumab and trastuzumab.

[0069] An antibody that "binds to extracellular subdomain II" of HER2 binds to residues in domain II (SEQ ID NO: 2) and optionally to residues in other subdomains of HER2, such as subdomains I and III (SEQ ID NOs: 1 and 3, respectively). Preferably, the antibody that binds to extracellular subdomain II binds to the junction between extracellular subdomains I, II, and III of HER2. In one embodiment, the antibody that binds to extracellular subdomain II is pertuzumab or a variant thereof.

[0070] For the purposes of this specification, “pertuzumab” and “rhuMAb 2C4” are used interchangeably and refer to antibodies containing the variable light chain and variable heavy chain amino acid sequences of SEQ ID NOs. 7 and 8, respectively. If pertuzumab is an intact antibody, it preferably comprises an IgG1 antibody, and in one embodiment, contains the light chain amino acid sequence of SEQ ID NOs. 11 or 15 and the heavy chain amino acid sequence of SEQ ID NOs. 12 or 16. The antibody is optionally produced by recombinant Chinese hamster ovary (CHO) cells. The terms “pertuzumab” and “rhuMAb 2C4” as used herein encompass biosimilar versions of the drug relating to the USAN or International Nomenclature (INN): pertuzumab.

[0071] Antibodies that "bind to extracellular subdomain IV" of HER2 bind to residues in domain IV (SEQ ID NO: 4) and, optionally, to residues in other subdomains of HER2. In one embodiment, the antibody that binds to extracellular subdomain IV is trastuzumab or a variant thereof.

[0072] For the purposes of this specification, “trastuzumab” and “rhuMAb4D5” are used interchangeably and refer to antibodies containing variable light-chain and variable heavy-chain amino acid sequences within the range of SEQ ID NOs: 13 and 14, respectively. If trastuzumab is an intact antibody, it preferably comprises an IgG1 antibody and, in one embodiment, contains 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 produced by Chinese hamster ovary (CHO) cells. In this specification, the terms “trastuzumab” and “rhuMAb4D5” encompass biosimilar versions of the drug relating to the United States generic name (USAN) or International generic name (INN): trastuzumab.

[0073] The term “combination drug” is used herein to refer to a single ready-to-use pharmaceutical formulation containing two or more active ingredients, for example, a single ready-to-use pharmaceutical formulation containing pertuzumab and trastuzumab formulated together for subcutaneous (SC) administration.

[0074] The terms “fixed-dose combination formulation” or “FDC” are used herein to refer to a single ready-to-use pharmaceutical formulation containing two or more active ingredients, for example, a single ready-to-use pharmaceutical formulation containing pertuzumab and trastuzumab formulated together for subcutaneous (SC) administration. “Pertuzumab-trastuzumab FDC” contains pertuzumab, trastuzumab, and optionally hyaluronidase.

[0075] The term "hyaluronidase" or "hyaluronidase enzyme" refers to a group of generally neutral or acid-active enzymes found throughout the animal kingdom. Hyaluronidases differ in terms of substrate specificity and mechanism of action (International Publication No. 2004 / 078140). There are three general classes of hyaluronidases: 1. Mammalian hyaluronidases (EC 3.2.1.35), which are endo-β-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as their main end products. They possess both hydrolytic and transglycosidase activity and can degrade hyaluronan and chondroitin sulfate (CS), generally C4-S and C6-S. 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan to varying degrees into CS and DS. These are endo-β-N-acetylhexosaminidases that operate primarily by beta-elimination reactions that produce disaccharide end products. 3. Hyaluronidases from leeches, other parasites, and crustaceans (EC3.2.1.36) are endo-beta-glucuronidases that produce tetrasaccharide and hexasaccharide end products via hydrolysis of β1-3 linkages. Mammalian hyaluronidases can be further divided into two groups: neutral-active enzymes and acid-active enzymes. Hyaluronidase-like enzymes can also be characterized by glycosylphosphatidylinositol anchors, such as human HYAL2 and human PH20, which are commonly locked to the plasma membrane [Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA, 2003; Vol. 100 (No. 8): pp. 4580-4585; Phelps et al., Science 1988; Vol. 240 (No. 4860): pp. 1780-1782], as well as those that are generally soluble, such as human HYAL1 [Frost, IG et al., "Purification, cloning, and expression of human plasma hyaluronidase", Biochem. Biophys. Res. Commun. 1997; Vol. 236 (No. 1): pp. 10-15].Bovine PH20 binds very loosely to the plasma membrane and is not fixed via a phospholipase-sensitive anchor [Lalancette et al., Biol. Reprod., 2001; Vol. 65 (No. 2): pp. 628-636]. This unique characteristic of bovine hyaluronidase allows for the use of soluble bovine testicular hyaluronidase enzymes (Wydase®, Hyalase®) as extracts for clinical use. Other PH20 species are generally lipid-anchored enzymes that do not become soluble without the use of detergents or lipases. For example, human PH20 is fixed to the plasma membrane via a GPI anchor. Naturally occurring macaque sperm hyaluronidase is found in both soluble and membrane-bound forms. The 64kDa membrane-bound form is enzymatically active at pH 7.0, while the 54kDa form is active only at pH 4.0 [Cherr et al., Dev. Biol., 1996; 10th edition; Vol. 175 (No. 1): pp. 142-153]. International Publication No. 2006 / 091871 describes a soluble hyaluronidase glycoprotein (sHASEGP) that facilitates the administration of therapeutic agents into subcutaneous tissue. By rapidly depolymerizing HA in the extracellular space, sHASEGP reduces interstitial viscosity, thereby increasing water conductivity and enabling the safe and comfortable administration of larger volumes into subcutaneous tissue. The preferred hyaluronidase enzyme is human hyaluronidase enzyme, most preferably recombinant human hyaluronidase enzyme known as rHuPH20 (bor hyaluronidase alpha). rHuPH20 is a member of the family of neutral and acid-active β-1,4 glycosyl hydrolases that depolymerize hyaluronan by hydrolysis of the β-1,4 linkage between the C1 position of N-acetylglucosamine and the C4 position of glucuronic acid. Hyaluronidase products approved in EU member states include Hylase® "Dessau" and Hyalase®. Animal-derived hyaluronidase products approved in the United States include Vitrase®, Hydase®, and Amphadase®.

[0076] rHuPH20 is the first and only recombinant human hyaluronidase enzyme currently available for therapeutic use. The amino acid sequence of rHuPH20 (HYLENEX®) is well known and is available under CAS registry number 75971-58-7. Its approximate molecular weight is 61 kDa. In one embodiment, pertuzumab / trastuzumab FDC optionally contains hyaluronidase at a concentration of 2000 U / mL.

[0077] In this specification, “loading” generally refers to an initial dose of the therapeutic agent administered to a patient, followed by one or more maintenance doses. The loading dose (LD) of pertuzumab / trastuzumab FDC includes 40 mg / mL of trastuzumab, 80 mg / mL of pertuzumab, and 2000 U / mL of rHuPH20.

[0078] In this specification, the “maintenance” dose refers to one or more doses of the therapeutic agent administered to a patient over the course of treatment. Typically, the maintenance dose is administered at intervals, for example, approximately weekly, approximately every two weeks, approximately every three weeks, or approximately every four weeks, preferably every three weeks. The maintenance dose (MD) of pertuzumab / trastuzumab FDC comprises 60 mg / mL of trastuzumab, 60 mg / mL of pertuzumab, and 2000 U / mL of rHuPH20.

[0079] As used herein, “capture reagent” refers to any agent (e.g., an anti-HER2 antibody) that can bind to the analyte. Preferably, “capture reagent” refers to any agent that is specifically bound by the anti-HER2 antibody in a fixed-dose combination of two anti-HER2 antibodies. In order to specifically analyze the binding of one of the two anti-HER2 antibodies in the fixed-dose combination, the capture reagent must be specific to that antibody, for example, the antibody to be analyzed should have a higher binding affinity and / or specificity to the capture reagent than the second anti-HER2 antibody of the FDC. In one embodiment, the capture reagent provided for the assay is a modified HER2 ECD.

[0080] A "modified HER2 ECD" is a genetically engineered protein or peptide containing one or more recombinant HER2 ECD subdomains. The HER2 ECD is modified to bind to one of the anti-HER2 antibodies assessed in the FDC, but not to a second anti-HER2 antibody in the FDC. This is achieved by either omitting the HER2 ECD subdomain to which the second anti-HER2 antibody binds, or by replacing it with a structurally similar subdomain that does not bind to any anti-HER2 antibody. Preferably, the modified HER2 ECD is constructed to closely mimic the natural HER2 ECD. The subdomains may be full length or shortened by a few amino acids at the N or C terminus. The inventors of this invention have found that the three-dimensional structural integrity of the HER2 ECD is maintained or improved by using one or more recombinant HER2 ECD subdomains shortened by approximately 4-5 amino acids at the C terminus.

[0081] In this specification, “Fc domain” is used to define the C-terminal domain of an immunoglobulin heavy chain. Fc domains can originate from various sources, such as mouse, rat, goat, or human. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc domain is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. Unless otherwise indicated, in this specification, the numbering of residues in immunoglobulin heavy chains is the EU index numbering, as expressed herein by reference, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). “Kabat's EU index” refers to the residue numbering of the human IgG1 EU antibody.

[0082] When used herein, the term "detectable antibody" refers to an antibody conjugated to a drug or detectable label capable of generating a detectable signal, which can be used to assess the presence and / or amount of the analyte to be detected (i.e., an anti-HER2 antibody).

[0083] The term "label" or "detectable label" refers to any chemical group or moiety 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, and electroactive species. Alternatively, a detectable label may signal its presence by participating in a specific binding reaction. Examples of such labels include haptens, antibodies, biotin, streptavidin, His tags, nitrilotriacetate, glutathione S-transferase, and glutathione.

[0084] The term “detection means” refers to the part or technique used to detect the presence of a detectable antibody via the signal reporting read in the assay herein. “Photoluminescence” is the process by which a material emits light after absorbing light (electromagnetic radiation, or alternatively referred to as EMR). Fluorescence and phosphorescence are two different types of photoluminescence. The process of “chemiluminescence” involves the creation of a luminescent species by a chemical reaction. “Electrochemiluminescence” or “ECL” is the process by which a species, for example, the antibody of interest, emits light when exposed to the electrochemical energy of that species under appropriate ambient chemical conditions.

[0085] As used herein, the term "ELISA" (also known as enzyme-linked immunosorbent assay) primarily refers to a biochemical technique used to detect the presence of antibodies in biological samples. For the purposes of this application, the ELISA technique is used for the detection and quantification of anti-HER2 antibodies in a fixed-dose formulation. Typically for ELISA-based assays, the capture reagent is immobilized or immobilizable.

[0086] In this specification, “potency” refers to the therapeutic activity or intended biological effect of a biotherapeutic drug. The potency of a biotherapeutic drug can be determined by measuring or quantifying the biological activity of the active ingredient of the biotherapeutic drug.

[0087] In this specification, “biological activity” of a monoclonal antibody refers to the antibody’s ability to bind to an antigen and produce a measurable biological response that can be measured in vitro or in vivo. In one embodiment, biological activity refers to the ability to bind to a capture reagent in a binding assay provided herein. In one embodiment, the binding of an anti-HER2 antibody in a FDC correlates with the ability of the anti-HER2 antibody in a single antibody formulation to inhibit the proliferation of a human breast cancer cell line. 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.

[0088] In this specification, the term "antibody" is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity.

[0089] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the smallest sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is an antibody in which residues in the hypervariable region of a human immunoglobulin (recipient antibody) are replaced with residues in the hypervariable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capability. In some cases, residues in the framework region (FR) of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies also optionally include the constant region (Fc) of an immunoglobulin, typically at least a portion of the constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321: pp. 522-525 (1986); Riechmann et al., Nature 332: pp. 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: pp. 593-596 (1992). Specific examples of humanized HER2 antibodies include trastuzumab (HERCEPTIN®), as defined herein and listed in Table 3 of U.S. Patent No. 5,821,337, expressly incorporated herein by reference; and humanized 2C4 antibodies, such as pertuzumab, as described and defined herein.

[0090] In this specification, an "intact antibody" comprises two antigen-binding regions and one Fc region. Preferably, the intact antibody has a functional Fc region.

[0091] An "antibody fragment" preferably comprises a portion of an intact antibody, including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0092] "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 a heavy chain by one disulfide covalent bond, although the number of disulfide linkages differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H Each light chain has a variable domain (V) at one end. L It has a constant domain at the 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. Certain amino acid residues are thought to form a contact surface between the light chain variable domain and the heavy chain variable domain.

[0093] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally consists of amino acid residues in the "complementarity-determining region" 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 in the "hypervariable loop" (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; Chothia and Lesk This includes J.Mol.Biol.196:pp.901-917 (1987). "Framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein.

[0094] Depending on the amino acid sequence of the heavy chain constant domain, intact antibodies can be assigned different "classes." There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

[0095] A "naked antibody" is an antibody that is not conjugated with a heterologous molecule, such as a cytotoxic moiety or radiolabeling.

[0096] Affinity-matured antibodies are those that have one or more modifications within one or more hypervariable regions, where these modifications result in improved affinity for the antigen compared to parent antibodies without these modifications. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992), describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues is described in Barbas et al., Proc Nat. Acad. Sci, USA vol. 91: pp. 3809-3813 (1994); Schier et al., Gene vol. 169: pp. 147-155 (1995); Yelton et al., J. Immunol. vol. 155: pp. 1994-2004 (1995); Jackson et al., J. Immunol. vol. 154 (no. 7): pp. 3310-339 (1995); and Hawkins et al., J. Mol. Biol. vol. 226: pp. 889-896 (1992).

[0097] A "vial" is a container suitable for holding liquids or lyophilized preparations. In one embodiment, the vial is a disposable vial, for example, a 10 mL or 20 mL disposable vial with a stopper, for example, a 10 mL disposable glass vial with a 20 mm stopper.

[0098] As used herein, "elution" refers to the removal of a target protein (e.g., an antibody) from a cation exchange material by modifying the ionic strength of the buffer surrounding the cation exchange material so that the buffer competes with the charged sites of the cation exchange material.

[0099] As used herein, the term "chromatography" refers to a method of separating a solute of interest in a mixture, for example, a protein of interest, from other solutes in the mixture by percolation of the mixture through an adsorbent that adsorbs or retains the solute somewhat strongly due to its properties, such as pi, hydrophobicity, size, and structure, under specific buffering conditions of the method.

[0100] The terms "ion exchange" and "ion exchange chromatography" refer to chromatographic methods in which an ionizable solute of interest (e.g., antibodies to FDCs, and their acidic and basic variants) interacts with a reverse-charged ligand (e.g., covalently) linked to a solid-phase ion exchange material, under appropriate pH and conductivity conditions such that the solute of interest interacts with the charged compound more nonspecifically than solute impurities or contaminants in the mixture.

[0101] "Ion exchange chromatography" specifically includes cation exchange (CEX) chromatography, anion exchange chromatography, and mixed-mode chromatography.

[0102] A "cation exchange material" or "CEX material" is a solid phase that is negatively charged and has free cations for exchange with cations in an aqueous solution passing through or through the solid phase. Any load ligand can be used bound to a solid phase suitable for forming a cation exchange material, such as carboxylates, sulfonates, etc., as described below. Commercially available cation exchange materials include, for example, those with sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S from GE Healthcare, SP Sepharose Fast Flow™, SP Sepharose High Performance, Toyopearl SP-650S and SP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic HyperD S, Trisacryl M and LS SP from Pall Technologies, Spherodex LS SP); those with sulfoethyl-based groups (e.g., Fractogel SE from EMD, Poros S-10 and S-20 from Applied Biosystems); those with sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Poros HS-20 and HS 50 from Applied Biosystems); and those with sulfisobutyl-based groups (e.g., Fractogel EMD from EMD). SO3); those with a sulfoethyl-based group (e.g., SE52, SE53 and Express-Ion S from Whatman); those with a carboxymethyl-based group (e.g., CM Sepharose Fast Flow from GE Healthcare, Biochrom Labs Inc.)Hydrocell CM from [company name], Macro-Prep CM from BioRad, Ceramic HyperD CM from Pall Technologies, Trisacryl M CM, Trisacryl LS CM, 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); those with sulfonic acid and carboxylic acid-based groups (e.g., BAKERBOND Carboxy-Sulfon from JTBaker); those with carboxylic acid-based groups (e.g., WP CBX from JT Baker, DOWEX MAC-3 from Dow Liquid Separations, Amberlite Weak Cation Exchangers, DOWEX Weak Cation Exchanger and Diaion Weak Cation Exchangers from Sigma-Aldrich, Fractogel EMD from EMD) Examples include, but are not limited to, those having a COO-- group; a sulfonic acid-based group (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S from JTBaker, WP Sulfonic, Sartobind S membrane from Sartorius, Amberlite Strong Cation Exchangers, DOWEX Strong Cation, and Diaion Strong Cation Exchanger from Sigma-Aldrich); and those having an orthophosphate-based group (e.g., PI 1 from Whatman).

[0103] Depending on the chemical properties of the charged group / substituent, "ion exchange chromatography materials" can be classified as strong or weak ion exchange materials based on the strength of the covalently charged substituent. "Strong cation exchange materials" or "(SCX) materials," as used herein, have sulfonic acid-based groups, such as sulfonates, sulfopropyl groups, sodium polystyrene sulfonate, or polyAMPS (poly(2-acrylamide-2-methyl-1-propanesulfonic acid)).

[0104] The "isoelectric point" or "pI" of a protein or antibody corresponds to the pH value at which the effective charge of the protein or antibody is neutral. The pI can be determined by 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 / ), which calculates the pI based on the amino acid sequence of the protein or antibody. The theoretical pI of trastuzumab is 8.4, and the theoretical pI of pertuzumab is 8.7.

[0105] The "mobile phase" is a liquid or gas that flows through the chromatography system, moving the materials to be separated in the stationary phase at different rates. Preferably, the mobile phase is a liquid. In one example, the mobile phase may be a loading buffer ("mobile phase A") or an elution buffer ("mobile phase B").

[0106] The "loading buffer" provides conditions that ensure the target molecule effectively interacts with the ligand of the ion-exchange chromatography material, is retained in the affinity medium, and all other molecules are washed away from the column.

[0107] Using the "elution buffer," unbound proteins are first washed out, and then the charged mutants and native antibodies are released from the ligand at higher concentrations.

[0108] In this specification, the terms "major species antibody" or "natural antibody" refer to the antibody amino acid sequence structure in the composition that is quantitatively dominant in the composition. With respect to a fixed-dose combination of two anti-HER2 antibodies, the two major species antibodies are part of the composition. Thus, in one embodiment, the major species antibodies are an antibody that binds to extracellular subdomain II of HER2 and an antibody that binds to extracellular subdomain IV. In one embodiment, the major species antibodies of FDC are pertuzumab and trastuzumab.

[0109] A "charge variant" is a variant of a major species antibody that has an overall different charge from the major species antibody. Examples of charge variants are acidic and basic variants.

[0110] "Acid variants" are variants of the major species antibody that are more acidic than the major species antibody. Acid variants acquire a negative charge or lose a positive charge compared to the major species antibody. Such acid variants can be degraded using separation methods that separate proteins according to their charge, such as ion exchange chromatography. When separated by cation exchange chromatography, acid variants of the major species antibody elute earlier than the main peak. Acid variants of pertuzumab and trastuzumab can be separated and quantified by ion exchange chromatography as described herein. Examples of acidic pertuzumab variants are pertuzumab deamidated with heavy chain asparagine at position 391 (HC-Asn-391), pertuzumab Fc sialic acid variant, and pertuzumabridin glycated variant. Examples of acidic trastuzumab variants are trastuzumab deamidated with LC-Asn-30 and trastuzumab deamidated with HC-Asn-55.

[0111] A "basic variant" is a variant of the major species antibody that is more basic than the major species antibody. Basic variants gain or lose a positive charge compared to the major species antibody. Such basic variants can be degraded using separation methods that separate proteins according to their charge, such as ion-exchange chromatography. Basic variants of major species antibodies elute later than the main peak when separated by cation-exchange chromatography. Basic variants of pertuzumab and trastuzumab can be separated and quantified by ion-exchange chromatography as described herein.

[0112] As used herein, the term “gradient” refers to a change in the properties of the mobile phase while a chromatographic sample is flowing through it. In a “continuous gradient,” one or more conditions of the mobile phase, e.g., pH, ionic strength, salt concentration, and / or mobile phase flow, change continuously, i.e., increase or decrease. The 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 change incrementally, i.e., stepwise, in contrast to linear change.

[0113] The term "RP-UHPLC" refers to reversed-phase ultrahigh-performance liquid chromatography. HPLC is used to separate compounds based on their polarity and their interaction with the stationary phase of the column. Reverse-phase chromatography is an elution procedure used in liquid chromatography where the mobile phase is significantly more polar than the stationary phase.

[0114] As used herein, "RP-HPLC phenyl column" refers to a column having hydrophobic phenyl groups present in 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 covalently bonded to a silica surface or diphenyl phase. Some phenyl columns have phenyl groups with alkyl spacers between the phenyl groups and the silica surface. Stereoselectivity and aromatic selectivity can be enhanced by increasing the length of the alkyl spacers. RP-HPLC phenyl columns differ in the number of aromatic groups (monophenyl vs. biphenyl), the length of the alkyl spacers between the silica surface and the phenyl groups, the properties of the substituents on the bonded ligands (typically methyl, or the more sterically bulky isobutyl groups), the inclusion of oxygen atoms in the linker to activate the π-electron system of the aromatic ring, and finally, whether or not the silica stationary surface is end-capped. For example, an RP-HPLC phenylcalm may have the following groups: ethylphenyl with a methyl side group and an end-capped silica surface, a phenylhexyl phase with an extended (hexyl) ligand spacer methyl side group, an ethylphenyl ligand with a sterically protected (isobutyl) side group, a hexyl biphenyl with a methyl side group, a biphenyl phase with a methyl side group, and an oxygen-activated phenylethylphenyl phase with a methyl side group. HPLC columns with stationary phases modified with phenyl (e.g., monophenyl, biphenyl, diphenyl, phenylhexyl, phenylpropyl) are readily available from most major column suppliers, such as Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, Agilent Zorbax RRHD 300-Diphenyl, and Agilent AdvanceBio RP mAb Diphenyl.

[0115] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth.

[0116] "Advanced" cancer is cancer that has spread outside of its original site or organ, either by local invasion ("locally advanced") or by metastasis ("metastatic"). Therefore, the term "advanced" cancer encompasses both locally advanced and metastatic diseases.

[0117] "Metastatic" cancer refers to cancer that has spread from one part of the body (for example, the breast) to another part of the body.

[0118] "Refractory" cancer is cancer that progresses despite being administered antitumor drugs, such as chemotherapy, or biological therapies, such as immunotherapy. An example of a refractory cancer is platinum-resistant cancer.

[0119] A "recurrent" cancer is a cancer that has grown again, either in its original site or a distal site, after responding to initial therapy, such as surgery.

[0120] "Local recurrence" cancer is cancer that recurs in the same location where it was previously treated, after treatment.

[0121] "Non-resectable" or "unresectable" cancers cannot be removed (surgery) by surgery.

[0122] In this specification, “early-stage breast cancer” refers to breast cancer that has not spread beyond the breast or axillary lymph nodes. Such cancers are generally treated with neoadjuvant or adjuvant therapy.

[0123] "Neoadjuvant therapy," "neoadjuvant treatment," or "neoadjuvant administration" refers to systemic therapy given before surgery.

[0124] "Adjuvant therapy," "adjuvant treatment," or "adjuvant administration" refers to systemic therapy given after surgery.

[0125] In this specification, “patient” or “subject” means a human patient. A patient may be a “cancer patient,” that is, a cancer patient who has or is at risk of having one or more manifestations of cancer, particularly breast cancer.

[0126] A "patient population" refers to a group of cancer patients. Such a population can be used to demonstrate the statistically significant efficacy and / or safety of a drug, such as pertuzumab and / or trastuzumab.

[0127] A "relapsed" patient is one who has signs or symptoms of cancer after achieving remission. On an optional basis, patients who have relapsed after adjuvant therapy or neoadjuvant therapy are included.

[0128] A cancer or biological specimen that "shows HER expression, amplification, or activation" is one that expresses (including overexpression) the HER receptor, has an amplified HER gene, and / or otherwise demonstrates HER receptor activation or phosphorylation in a diagnostic test.

[0129] A cancer or biological specimen that "shows HER activation" is one that exhibits activation or phosphorylation of the HER receptor in a diagnostic test. Such activation can be determined directly (e.g., by measuring HER phosphorylation by ELISA) or indirectly (e.g., by gene expression profiling or by detecting HER heterodimers, as described herein).

[0130] Cancer cells with "HER receptor overexpression or amplification" are cancer cells that have significantly higher levels of HER receptor protein or gene compared to non-cancerous cells of the same tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. HER receptor overexpression or amplification can be determined in diagnostic or prognostic assays by assessing the increased level of HER protein present on the cell surface (e.g., via immunohistochemical assay, IHC). Alternatively, or in addition, the level of HER-coding nucleic acids in cells can be measured via, for example, fluorescence in situ hybridization (FISH; see International Publication No. 98 / 45479, October 1998) and chromogenic in situ hybridization (CISH; see, for example, Tanner et al., Am.J. Pathol. Vol. 157 (No. 5): pp. 1467-1472 (2000); Bella et al., J. Clin. Oncol. Vol. 26: (Appendix May 20; Abstract 22147) (2008)), Southern blotting, or polymerase chain reaction (PCR) techniques, such as in situ hybridization (ISH) including quantitative real-time PCR (qRT-PCR). Furthermore, HER receptor overexpression or amplification can also be studied by measuring the shed antigen in biological fluids, such as serum (e.g., by measuring the extracellular domain of HER receptors (see, for example, U.S. Patent No. 4,933,294 issued June 12, 1990; International Publication No. 91 / 05264 published April 18, 1991; U.S. Patent No. 5,401,638 issued March 28, 1995; and Sias et al., J.Immunol.Methods Vol. 132: pp. 73-80 (1990)). In addition to the assays described above, various in vivo assays are available to those skilled in the art. For example, cells in a patient's body can be exposed to antibodies that are optionally labeled with a detectable label, such as in situ radioactivity, or biopsies taken from patients previously exposed to this antibody can be analyzed.

[0131] "HER2-positive" cancers include cancer cells that have higher-than-normal levels of HER2. Optionally, HER2-positive cancers have an immunohistochemistry (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 of ≥2.0.

[0132] "HER2 mutation" cancers include cancer cells that have HER2-activating mutations, including kinase domain mutations that can be confirmed, for example, by next-generation sequencing (NGS) or real-time polymerase chain reaction (RT-PCR). Specifically, "HER2 mutation" cancers include cancers characterized by insertions of HER2 into exon 20, deletions around amino acid residues 755-759 of HER2, any of the mutations G309A, G309E, S310F, D769H, D769Y, V777L, P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov 2013; vol. 3: pp. 1-14), as well as identical non-synonymous putative activating mutations (or indels) previously reported in the COSMIC database found in two or more unique specimens. For further details, see, for example, Stephens et al., Nature 2004; Vol. 431: p. 5256; Shigematsu et al., Cancer Res 2005; Vol. 65: p. 16426; Buttitta et al., Int J Cancer 2006; Vol. 119: p. 258691; Li et al., Oncogene 2008; Vol. 27: p. 470211; Sequist et al., J Clin Oncol 2010; Vol. 28: p. 307683; Arcila et al., Clin Cancer Res 2012; Vol. 18: p. 49108; Greulich et al., Proc Natl Acad Sci USA 2012; Vol. 109: p. 1447681; and Herter-Sprie et al., Front Oncol 2013; Vol. 3: p. 110.

[0133] In this specification, “antineoplastic agent” refers to a drug used to treat cancer. In this specification, non-exclusive examples of antineoplastic agents include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, antihormone compounds, cytokines, EGFR-targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and antibodies, cytotoxic agents, antibodies that induce apoptosis, COX inhibitors, farnesyltransferase inhibitors, antibodies that bind to the carcinoembryonic protein CA125, HER2 vaccines, Raf or ras inhibitors, liposomal doxorubicin, topotecan, taxanes, bi-tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib, and bevacizumab.

[0134] "Treatment" refers to both therapeutic treatment and preventive or protective measures. Those requiring treatment include individuals who already have cancer, as well as those for whom cancer should be prevented. Therefore, patients treated in this specification may have been diagnosed with cancer, may be predisposed to cancer, or may be prone to developing cancer.

[0135] The term "effective amount" refers to the amount of a drug that is effective in treating cancer in a patient. An effective amount of a drug can reduce the number of cancer cells, reduce tumor size, inhibit cancer cell infiltration into peripheral organs (i.e., delay and preferably stop it to some extent), inhibit tumor metastasis (i.e., delay and preferably stop it to some extent), inhibit tumor growth to some extent, and / or reduce one or more cancer-related symptoms to some extent. The drug can be cytostatic and / or cytotoxic to the extent that it can prevent the growth of existing cancer cells and / or kill them. An effective amount can extend progression-free survival (e.g., measured by Response Evaluation Criteria in Solid Tumors, RECIST or CA-125 changes), result in an objective response (including partial response, PR or complete response, CR), increase overall survival, and / or improve one or more symptoms of cancer (e.g., assessed by FOSI).

[0136] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. This term is intended to include radioisotopes (e.g., radioisotopes of At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、and Lu), chemotherapeutic agents, and toxins, such as low molecular weight toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, including fragments and / or variants thereof.

[0137] "Chemotherapy" is the use of chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents used in chemotherapy include alkylating agents, e.g., thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa and uredopa; ethyleneimines and methylamelamine, including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK286 (TELCYTA®); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-rapa Con; Lapacol; Colchicine; Betulinic acid; Camptothecin (including synthetic analogs Topotecan (HYCAMTIN®), CPT-11 (Irinotecan, CAMPTOSAR®), Acetylcamptothecin, Scopolectin and 9-Aminocamptothecin); Briostatin; Callystatin; CC-1065 (including synthetic analogs Adzelesin, Carzelesin and Biseresin); Podophyllotoxin; Podophyllinic acid; Teniposide; Cryptophycin (especially Cryptophycin 1 and Cryptophycin 8); Dorastatin; Duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); Erytherobin; Pancratistatin; Sarcodictyin; Spongistatin;Nitrogen mustards, e.g., chlorambucil, chlornaphazine, chorophosphamide, estramustine, ifosfamide, mechloretamine, mechlorethamine oxide hydrochloride, melphalan, nobembitin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, e.g., carmustine, chlorozotocin, photemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, e.g., clodronate;Antibiotics, such as engine antibiotics (e.g., calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, Chem Intl. Ed. Engl. Vol. 33: pp. 183-186 (1994))), and anthracyclines, such as annamycin, AD32, alcarubicin, daunorubicin, doxorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, barurubicin, KRN5500, menogalil, dynemycin including dynemicin A, esperamicin, neocardinostatin chromophores and related pigment protein engine antibiotic chromophores, and aclasinomycin. sin), actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin and Mitomycins such as deoxidexorubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin and zorubicin; folate analogs, e.g., denopterin, pteropterin and trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine and thioguanine;Pyridine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, and trilostane; folate supplements, e.g., folic acid (leucovorin); acegraton; folate antagonists; anti-folate anti-neoplastic agents. Agents, for example, dihydrofolate reductase inhibitors such as ALIMTA®, LY231514 pemetrexed, methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and their prodrugs, for example UFT, S-1, and capecitabine, as well as thymidylate synthase inhibitors and glycinamide ribonucleotide transformylase inhibitors, for example larcitrexed (TOMUDEX®, TDX); dihydropyrimidine dehydrogenase inhibitors, for example enyluracil; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabucil; bisantrene; edatraxate; defofamine; demecoltin; diaziquone; elfornithine; elliptinium acetate acetate; epotilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids, e.g., mytansin and ansamitocin; mitoglucon; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK7 polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane;Rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); daca Luvazin; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxane; Chlorambucil; Gemcitabine (GEMZAR®); 6-Thiogunine; Mercaptopurine; Platinum; Platinum analogs or platinum-based analogs, e.g., cisplatin, oxaliplatin and carboplatin; Vinblastine (VELBAN( (Registered Trademark)); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloids; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS2000; difluorometholornithine (DMFO); retinoids, e.g., retinoic acid; any pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the above, e.g., CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for the therapeutic regimen of oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin.

[0138] Furthermore, this definition includes anti-hormone agents such as anti-estrogen drugs and selective estrogen receptor modulators (SERMs) that act to regulate or inhibit the hormonal effects on tumors, such as tamoxifen (including NOLVADEX® tamoxifen), raloxifen, doroxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone and FARESTON® toremifene; aromatase inhibitors; and anti-androgen drugs, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (1,3-dioxolannucleate). Examples include: leosidocytosine analogs; antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase I inhibitor; ABARELIX® rmRH; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0139] Taxanes are chemotherapy drugs that inhibit mitosis and interfere with microtubules. Examples of taxanes include paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, NJ); albumin-modified nanoparticle formulations of paclitaxel or nab-paclitaxel without cremofol (ABRAXANE®; American Pharmaceutical Partners, Schaumberg, Illinois); and docetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France).

[0140] Anthracyclines are a type of antibiotic derived from the fungus Streptococcus peucetius, and include, for example, daunorubicin, doxorubicin, epirubicin, and any other anthracycline chemotherapeutic agents, including those listed above.

[0141] Anthracycline-based chemotherapy refers to a chemotherapy regimen consisting of or containing one or more anthracyclines. Examples include, but are not limited to, 5-FFU, epirubicin, and cyclophosphamide (FEC); 5-FU, doxorubicin, and cyclophosphamide (FAC); doxorubicin, and cyclophosphamide (AC); epirubicin, and cyclophosphamide (EC); and intensive doxorubicin, and cyclophosphamide (ddAC).

[0142] For the purposes of this specification, “carboplatin-based chemotherapy” refers to a chemotherapy regimen consisting of or containing one or more carboplatins. An example is TCH (docetaxel / TAXOL®, carboplatin, and trastuzumab / HERCEPTIN®).

[0143] "Aromatase inhibitors" inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland. Examples of aromatase inhibitors include 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® borozole, FEMARA® letrozole, and ARIMIDESEX® anastrozole. In one embodiment, the aromatase inhibitor as used herein is letrozole or anastrozole.

[0144] "Antimetabolites" refer to the use of drugs that are structurally similar to their metabolites but cannot be used by the body due to their production method. Many antimetabolites interfere with the production of nucleic acids, RNA, and DNA. Examples of antimetabolites include gemcitabine (GEMZAR®), 5-fluorouracil (5-FU), capecitabine (XELODA®), 6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, larcitrexed, arabinosylcytosine (ARA-C cytarabine, CYTOSAR-U®), dacarbazine (DTIC-DOME®), azocytosine, deoxycytosine, pyridmidene, fludarabine (FLUDARA®), cladribine, and 2-deoxy-D-glucose.

[0145] "Chemotherapy-resistant" cancer means that the cancer is progressing while the patient is receiving a chemotherapy regimen (i.e., the patient is "chemotherapy-refractory"), or that the patient's cancer is progressing within 12 months (for example, within 6 months) after the completion of a chemotherapy regimen.

[0146] The term "platin" is used herein to refer to platinum-based chemotherapy, including but not limited to cisplatin, carboplatin, and oxaliplatin.

[0147] The term "fluoropyrimidine" is used herein to refer to antimetabolite chemotherapy, including, but not limited to, capecitabine, phloxuridine, and fluorouracil (5-FU).

[0148] In this specification, a “constant” or “flat” dose of a therapeutic agent refers to the dose administered to a human patient regardless of the patient’s body weight (WT) or body surface area (BSA). Therefore, a constant or flat dose is equivalent to a mg / kg dose or mg / m² dose. 2 It is not provided as a dosage, but rather as an absolute amount of the therapeutic agent.

[0149] II. Assays Combinations of therapeutic monoclonal antibodies (mAbs) and fixed-dose combination agents (FDCs) increase drug complexity and present challenges for characterization and product quality control. This challenge is exacerbated when the combined antibodies have similar physicochemical properties, such as similar isoelectric points, sequence similarity, and no significant differences in size. Furthermore, each of the combined antibodies may 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 fixed-dose combination agents. Described herein are analytical methods for determining critical quality attributes (CQAs) of a fixed-dose combination of two anti-HER2 antibodies.

[0150] In one embodiment, these assays are suitable for analyzing a fixed-dose combination of two anti-HER2 antibodies, trastuzumab and pertuzumab. Trastuzumab and pertuzumab have more than 93% sequence identity, differing by only 30 Da, and both have a molecular weight of approximately 148 kDa. Furthermore, both 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 known analytical methods cannot be applied to this combination. In addition, the assays developed for the test strategy take into account that the trastuzumab-pertuzumab fixed-dose combination is provided in two different doses, namely a loading dose and a maintenance dose, with different ratios of pertuzumab SC and trastuzumab.

[0151] (i) Efficacy Test Potency is a CQA included in the control system during the release and safety testing of biotherapeutic drugs, including therapeutic monoclonal antibodies. Potency monitors the cumulative effect of product quality attributes on biological activity and can potentially affect safety and efficacy; that is, high potency may raise safety concerns, while low potency may raise considerations regarding efficacy. Ideally, a potency assay represents the mechanism of action of the product (i.e., the relevant therapeutic activity or intended biological effect). In accordance with the U.S. Food and Drug Administration's (FDA) "Guidance for Industry on Potency Tests for Cellular and Gene Therapy Products," a traditional method for assessing the potency of a biological product is to develop a quantitative biological assay (bioassay) that measures the activity of the product in relation to its specific ability to produce a given outcome. A bioassay can provide a measure of potency by evaluating the active ingredients of the product within a biological system. A bioassay can include in vivo animal studies, in vitro organ, tissue, or cell culture systems, or any combination thereof. A widely used example of a bioassay for determining or quantifying titer is a cell-based assay. Two specific cell-based assays (antiproliferative assays) designed to measure inhibition of cell growth, specifically for pertuzumab or trastuzumab, were assessed for their suitability to control the bioactivity of a pertuzumab-trastuzumab constant-dose combination. This assessment demonstrated that the assays are unsuitable for the constant-dose combination due to limitations that hinder the control of relevant changes in the product quality of the individual antibodies when combined. Due to the nature of the combination of two antibodies that bind to the same receptor and inhibit similar signaling pathways, there are no alternative HER2-expressing cell lines that can overcome these limitations.

[0152] In trastuzumab and pertuzumab, which bind to the same receptor and inhibit similar signaling pathways in target cells, the effects on downstream signaling, gene expression, and proliferation of HER2-expressing target cells are mediated by their ability to bind to the corresponding epitope of HER2. Therefore, potential molecular changes in antibodies that affect the titer of inhibiting HER2-driven cell growth can be observed at the binding level. This hypothesis was assessed in a comparative study of selected product variants (charge and size variants, as well as CDR affinity mutants), as shown in the examples herein. This study confirmed that the binding differences detected by the binding assays provided herein, with the exception of size variants, reflected the changes observed in the antiproliferative activity of most of the product variants tested. Given the ability of the interference and binding assays in the antiproliferative assays provided herein to detect single-antibody quality changes affecting titer, the novel binding assays provided herein are considered the best assays capable of controlling for relevant changes in product quality affecting target binding and RER2 signaling.

[0153] In one embodiment, the pertuzumab / trastuzumab FDC drug is tested by a binding assay that specifically measures HER2 binding to pertuzumab or trastuzumab to determine its potency. Although both trastuzumab and pertuzumab target HER2, they bind to distinct, non-overlapping epitopes of the HER2 extracellular domain (ECD). Trastuzumab recognizes subdomain IV, which is the near-membrane region, while pertuzumab recognizes subdomain II, which is the dimerization region (Rocca A, Andreis D, Fedeli A et al., Pharmacokinetics, pharmacodynamics and clinical efficacy of pertuzumab in breast cancer therapy. Expert Opin Drug Metab Toxicol 2015; Vol. 11: pp. 1647-1663). Binding of trastuzumab to 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; Vol. 15: pp. 429-440), preventing proteolytic cleavage of the ECD and thereby prohibiting the subsequent constitutive activation of the related intracellular signaling pathway (Molina MA, Codony-Servat J, Albanell J et al., "Trastuzumab (Herceptin), a humanized anti-HER2 receptor monoclonal antibody, inhibits basal and activated HER2 ectodomain cleavage in breast cancer cells." Cancer Research 2001; Vol. 61: pp. 4744-49).As a result, trastuzumab inhibits the proliferation of HER2-overexpressing human tumor cells, as demonstrated in both in vitro assays and in animals. Pertuzumab binding to HER2 subdomain II blocks ligand-dependent heterodimerization of HR2 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; Vol. 5: pp. 317-328; Adams CW, Allison DE, Flagella K et al., Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 2006; Vol. 55: pp. 717-727; 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; Vol. 1130: pp. 280-286). As a result, pertuzumab inhibits ligand-initiated intracellular signaling, induces inhibition of cell growth, and induces apoptosis in HER2-overexpressing human tumor cells.

[0154] Pertuzumab and trastuzumab have a complementary mechanism that allows them to bind to distinct, non-overlapping epitopes of the HER2 ECD without competing with each other, thereby disrupting HER2 signaling. This results in enhanced antiproliferative activity in vitro and in vivo when pertuzumab and trastuzumab are administered as a combination drug (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; vol. 69: pp. 9330-9336). In one embodiment, the antiproliferative activity and HER2 signaling of the FDC drug are determined using two distinct HER2 binding assays to ensure control of the respective quality of the two antibodies in the pertuzumab-trastuzumab FDC drug.

[0155] In one embodiment, a binding assay for a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Contact the FDC with the capture reagent to confirm that the capture reagent is a modified HER2 ECD. b. Contacting the sample with a detectable antibody, c. Quantifying the level of antibody bound to the capture reagent using a detection method for detectable antibodies, A binding assay is provided, including the following.

[0156] A combination of two anti-HER2 antibodies in fixed doses is brought into contact with a capture reagent and incubated so that the capture reagent can capture or bind to one of the target anti-HER2 antibodies and be detected in the detection 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 of the anti-HER2 antibodies to be assessed in the FDC binds, but a second anti-HER2 antibody in the FDC does not bind. This is achieved by either omitting the HER2 ECD subdomain to which the second anti-HER2 antibody binds, or by replacing it with a structurally similar subdomain that does not bind to any of the anti-HER2 antibodies. The structurally similar subdomain can be any subdomain that does not interfere with the three-dimensional structure of the modified HER2 ECD when incubated with the modified HER2 EDC. Examples of structurally similar subdomains are the corresponding subdomains of EGFR, HER3, or HER4. Preferably, the modified HER2 ECD has a three-dimensional structure that closely mimics the native HER2 ECD. The subdomain may be full length or shortened by a few amino acids at the N or C terminus. The inventors of this invention have found that the structural integrity of the HER2 ECD can be preserved or improved by using one or more recombinant HER2 ECD subdomains that are shortened by about 4-5 amino acids at the C terminus.

[0157] In one embodiment, the modified HER2 ECD fuses with a peptide or protein to facilitate the immobilization of the capture reagent onto a solid substrate. Suitable examples of peptides or proteins include biotin, bovine serum albumin (BSA), and Fc domains. One modified HER2 extracellular domain fuses with an Fc domain. In one embodiment, the Fc domain is from a different species than the Fc domain of the anti-HER2 antibody being analyzed. For example, if the anti-HER2 antibody being analyzed contains a human Fc domain, the capture reagent should contain a non-human Fc domain, such as from a mouse, porcupine, rat, or rabbit. In one embodiment, the Fc domain of the recombinant HER2 ECD subdomain is a mouse Fc domain. In one embodiment, the Fc domain contains SEQ ID NO: 35.

[0158] In the next step, the sample containing the capture reagent and the captured anti-HER2 antibody is incubated with a detectable antibody. The detectable antibody binds to any of the conjugated anti-HER2 antibodies of interest. In the next step, a detection means is used to detect the label of the detectable antibody, thereby detecting the presence or amount of the anti-HER2 antibody of interest present in the FDC.

[0159] In one embodiment, the fixed-dose formulation includes an antibody that binds to HER2 extracellular subdomain II and an antibody that binds to HER2 extracellular subdomain IV. In one embodiment, the antibody that binds to HER2 extracellular subdomain II is pertuzumab. In one embodiment, the antibody that binds to HER2 extracellular subdomain IV is trastuzumab. In one embodiment, the fixed-dose formulation includes pertuzumab and trastuzumab. In one embodiment, the fixed-dose formulation additionally includes hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one preferred embodiment, the hyaluronidase is rHUPH20. The pertuzumab-trastuzumab FDC drug is offered in two different doses, namely loading dose (LD) and maintenance dose (MD). The LD and MD have the same total protein content but differ in the ratio of pertuzumab SC to trastuzumab. In one embodiment, the LD of pertuzumab-trastuzumab FDC is analyzed using a binding assay. In one embodiment, the pertuzumab-trastuzumab FDC containing 40 mg / mL of trastuzumab and 80 mg / mL of pertuzumab is analyzed using a binding assay. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains rHuPH20 at 2000 U / mL. In one embodiment, the MD of pertuzumab-trastuzumab FDC is analyzed using a binding assay. In one embodiment, the pertuzumab-trastuzumab FDC containing 60 mg / mL of trastuzumab and 60 mg / mL of pertuzumab is analyzed using a binding assay. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains rHuPH20 at 2000 U / mL.

[0160] In one embodiment, the binding of pertuzumab and trastuzumab is determined by two separate binding assays.

[0161] The pertuzumab binding assay determines specific biological activity as the ability of pertuzumab to specifically bind to the epitope of a recombinant HER2 capture reagent. In one embodiment, pertuzumab binding 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.

[0162] In one embodiment, the modified HER2 ECD includes HER2 ECD subdomains I, II, and III or a portion thereof. In one embodiment, the modified HER2 ECD includes human HER2 ECD subdomains I, II, and III or a portion thereof. In one embodiment, the modified HER2 ECD does not include subdomain IV. The inventors have found that when the modified HER2 ECD includes recombinant subdomain III which is shortened at the C-terminus, it can be produced with a three-dimensional structure similar to that of the natural HER2 ECD. In one such embodiment, the modified HER2 ECD includes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34. In one embodiment, the modified HER2 ECD includes SEQ ID NO: 24. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with SEQ ID NO: 24.

[0163] In one embodiment, recombinant HER2 extracellular subdomains I, II, and III are fused to an Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In any of the above embodiments, the capture reagent for assessing pertuzumab binding does not contain HER2 extracellular subdomain IV. In one embodiment, the capture reagent includes 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 to SEQ ID NO: 25. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 26. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 27.

[0164] In one embodiment, trastuzumab binding is quantified. In one such embodiment, the capture reagent comprises recombinant HER2 extracellular subdomain IV or a portion thereof. In one such embodiment, the capture reagent comprises human recombinant HER2 extracellular subdomain IV. In one embodiment, the capture reagent comprises SEQ ID NO: 28 or SEQ ID NO: 4.

[0165] In one embodiment, the capture reagent comprises recombinant HER2 extracellular subdomains I, III, and IV. In one embodiment, the capture reagent comprises human HER2 extracellular subdomains I, III, and IV. In one embodiment, the capture reagent comprises recombinant HER2 extracellular subdomains I, III, and IV, as well as subdomain II of EGFR. In one embodiment, the capture reagent comprises recombinant human HER2 extracellular subdomains I, III, and IV, as well as recombinant human subdomain II of EGFR. The inventors have found that modified HER2 ECDs can be produced with a three-dimensional structure similar to that of native HER2 ECDs when they contain recombinant HER2 extracellular subdomains I and IV, both shortened at the C-terminus. 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.

[0166] In one embodiment, the modified HER2 ECD includes sequence number 29. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity with sequence number 29.

[0167] In one embodiment, recombinant HER2 extracellular subdomains I, III, and IV, as well as EGFR subdomain II, are fused to an Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In any of the above embodiments, the capture reagent for assessing trastuzumab binding does not contain HER2 subdomain II. In one embodiment, the capture reagent includes 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 to SEQ ID NO: 30. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 31. In one embodiment, the modified HER2 ECD has at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 32.

[0168] In any of the embodiments described above, the detectable antibody includes a label that enables detection by various methods. These labels include a directly detectable portion, such as a fluorescent dye label, a chemiluminescent label, and a radioactive label, as well as a portion that must be reacted or derivatized for detection, such as an enzyme. Examples of such labels include the radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, ruthenium, dansyl, umbelliferone, luceriferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, HRP, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, such as g Examples include glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, enzymes that oxidize pigment precursors using hydrogen peroxide, such as HRP, heterocyclic oxidases coupled with lactoperoxidase or microperoxidase, such as uricase and xanthine oxidase, biotin (detectable by avidin, streptavidin, streptavidin-HRP and streptavidin-β-galactosidase using MUG, spin labeling, bacteriophage labeling, and stable free radicals).

[0169] A preferred label for detectable antibodies is horseradish peroxidase (HRP). Substrates commonly used for HRP fall into different categories, depending on whether they produce colored, fluorescent, or luminescent derivatives, and include dye-producing substrates (e.g., aminoethylcarbazole (AEC), 3,3'-diaminobenzidine tetrahydrochloride (DAB), chloronaphthol combined with diaminobenzidine (CN / DAB), tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)), fluorescent substrates (e.g., ADHP), and chemiluminescent substrates (e.g., enhanced chemiluminescence (ECL)). The preferred substrate is ABTS.

[0170] In one embodiment, the detectable antibody targets the F(ab')2 portion of human IgG. In another embodiment, the detectable antibody targets the F(ab')2 portion of an anti-HER2 antibody.

[0171] In one embodiment, the binding assay is enzyme-linked immunosorbent assay (ELISA). In ELISA, the capture reagent is bound to a solid substrate. The solid phase used for immobilization may be any inert support or carrier that is essentially water-insoluble and useful for immunoassay assays, such as surfaces, particles, or porous matrices. Examples of commonly used supports include small sheets, SEPHADEX® gels, polyvinyl chloride, plastic beads, and assay plates or test tubes made from polyethylene, polypropylene, polystyrene, etc., including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive, water-insoluble matrices, such as the cyanide-activated carbohydrates and reaction substrates described in U.S. Patents No. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440, are suitably used for immobilizing the capture reagent. In a preferred embodiment, the immobilized capture reagent is coated onto a microtiter plate, and in particular, the preferred solid phase used is a multi-well microtiter plate that can be used to analyze several samples at once. A preferred microtiter plate is a plate having a highly charged polystyrene surface with a high affinity for molecules having polar or hydrophilic groups and having high binding ability to proteins. Most preferred are the MICROTEST® or MAXISORP® 96-well ELISA plates, for example, those marketed as NUNC MAXISORP® or IMMULON®.

[0172] The 96-well plate is preferably coated with the capture reagent for at least 30, 40, 50, 60 minutes, about 20–80 minutes, or about 30–60 minutes. The 96-well plate is more preferably coated with the capture reagent at a temperature of about 4–20°C, more preferably about 2–8°C. The plates may be stacked and coated prior to the assay, and the assay can then be performed on several samples simultaneously in a manual, semi-automatic, or automated manner, for example, by robotics.

[0173] The amount of capture reagent used is sufficient to produce a good signal, but not in excess molars compared to the maximum exposure level of the antibody of interest in the sample. In one embodiment, the coating reagent concentration is about 0.5 μg / mL to 5 μg / mL, preferably about 1 μg / mL to 1.5 μg / mL.

[0174] The coated plates are then typically treated with a blocking agent that nonspecifically binds to the binding sites and saturates them, thereby preventing undesirable binding of free ligands to excess areas in the plate wells. Examples of blocking agents suitable for this purpose include, for example, gelatin, bovine serum albumin, egg albumin, casein, and skim milk. The blocking treatment is typically carried out at room temperature for about 1 to 4 hours, about 1 to 3 hours, preferably about 1 to 1.5 hours.

[0175] After coating and blocking, the standard or FDC sample to be analyzed is added to the coated plate with a standard diluent. In one embodiment, progressively increasing concentrations of pertuzumab / trastuzumab FDC (standard, product control, and sample to be analyzed) are added to the coated plate.

[0176] The incubation conditions for the FDC sample and the immobilized capture reagent are selected to maximize assay sensitivity, minimize dissociation, and ensure that the anti-HER2 antibody assessed in the FDC sample binds to the immobilized capture reagent. Preferably, incubation is achieved at a nearly constant temperature in the range of about 0°C to about 40°C, preferably at room temperature or near room temperature. The incubation time is generally about 10 hours or less. Preferably, to maximize the binding of the anti-HER2 antibody assessed in the FDC sample to the capture reagent, the incubation time is about 0.5 to 3 hours, more preferably about 1 to 1.5 hours, at room temperature or near room temperature.

[0177] An immobilized capture reagent containing any conjugated anti-HER2 antibody is brought into contact with a detectable antibody, preferably at a temperature of about 20-40°C, more preferably at room temperature. The exact temperature and time of contact between the two depend mainly on the detection method used.

[0178] In another embodiment, the binding assay is electrochemiluminescence (ECL).

[0179] In one embodiment, the binding assay is used to analyze the titer of one of the anti-HER2 antibodies. Therefore, in one embodiment, the binding assay is d. A step of correlating the level of the antibody bound to the capture reagent with the biological activity of the antibody. It also includes the following.

[0180] In one embodiment, the dose-response curve generated by the sample is compared with a standard dose-response curve. In one embodiment, the standard titer is quantified by separately correlating the results obtained in the binding assay with the biological activity of the isolated antibody in the cell-based assay.

[0181] In one embodiment, nonlinear four-parameter dose-response curves generated by the sample and the standard are compared. Once the similarity criteria between the standard and sample dose-response curves are assessed, the relative titer of the sample is calculated using four-parameter parallel line analysis based on the concentration shift between the standard and sample dose-response curve fits.

[0182] In one embodiment, the binding assay is for batch dispensing of a fixed-dose combination of pertuzumab and trastuzumab. In one embodiment, the binding assay is for determining the shelf life of a fixed-dose combination of pertuzumab and trastuzumab. In one such embodiment, the pertuzumab-trastuzumab FDC is analyzed by the binding assay of the above embodiment at several points in time during storage.

[0183] (ii) Analysis of charge variants In one embodiment, a method is provided for evaluating a fixed-dose composition comprising pertuzumab and trastuzumab, comprising assessing the amounts of charge variants of pertuzumab and trastuzumab in the composition. In one embodiment, the fixed-dose formulation additionally comprises hyaluronidase. In one embodiment, the method is ion-exchange chromatography. Ion-exchange chromatography (IEX) is widely used for detailed characterization of therapeutic proteins and can be considered a 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 according to charge heterogeneity. Separation is brought about by the reversible adsorption of charged solute molecules to ion-exchange groups of opposite charge immobilized on a column packing material. Adsorption of molecules to a solid support is driven by ionic interactions between two parts. The strength of the interaction is determined by the number and position of charges in the molecule and the stationary phase. IEX is typically an extraction method in which specificization is set particularly by the distribution of acidic, major, and basic species of mAbs, respectively. These charged species are considered product-related impurities that can affect the titer. Furthermore, it is one of the few methods that can characterize a protein by confirming that it is natural and undenatured. IEX can also be used as a characterization method for certain biological products and is a standard test for justifying stability and shelf life.

[0184] Analyzing the distribution of charge variants in fixed-dose combinations of two anti-HER2 antibodies with very similar isoelectric points, such as trastuzumab and pertuzumab, requires a specific ion-exchange chromatography protocol that allows for precise isolation of all related species.

[0185] In one embodiment, a method is provided for evaluating a fixed-dose composition containing pertuzumab and trastuzumab, comprising assessing the amounts of charge variants of pertuzumab and trastuzumab in the composition. In one embodiment, the fixed-dose formulation additionally includes hyaluronidase. In one embodiment, the method is ion-exchange chromatography. In one particular embodiment, the method is cation-exchange chromatography. In cation-exchange chromatography, when applied to a pertuzumab / trastuzumab fixed-dose formulation (FDC), positively charged molecules are retained in the load-charged stationary phase. Acidic species elute with a shorter retention time than basic species.

[0186] After equilibrating the column and sample application, the FDC anti-HER2 antibodies pertuzumab and trastuzumab are adsorbed to the column ligand. The column is then washed to remove unadsorbed proteins, and elution is performed by changing the ionic strength of the mobile phase while maintaining the pH within a pre-defined range. In one embodiment, the pH is kept constant.

[0187] The ionic strength is varied by applying a gradient of gradually increasing salt concentrations, which can be either a stepped or continuous gradient. The inventors of the present invention found that the pH range of the loading buffer (mobile phase A) and elution buffer (mobile phase B) is important in the analysis of charge variants of the FDCs of two anti-HER2 antibodies, pertuzumab and trastuzumab. The best separation of charge variants is obtained by a pre-specified pH range of 7.5–7.65 for the loading buffer (mobile phase A) and a pre-specified pH range of 7.5–7.7 for the elution buffer (mobile phase B). In one embodiment, the pH is kept constant. In one embodiment, the constant pH value of the loading buffer is 7.5, 7.55, 7.6, or 7.65. In one embodiment, the constant pH value of the elution buffer is 7.5, 7.55, 7.6, 7.65, or 7.7.

[0188] After elution, the column is re-equilibriumated with loading buffer (mobile phase A).

[0189] In one embodiment, a fixed-dose pertuzumab / trastuzumab combination is brought into contact with a cation exchange material, and charge variants and innate antibodies are eluted using a salt gradient while maintaining the pH of the mobile phase within a pre-defined range. In one embodiment, the salt gradient is a continuous salt gradient. In one embodiment, the pH of the loading buffer mobile phase (mobile phase A) is pH 7.5 to pH 7.65. In one embodiment, the pH of the elution buffer mobile phase (mobile phase B) is pH 7.5 to pH 7.7.

[0190] In one embodiment, the salt gradient is a sodium chloride gradient. In one embodiment, the salt gradient is a sodium chloride gradient, and the pH of the elution buffer mobile phase (mobile phase B) is pH 7.5 to pH 7.7.

[0191] In one embodiment, a method for evaluating a fixed-dose composition containing pertuzumab and trastuzumab, a. Using a loading buffer, the antibody is bound to the ion exchange material, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. Elute the antibody with elution buffer, ensuring the pH of the elution buffer is approximately 7.5 to 7.7. A method including this is provided.

[0192] In one embodiment, elution in step b is carried out 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 include MES (2-ethanesulfonic acid), ACES (N-(2-acetamide)-2-aminoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate buffer, MOPS (3-(N-morpholino)propanesulfonic acid), TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), CAPSO (N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), and TPP (tris, phosphate, piperazine). Preferred buffers are ACES and HEPES.

[0193] In one embodiment, the sodium chloride concentration of the elution buffer (mobile phase B) is approximately 180-220 mM NaCl, approximately 200 mM NaCl, approximately 180 mM NaCl, approximately 190 mM NaCl, approximately 210 mM NaCl, or approximately 220 mM NaCl.

[0194] In one embodiment, the ion exchange material is a cation exchange material. To further optimize the method of the present invention, the inventors have found that the separation of charge variants is improved when a strong cation exchange column material is used. In a preferred embodiment, the method is carried out using a non-porous SCX column having a sulfonate group, with Na+ used as the counterion for elution. Thus, in one embodiment, the cation exchange material has a sulfonate group. In one such embodiment, the cation exchange material is a strong cation exchange (SCX) column having a sulfonate group, and the elution buffer contains sodium. In one such embodiment, the elution buffer contains sodium ions. In one embodiment, the SCX column is non-porous. Preferred cation exchange columns useful herein include the YMC Bio Pro SP-F column, MabPac SCX-10, Waters BioResolve SCX mAb, Sepax Proteomix SCX-NP1.7, or the non-porous Agilent Bio SCX.

[0195] In one embodiment, steps a and b of the above method are carried out at a temperature of 32°C to 40°C or about 36°C.

[0196] In one embodiment, ion exchange chromatography is performed with a total protein load of approximately 50 μg to 149 μg or approximately 51 μg to 153 μg. In one embodiment, ion exchange chromatography is performed by loading the total protein with a pertuzumab / trastuzumab FDC load of approximately 50 μg to 149 μg. In one embodiment, ion exchange chromatography is performed by loading the total protein with a maintenance dose of approximately 51 μg to 153 μg of pertuzumab / trastuzumab FDC. In one embodiment, the total protein loaded into ion exchange chromatography is approximately 100 μg.

[0197] In one embodiment, a method for evaluating a fixed-dose composition containing pertuzumab and trastuzumab, a. Using a loading buffer, the antibody is bound to the ion exchange material, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. Elute the antibody with elution buffer, ensuring the pH of the elution buffer is approximately 7.5 to 7.7. c. Selective detection of charge variants of pertuzumab and trastuzumab in the composition, A method including this is provided.

[0198] In one embodiment, acidic, native, and basic variants of trastuzumab and pertuzumab in a fixed-dose formulation are selectively detected.

[0199] In one embodiment, ion exchange chromatography is performed with a fixed-dose combination of pertuzumab and trastuzumab, which has been digested with carboxypeptidase B before being loaded onto the chromatography column.

[0200] In one embodiment, the fixed-dose combination of pertuzumab and trastuzumab additionally contains hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab-trastuzumab FDC contains about 2000 U / mL of rHuPH20. The pertuzumab-trastuzumab FDC drug is offered in two different doses, namely a loading dose (LD) and a maintenance dose (MD). The LD and MD have the same total protein content but differ in the ratio of pertuzumab SC to trastuzumab. In one embodiment, the method is useful for determining charge variants of the loading doses of pertuzumab and trastuzumab FDC. In one embodiment, charge variants of both pertuzumab and trastuzumab are determined simultaneously, i.e., by the same method. In one embodiment, the method is used to analyze charge variants of pertuzumab-trastuzumab FDC containing 40 mg / mL trastuzumab and 80 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains 2000 U / mL of rHuPH20. In one embodiment, the method is useful for determining the maintenance dose charge variants of pertuzumab and trastuzumab FDC. In one embodiment, charge variants of both pertuzumab and trastuzumab are determined simultaneously, i.e., by the same method. In one embodiment, the method is used to analyze charge variants of pertuzumab-trastuzumab FDC containing 60 mg / mL trastuzumab and 60 mg / mL pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains 2000 U / mL of rHuPH20.

[0201] In one embodiment, the innate antibodies, as well as their acidic and basic variants, are eluted over a salt gradient of 1–100% (solvent B) over at least 44 minutes. In another embodiment, the salt gradient is increased from 1 to 47% solvent B over 43 minutes. In yet another embodiment, the salt gradient is increased from approximately 1.8 to 103.4 mM NaCl.

[0202] In one embodiment, the mobile phase of ion exchange chromatography contains ACES buffer. In one embodiment, mobile phases A and B contain ACES buffer. In one embodiment, solvent A of ion exchange chromatography contains about 10–50 mM, about 15–25 mM, about 18–22 mM, or about 20 mM ACES. In one embodiment, solvent B of ion exchange chromatography contains 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 contains about 20 mM ACES.

[0203] (ii) Content / protein content assay UV spectroscopy is a typical method for determining the total protein content of a pharmaceutical sample. However, a different approach is required for a fixed-dose combination drug (FDC) containing two anti-HER2 antibodies, because conventional methods do not allow for separate and quantitative analysis of the protein content of each anti-HER2 antibody in the FDC. Different chromatographic methods were tested, such as hydrophobic interaction (HIC) and reversed-phase chromatography (RPC). For the separate quantitative analysis of protein content in pertuzumab-trastuzumab FDCs, reversed-phase chromatography proved to be the most suitable method.

[0204] Reverse-phase ultrahigh-performance liquid chromatography (RP-UHPLC, RPC) separates molecules in solution according to their hydrophobicity. Separation is achieved by the reversible hydrophobic adsorption of molecules to a nonpolar stationary phase in a column. Adsorption of molecules to a solid support is driven by a two-part hydrophobic / nonpolar interaction. The strength of the interaction is determined by the number and position of functional groups in the molecule and the stationary phase. In reverse-phase chromatography, nonpolar molecules elute from the stationary phase with longer retention times than polar molecules. Since the two anti-HER2 antibodies, trastuzumab and pertuzumab, have over 93% sequence identity and differ by only 30 Da in total, we developed a robust method that provides reliable overall resolution and peak separation without significant carryover contamination (i.e., carryover contamination should not exceed 0.2% in subsequent analyses). In addition, the content assay developed for the test strategy takes into account that the trastuzumab-pertuzumab constant-dose combination is provided in two different doses, namely loading and maintenance doses, with different ratios of pertuzumab SC and trastuzumab. The inventors have found that phenyl-based columns yield particularly robust results, and that the most important parameters for a robust method are column temperature and flow rate. Phenyl-based RP-UHPLC columns are known in the art and can have the following groups: ethylphenyl with a methyl side group and an end-capped silica surface, a phenylhexyl phase with an extended (hexyl) ligand spacer methyl side group, an ethylphenyl ligand with a sterically protected (isobutyl) side group, a hexyl biphenyl with a methyl side group, a biphenyl phase with a methyl side group, and an oxygen-activated phenylethylphenyl phase with a methyl side group. HPLC columns with stationary phases modified with phenyl (e.g., monophenyl, biphenyl, diphenyl, phenylhexyl, phenylpropyl) are readily available from most major column suppliers. An example of a phenylcalm useful in this specification is the Agilent Zorbax RRHD 300-Diphenly column. In one embodiment, the column is a 2.1 × 100 mm column.

[0205] Provided herein is a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Prepare the RP-HPLC phenyl column, b. Loading an RP-HPLC column with a fixed-dose combination of two anti-HER2 antibodies (FCD), c. Separate two anti-HER2 antibodies at a flow rate of 0.2-0.4 mL / min, with a column temperature of 64°C-76°C. This method includes [something].

[0206] The separation principle of RP-HPLC is based on the hydrophobic association of polypeptide solutes and hydrophobic ligands on the surface of a chromatography resin. An RP-HPLC column is typically part of a UHPLC system that includes a vacuum degasser, an autosampler with a sample condenser, a column heater, and a UV / VIS detector in series. Suitable examples of UHPLC systems include the Waters Aquity and Thermo Ultimate 3000 RS.

[0207] The FDCs of the two anti-HER2 antibodies are loaded onto the column by injecting the sample into the RP-HPLC system. Typically, the sample is diluted to a concentration of 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 trace species without saturating the detector signal. In one embodiment, the sample is diluted with a formulation buffer. 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 solution due to the use of a different diluent is eliminated. No relevant interference from the formulation buffer was observed when using the RP-HPLC method. 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.

[0208] Proteins are bound to an RP-HPLC column in an aqueous mobile phase and eluted from the column by increasing the hydrophobicity of the mobile phase. Proteins are then separated according to their hydrophobicity. In one embodiment of a method for analyzing the protein content of a fixed-dose formulation (FCD) of two anti-HER2 antibodies, the separation in step c) is achieved by a water-2-propanol / acetonitrile gradient. In one such embodiment, proteins are bound to a column in an aqueous phase (eluent A) containing water:2-propanol (98:2) + 0.1% trifluoroacetic acid (TFA) and eluted by increasing concentrations of an organic phase containing acetonyl. In one such embodiment, the organic layer (eluent B) contains 2-propanol:acetonyl:eluent A (70:20:10). Thanks to the type of phenyl-based column, improved specificity was achieved, and novel species were detected by 2-propanol alone but not by pure acetonitrile. Different specificities were achieved because the phenyl-based column interacted with the analyte via traditional hydrophobic interactions and additionally via π-π interactions. Literature has shown that pure acetonitrile inhibits these interactions, while 2-propanol does not (Yang, M., Fazio, S., Munch, D., and Drumm, P. Impact of methanol and acetonitrile on separations based on π-π interactions with a reversed-phase phenyl column. Journal of Chromatography A, Vol. 1097, pp. 124-129). However, considering the high viscosity of 2-propanol and the associated increase in back pressure, 20% acetonitrile was added to reduce the back pressure of the system.

[0209] In one embodiment, the aqueous mobile phase comprises 70% eluent A and 30% eluent B, where 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 layer (eluent B) is increased to 55% eluent A and 45% eluent B. In one embodiment, the gradient is increased to 45% solvent B over 15 minutes.

[0210] In one embodiment, the organic layer (eluent B) is increased to 10% eluent A and 90% eluent B. In one embodiment, the gradient is increased to 90% solvent B over 20 minutes.

[0211] Flow rates of 0.4 and 0.2 mL / min were tested and found not to have a significant effect on the method performed. In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, the flow rate in step c) is approximately 0.3 mL / min.

[0212] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, the antibodies are separated over a period of 10-20 minutes. In one such embodiment, the antibodies are separated over a period of 15 minutes. In one embodiment, the antibodies are separated over a period of 15 minutes at a flow rate of approximately 0.3 mL / min.

[0213] In addition to the loading and elution (separation) steps, RP-HPLC purification may include additional steps such as equilibration, washing, and regeneration. In one embodiment, RP-HPLC phenylcalm is equilibrated with 70% eluent A and 30% eluent B, where 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, RP-HPLC phenylcalm is washed with 10% eluent A and 90% elution mobile phase, where 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).

[0214] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, the column temperature is 70°C ± 2°C. Compared to room temperature, a column temperature of 70°C leads to high reproducibility, eliminates tailing effects, exhibits low back pressure in the system, and shows good resolution and separation in the overall results. Several column temperatures were tested, and 70°C showed an improved peak pattern while simultaneously not reaching the maximum temperature possible by the system and column type. Temperatures of 64°C and 76°C, and 66°C and 74°C were tested respectively and found not to have a significant effect on the method performed.

[0215] In one embodiment of a method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, the phenyl column is selected from the group consisting of Agilent Zorbax RRHD 300-Diphenyl column, Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, BioResolve RP mAb Polyphenyl, and Agilent AdvanceBio RP mAb Diphenyl. In one embodiment, the phenyl column is an Agilent Zorbax RRHD 300-Diphenyl column. In another embodiment, the phenyl column is a BioResolve RP mAb Polyphenyl column.

[0216] In one embodiment, the protein is detected by UV light. In one embodiment, the detection wavelength is 280 nm.

[0217] In one embodiment, the fixed-dose combination formulation comprises pertuzumab and trastuzumab. In one embodiment, the fixed-dose combination formulation of pertuzumab and trastuzumab additionally comprises hyaluronidase. In one such embodiment, the hyaluronidase is recombinant human hyaluronidase. In one embodiment, the hyaluronidase is rHuPH20. In one embodiment, the pertuzumab-trastuzumab FDC contains about 2000 U / mL of rHuPH20. The pertuzumab-trastuzumab FDC drug is offered in two different doses, namely a loading dose (LD) and a maintenance dose (MD). The LD and MD have the same total protein content but differ in the ratio of pertuzumab SC to trastuzumab. In one embodiment, a method is useful for determining the protein content of the loading dose of pertuzumab and trastuzumab FDC. In one embodiment, the protein content of both pertuzumab and trastuzumab is determined simultaneously, i.e., by the same method. In one embodiment, the method is used to analyze the protein content of pertuzumab-trastuzumab FDC containing 40 mg / mL of trastuzumab and 80 mg / mL of pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains 2000 U / mL of rHuPH20. In one embodiment, the method is useful for determining the protein content of maintenance doses of pertuzumab and trastuzumab FDC. In one embodiment, the protein content of both pertuzumab and trastuzumab is determined simultaneously, i.e., by the same method. In one embodiment, the method is used to analyze the protein content of pertuzumab-trastuzumab FDC containing 60 mg / mL of trastuzumab and 60 mg / mL of pertuzumab. In one embodiment, the pertuzumab-trastuzumab FDC additionally contains rHuPH20 at 2000 U / mL.

[0218] III. Anti-HER2 antibodies and compositions (i) Anti-HER2 antibody The HER2 antigen used for antibody production may be, for example, a soluble form of the extracellular domain of the HER2 receptor or a portion thereof containing the desired epitope. Alternatively, antibodies can be generated using cells that express HER2 on their cell surface (e.g., NIH-3T3 cells transformed to overexpress HER2; or cancer cell lines such as SK-BR-3 cells; see Stancovski et al., PNAS (USA) 88: pp. 8691-8695 (1991)). Other forms of the HER2 receptor useful for antibody production are apparent to those skilled in the art.

[0219] Various methods for producing monoclonal antibodies described herein are available in this technique. For example, monoclonal antibodies may be produced using the hybridoma method first described by Kohler et al., Nature, Vol. 256:p. 495 (1975), or by the recombinant DNA method (U.S. Patent No. 4,816,567).

[0220] The anti-HER2 antibody, trastuzumab, and pertuzumab used in this invention are commercially available.

[0221] U.S. Patent No. 6,949,245 describes the production of an exemplary humanized HER2 antibody that binds to HER2 and blocks ligand activation of the HER receptor.

[0222] Humanized HER2 antibodies include trastuzumab, as defined herein and listed in Table 3 of U.S. Patent No. 5,821,337, expressly incorporated herein by reference; and humanized 2C4 antibodies, such as pertuzumab, as defined herein.

[0223] The humanized antibodies described herein may, for example, include non-human hypervariable region residues incorporated into human variable weight domains, and may further include framework region (FR) substitutions at positions selected from the group consisting of 69H, 71H, and 73H using the variable domain numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody includes FR substitutions at two or all of the positions of 69H, 71H, and 73H.

[0224] In this specification, the exemplary humanized antibodies of interest comprise the variable weight domain complementarity-determining residues GFTFTDYTMX (SEQ ID NO: 17) (where X is preferably D or S); DVNPNSGGSIYNQRFKG (SEQ ID NO: 18); and / or NLGPSFYFDY (SEQ ID NO: 19), and optionally include amino acid modifications of these CDR residues, for example, modifications that essentially maintain or improve the affinity of the antibody. For example, antibody variants used in the methods of the present invention may have about 1 to about 7, or about 5, amino acid substitutions in the above variable weight CDR sequences. Such antibody variants can be prepared, for example, by affinity maturation as described below.

[0225] Humanized antibodies use the variable light domain complementarity-determining residue KASQDVSIGVA (SEQ ID NO: 20);SASYX 1 X 2 X 3 (X here) 1 is preferably R or L, and X 2 is preferably Y or E, and X 3(preferably T or S) (SEQ ID NO: 21); and / or QQYYIYPYT (SEQ ID NO: 22) may be included, for example, in addition to these variable heavy domain CDR residues in the preceding paragraph. Such humanized antibodies optionally include amino acid modifications of the above CDR residues, for example, the modifications essentially maintain or improve the affinity of the antibody. For example, the antibody variant of interest may have about 1 to about 7, or about 5, amino acid substitutions in the above variable light CDR sequence. Such antibody variants can be prepared, for example, by affinity maturation as described below.

[0226] This application also envisions an affinity-mature antibody that binds to HER2. The parent antibody may be a human antibody or a humanized antibody, and may, for example, contain the variable light sequence and / or variable heavy sequence of SEQ ID NOs. 7 and 8, respectively (i.e., contain the VL and / or VH of pertuzumab). The affinity-mature variant of pertuzumab preferably binds to the HER2 receptor with superior affinity to mouse 2C4 or pertuzumab (e.g., improved affinity from about 2x or 4x to about 100x or 1000x as assessed by ELISA). Exemplary variable heavy CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more of these residues (e.g., 2, 3, 4, 5, 6, or 7 of these residues). Examples of variable light CDR residues for modification include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or combinations of two or more of these residues (e.g., from two to three, four, five, or about ten of these residues).

[0227] Humanization of mouse 4D5 antibodies that generate humanized variants, including those with trastuzumab, is described in U.S. Patents 5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and 6,800,738, as well as in Carter et al., PNAS (USA), Vol. 89: pp. 4285-4289 (1992). HuMAb4D5-8 (trastuzumab) binds to the HER2 antigen three times more tightly than mouse 4D5 antibodies and possesses secondary immune function (ADCC) that enables targeted cytotoxic activity of the humanized antibody in the presence of human effector cells. HuMAb4D5-8 has been incorporated into the consensus framework for variable light (VL)κ subgroup I. L CDR residues and variable heavy chain (V H ) V incorporated into the consensus framework of subgroup III H The antibody contained a CDR residue. H FR substitutions at positions 71, 73, 78 and 93 (Kabat numbering of framework region (FR) residues), and V L It contains an FR substitution at position 66 (Kabat numbering of FR residues). Trastuzumab contains a non-A allotype human γ1Fc region.

[0228] Various forms of humanized antibodies or affinity-mature antibodies are intended. For example, the humanized antibody or affinity-mature antibody may be an antibody fragment. Alternatively, the humanized antibody or affinity-mature antibody may be an intact antibody, such as an intact IgG1 antibody.

[0229] (ii) Pertuzumab composition In one embodiment of the HER2 antibody composition, the composition comprises a mixture of a natural pertuzumab antibody and one or more variants thereof. Preferred embodiments of the natural pertuzumab antibody as used herein include the variable light and variable heavy amino acid sequences of SEQ ID NOs. 7 and 8, and most preferably include the light chain amino acid sequence of SEQ ID NO: 11 and the heavy chain amino acid sequence of SEQ ID NO: 12. In one embodiment, the composition comprises a mixture of the natural pertuzumab antibody and an amino acid sequence variant containing an amino-terminal leader extension. Preferably, the amino-terminal leader extension is located on the light chain of the antibody variant (e.g., one or two light chains of the antibody variant). The major species HER2 antibody or antibody variant may be a full-length antibody or an antibody fragment (e.g., Fab of the F(ab=)2 fragment), but preferably both are full-length antibodies. The antibody variant as used herein may contain an amino-terminal leader extension on one or more of its heavy or light chains. Preferably, the amino-terminal leader extension is located on one or two light chains of the antibody. The amino-terminal leader extension preferably contains or consists of a VHS. The presence of the amino-terminal leader extension in the composition can be detected by a variety of analytical techniques, including, but not limited to, N-terminal sequencing analysis, assays for charge heterogeneity (e.g., cation exchange chromatography or capillary zone electrophoresis), and mass spectrometry. The amount of antibody variants in the composition generally ranges from an amount constituting the detection limit of any assay used to detect the variants (preferably N-terminal sequencing analysis) to an amount less than the amount of the major species antibody. Generally, about 20% or less of the antibody molecules in the composition (e.g., about 1% to about 15%, e.g., 5% to about 15%) contains the amino-terminal leader extension. The percentage of such an amount is preferably determined using quantitative N-terminal sequencing analysis or cation exchange analysis (preferably using a high-resolution weak cation exchange column, e.g., PROPAC WCX-10® cation exchange column). Apart from amino-terminal leader extension variants, further amino acid sequence modifications of major species antibodies and / or variants are being attempted, including, but are not limited to, antibodies containing a C-terminal lysine residue in one or both of the heavy chains, and deamidated antibody variants.

[0230] Furthermore, the major species antibody or variant may further include glycosylation modifications, non-limiting examples of which include antibodies containing a G1 or G2 oligosaccharide bound to the Fc region, antibodies containing a carbohydrate moiety bound to the light chain (e.g., one or two carbohydrate moieties bound to one or more lysine residues, e.g., glucose or galactose, bound to one or two light chains of the antibody), antibodies containing one or two non-glycosylated heavy chains, or antibodies containing a sialidated oligosaccharide bound to one or two heavy chains.

[0231] The composition can be recovered from genetically modified cell lines, such as Chinese hamster ovary (CHO) cell lines expressing HER2 antibodies, or prepared by peptide synthesis.

[0232] For further information regarding exemplary pertuzumab compositions, see U.S. Patent Nos. 7,560,111 and 7,879,325, and U.S. Patent Application No. 2009 / 0202546A1.

[0233] (iii) Trastuzumab composition Trastuzumab compositions generally comprise a mixture of major species antibodies (containing the light and heavy chain sequences of SEQ ID NOs. 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. Furthermore, for morphologies of trastuzumab that can be degraded by cation exchange chromatography, including peak A (Asn30 deamidated to Asp in both 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 morphology, or major species antibody); peak 4 (Asp102 isomerized to isoAsp in one heavy chain); and peak C (Asp102 succinimide (Asu) in one heavy chain), see Harris et al., J. Chromatography, B752: pp. 233-245 (2001).

[0234] (iv) Trastuzumab / pertuzumab composition in a fixed-dose combination This embodiment discloses extensive studies on various charge variants found in a fixed-dose trastuzumab / pertuzumab combination. Acceptable criteria were established based on clinical experience and the anticipated impact on the bioactivity / PK and safety / immunogenicity profiles. The compositions provided herein are considered to possess the bioactivity and PK required for safe biomedicines without additional immunogenicity and safety risks.

[0235] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 23% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 28% of pertuzumab native antibody, at least 16% of trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0236] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 23% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 38% of pertuzumab native antibody, at least 16% of trastuzumab native antibody, and less than 9% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0237] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 21% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 28% of pertuzumab native antibody, at least 23% of trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0238] In one embodiment, a composition comprising pertuzumab, trastuzumab, and their charge variants is analyzed by ion-exchange chromatography. In one embodiment, a composition comprising pertuzumab, trastuzumab, and their charge variants is analyzed using ion-exchange chromatography according to any of the embodiments described above. In one embodiment, the percentages of the native antibody and charge variants are equal to the peak region determined by ion-exchange chromatography according to any of the embodiments described above, and (i) pertuzumab variants deamidated with HC-Asn-391, pertuzumab sialic acid variants, pertuzumabridin glycated variants, trastuzumab deamidated with LC-Asn-30, and trastuzumab deamidated with HC-Asn-55 elute at peaks 1-3, and thus the percentages of these variants in the composition are equal to the peaks (ii) Pertuzumab native antibody elutes at peak 4, and therefore the percentage of pertuzumab native antibody in the composition is equal to the peak region of peak 4; (iii) Trastuzumab native antibody elutes at peak 7, and therefore the percentage of trastuzumab native antibody in the composition is equal to the peak region of peak 7; (iv) Trastuzumab having a single isomerization of HC-Asp-102 to isoaspartic acid on one of the heavy chains elutes at peak 8, and therefore the percentage of this variant in the composition is equal to the peak region of peak 8.

[0239] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, which, as determined by the method described in any of the above embodiments, includes a peak region of less than 23% of the sum of peaks 1-3, a peak region of at least 28% of peak 4 (pertuzumab natural antibody), a peak region of at least 16% of peak 7 (trastuzumab natural antibody), and a peak region of less than 12% of peak 8. In one embodiment, the method is a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. A step in which the antibody is eluted with elution buffer, and the pH of the elution buffer is approximately 7.5 to approximately pH 7.7, Includes.

[0240] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0241] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0242] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

[0243] In one embodiment, the method is carried out at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0244] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0245] In one embodiment, a composition comprising pertuzumab and trastuzumab is provided, which, as determined by the method described in any of the above embodiments, includes a peak region of less than 23% of the sum of peaks 1-3, a peak region of at least 38% of peak 4 (pertuzumab natural antibody), a peak region of at least 16% of peak 7 (trastuzumab natural antibody), and a peak region of less than 9% of peak 8. In one embodiment, the method is a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. Eluting the antibody with elution buffer where the pH of the elution buffer is from about 7.5 to about pH 7.7, and comprising.

[0246] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0247] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0248] In one embodiment, the method for evaluating a fixed-dose composition containing the above pertuzumab and trastuzumab is c. Selectively detecting the charge variants of pertuzumab and trastuzumab in the composition, additionally comprising.

[0249] In one embodiment, the method is carried out at a temperature of 32 - 40 °C. In one embodiment, the composition containing pertuzumab and trastuzumab additionally contains rHuPH20.

[0250] In one embodiment, the composition containing pertuzumab and trastuzumab contains trastuzumab at 40 - 60 mg / mL and pertuzumab at 60 - 80 mg / mL.

[0251] In one embodiment, a composition containing pertuzumab and trastuzumab is provided, and the composition, determined by the method described in any of the above embodiments, contains a peak area of less than 21% of the total of peaks 1 - 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. In one aspect, the method is a. A step in which antibodies are bound to the ion exchange material using a loading buffer, and the pH of the loading buffer is approximately 7.5 to approximately pH 7.65. b. A step in which the antibody is eluted with elution buffer, and the pH of the elution buffer is approximately 7.5 to approximately pH 7.7, Includes.

[0252] In one embodiment, the ion exchange material is a cation exchange material. In one embodiment, the cation exchange chromatography material is a strong cation exchange material. In one embodiment, the cation exchange material contains a sulfonate group.

[0253] In one embodiment, step b is carried out with a salt gradient. In one embodiment, the elution buffer contains sodium. In one embodiment, the elution buffer contains sodium chloride.

[0254] In one embodiment, a method for evaluating a fixed-dose composition containing the above-mentioned pertuzumab and trastuzumab is: c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. It also includes the following.

[0255] In one embodiment, the method is carried out at a temperature of 32-40°C. In one embodiment, the composition comprising pertuzumab and trastuzumab further comprises rHuPH20.

[0256] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0257] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 22% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 29.2% of pertuzumab native antibody, at least 21.8% of trastuzumab native antibody, and less than 5% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0258] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 22% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 39.4% of pertuzumab native antibody, at least 21.8% of trastuzumab native antibody, and less than 4.1% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0259] In one embodiment, a composition is provided comprising pertuzumab and trastuzumab, the composition comprising less than 19.8% of pertuzumab acidic variants selected from deamidation of HC-Asp-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 29.2% of pertuzumab native antibody, at least 31% of trastuzumab native antibody, and less than 5% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0260] In one embodiment, a composition comprising pertuzumab and trastuzumab contains 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab.

[0261] In a further embodiment of the present invention, the compositions provided herein are comprised of the following steps: a. A step of adding a predetermined amount of pertuzumab to a compounding container, b. A step of adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or a 1:2 ratio of trastuzumab to pertuzumab, The process involves adding c.rHuPH20, It is obtained by a method that includes the following.

[0262] In one embodiment, a 1:1 ratio of trastuzumab to pertuzumab results in a composition containing 60 mg / mL of trastuzumab and 60 mg / mL of pertuzumab. In one embodiment, a 1:2 ratio of trastuzumab to pertuzumab results in a composition containing 40 mg / mL of trastuzumab and 80 mg / mL of pertuzumab. In one embodiment, rHuPH20 is added to the composition to achieve a final concentration of rHuPH20 of 2000 U / mL.

[0263] IV. Recombinant HER2 extracellular domain The inventors have found that modified HER2 ECDs lacking subdomain IV can be produced with a three-dimensional structure similar to that of natural HER2 ECDs, provided they contain recombinant subdomain III which is shortened at the C-terminus. In one such embodiment, the modified HER2 ECD includes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34. In one embodiment, a modified HER2 ECD containing SEQ ID NO: 24 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 24 is provided.

[0264] In one embodiment, recombinant HER2 extracellular subdomains I, II, and III are fused to an Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or porcupine Fc domain. In one embodiment, a modified HER2 ECD containing 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 to SEQ ID NO: 25 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 26 is provided. In one embodiment, a modified HER2 ECD having 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 27 is provided.

[0265] In one embodiment, a modified ECD is provided that includes sequence number 33, sequence number 3, and sequence number 4. In one embodiment, the modified ECD includes sequence number 33, sequence number 36, sequence number 3, and sequence number 4.

[0266] The inventors have found that modified HER2 ECDs lacking subdomain II can be produced with a three-dimensional structure similar to that of natural HER2 ECDs if they contain a recombinant subdomain I that is shortened at the C-terminus and in which HER2 ECD subdomain II is replaced by EGFR subdomain II. In one embodiment, a modified HER2 ECD containing sequence number 29 is provided. In one embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to sequence number 29 is provided.

[0267] In one embodiment, recombinant HER2 extracellular subdomains I, III, and IV, and the subdomain II of EGFR are fused to the Fc domain. In one embodiment, the Fc domain is a mouse, rat, rabbit, or guinea pig Fc domain. In any of the above embodiments, the capture reagent for assaying the binding of trastuzumab does not contain the 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 to 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 to SEQ ID NO: 31 is provided. In one embodiment, a modified HER2 ECD having at least 99%, 98%, 97%, 96%, 95%, or 90% sequence identity to SEQ ID NO: 32 is provided.

[0268] The recombinant HER2 extracellular domain can be produced and purified by methods known in the art. In one embodiment, a method for making a recombinant HER2 extracellular domain is provided, the method comprising culturing a host cell comprising 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 the 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 a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0269] Suitable host cells for cloning or expressing recombinant HER2 extracellular domain coding vectors include prokaryotic or eukaryotic cells as described herein. For example, recombinant HER2 extracellular domains can be produced from bacteria. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. After expression, the recombinant HER2 extracellular domain may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0270] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for recombinant HER2 extracellular domain coding vectors. Suitable host cells for the expression of recombinant HER2 extracellular domains are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and may be used in combination with insect cells, particularly for transfection of 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 may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cells (e.g., 293 or 293T cells described by Graham, FL et al., J. Gen Virol. Vol. 36 (1977), pp. 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described by Mather, JP, Biol. Reprod. Vol. 23 (1980), pp. 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat kidney cells (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT060562); and TRI cells (e.g., Mather, JP et al., Annals). These are MRC5 cells and FS4 cells, as described in NYAcad.Sci. Vol. 383 (1982), pp. 44-68. Other useful mammalian host cell lines include Chinese hamster ovary (HO) cells, DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA Vol. 77 (1980), pp. 4216-4220); and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, e.g., Chinese hamster ovary (CHO) cell.

[0271] V. Kit The present invention also provides a kit for specifically quantifying the binding of an antibody to the HER2 extracellular subdomain II in a fixed-dose combination (FDC) of a first antibody and a second HER2 antibody that bind to the HER2 extracellular subdomain II. (a) A container containing proteins including SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34 as a capture reagent, (b) Instructions for quantifying the binding of antibodies that bind to the extracellular subdomain II of HER2, This is a kit that includes [the following items].

[0272] In one embodiment, the capture reagent contains 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.

[0273] In one embodiment, the capture reagent includes 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.

[0274] In one embodiment, the instructions include instructions that correlate the binding of a first antibody binding to the extracellular subdomain II of HER2 with its titer.

[0275] In one embodiment, the second antibody binds to a different epitope than the first antibody. In one embodiment, the second antibody is an antibody that binds to HER2 extracellular subdomain IV. In one embodiment, the first antibody is pertuzumab. In one embodiment, the second antibody is trastuzumab.

[0276] 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-trastuzumab FDC contains about 2000 U / mL of rHuPH20.

[0277] The present invention also provides a kit for specifically quantifying the binding of an antibody to the HER2 extracellular subdomain IV in a fixed-dose combination (FDC) of an antibody that binds to the HER2 extracellular subdomain IV and a second HER2 antibody. a. A container containing proteins including SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 4 as a capture reagent, (b) Instructions for quantifying the binding of an antibody that binds to the extracellular subdomain IV of HER2, This is a kit that includes [the following items].

[0278] In one embodiment, the capture reagent contains 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.

[0279] In one embodiment, the capture reagent includes 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.

[0280] In one embodiment, the instructions include instructions for correlating the binding of an antibody that binds to the HER2 extracellular subdomain IV with its titer.

[0281] In one embodiment, the second antibody binds to a different epitope than the first antibody. In one embodiment, the second antibody is an antibody that binds to the HER2 extracellular subdomain II. In one embodiment, the first antibody is trastuzumab. In one embodiment, the second antibody is pertuzumab.

[0282] 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-trastuzumab FDC contains about 2000 U / mL of rHuPH20.

[0283] VI. Manufacturing method In one embodiment, a method is provided for preparing a composition, comprising (1) producing a fixed-dose combination containing pertuzumab, trastuzumab, and one or more variants thereof, and (2) subjecting the composition thus produced to an analytical assay to evaluate the amount of variants, wherein the variants include (i) pertuzumab deamidated with HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumabridin glycated variant, trastuzumab deamidated with LC-Asn-30, trastuzumab deamidated with HC-Asn-55, (ii) a natural antibody of pertuzumab, (iii) a natural antibody of trastuzumab, and (vi) trastuzumab having a sole isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0284] In one embodiment, the mutant comprises (i) less than 23% of the following mutants: pertuzumab deamidated with HC-Asn-391, pertuzumab FC sialic acid mutant and pertuzumabridin glycation mutant, trastuzumab deamidated with LC-Asn-30, and trastuzumab deamidated with HC-Asn-55; (ii) at least 28% of pertuzumab native antibody; (iii) at least 16% of trastuzumab native antibody; and (iv) less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0285] In one embodiment, the mutant comprises (i) less than 23% of the following mutants: pertuzumab deamidated with HC-Asn-391, pertuzumab FC sialic acid mutant, pertuzumabridin glycated mutant, trastuzumab deamidated with LC-Asn-30, and trastuzumab deamidated with HC-Asn-55; (ii) at least 38% of pertuzumab native antibody; (iii) at least 16% of trastuzumab native antibody; and (iv) less than 9% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0286] In one embodiment, the mutant comprises (i) less than 21% of the following mutants: pertuzumab deamidated with HC-Asn-391, pertuzumab C sialic acid mutant and pertuzumabridin glycated mutant, trastuzumab deamidated with LC-Asn-30, and trastuzumab deamidated with HC-Asn-55; (ii) at least 28% of pertuzumab native antibody; (iii) at least 23% of trastuzumab native antibody; and (iv) less than 12% of trastuzumab having a single isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

[0287] In one embodiment, the analytical assay is ion-exchange chromatography. In one embodiment, the analytical assay is ion-exchange chromatography according to any of the above embodiments. In one embodiment, the percentage is equal to the peak region determined by ion-exchange chromatography according to any of the above embodiments, and (i) pertuzumab variants deamidated with HC-Asn-391, pertuzumab FC sialic acid variant, pertuzumabridin glycated variant, trastuzumab deamidated with LC-Asn-30, and trastuzumab deamidated with HC-Asn-55 elute at peaks 1-3, and thus the percentage of these variants in the composition is in peak region 1-3. (ii) Pertuzumab native antibody elutes at peak 4, and therefore the percentage of pertuzumab native antibody in the composition is equal to the peak region of peak 4; (iii) Trastuzumab native antibody elutes at peak 7, and therefore the percentage of trastuzumab native antibody in the composition is equal to the peak region of peak 7; (iv) Trastuzumab having a single isomerization of HC-Asp-102 to isoaspartic acid on one of the heavy chains elutes at peak 8, and therefore the percentage of this variant in the composition is equal to the peak region of peak 8.

[0288] In one embodiment, the amounts of the following additional variants are analyzed by analytical assay: (v) pertuzumab having N-terminal VHS in the heavy and light chains, pertuzumab having C-terminal lysine in the heavy chain, trastuzumab having deamidation of HC-Asn-392, trastuzumab having lysine glycosylation, and trastuzumab having increased Fc-sialic acid content.

[0289] In one embodiment, the analytical assay is ion-exchange chromatography. In one embodiment, the analytical assay is ion-exchange chromatography according to any of the above embodiments. In one embodiment, the percentage is equal to the peak region determined by ion-exchange chromatography according to any of the above embodiments, (vii) pertuzumab having N-terminal VHS in the heavy and light chains, pertuzumab having C-terminal lysine in the heavy chain, trastuzumab having deamidation of HC-Asn-392, trastuzumab having lysine glycosylation, and trastuzumab having increased Fc-sialic acid content elute at peaks 5-6, and thus the percentage of these variants in the composition is equal to the peak region of peaks 5-6.

[0290] In one embodiment, the amount of the following additional variant is analyzed by an analytical assay: (vi) trastuzumab having sole isomerization of HC Asp 102 to succinimide on one of its heavy chains and having trastuzumab Fc oxidation.

[0291] In one embodiment, the analytical assay is ion-exchange chromatography. In one embodiment, the analytical assay is ion-exchange chromatography according to any of the above embodiments. In one embodiment, the percentage is equal to the peak region determined by ion-exchange chromatography according to any of the above embodiments, and (vi) trastuzumab having sole isomerization of HC Asp102 to succinimide on one heavy chain and trastuzumab Fc oxidation elutes at peaks 9-10. Therefore, the percentage of these variants in the composition is equal to the peak region of peaks 9-10.

[0292] In one embodiment, the method is for preparing a composition further comprising rHuPH20. In one embodiment, the composition comprises 2000 U / ml of rHuPH20. In one embodiment, the method is for preparing a composition comprising 40-60 mg / mL of trastuzumab and 60-80 mg / mL of pertuzumab. In one embodiment, the composition comprises 40 mg / mL of trastuzumab and 80 mg / mL of pertuzumab. In one embodiment, the composition comprises 60 mg / mL of trastuzumab and 60 mg / mL of pertuzumab.

[0293] In one embodiment, step (1) of the manufacturing method described above is the following step: a. A step of adding a predetermined amount of pertuzumab to a compounding container, b. A step of adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or a 1:2 ratio of trastuzumab to pertuzumab, The process involves adding c.rHuPH20, Includes.

[0294] In one embodiment, a 1:1 ratio of trastuzumab to pertuzumab results in a composition containing 60 mg / mL of trastuzumab and 60 mg / mL of pertuzumab. In one embodiment, a 1:2 ratio of trastuzumab to pertuzumab results in a composition containing 40 mg / mL of trastuzumab and 80 mg / mL of pertuzumab.

[0295] In one embodiment, rHuPH20 is added to the composition to achieve a final concentration of rHuPH20 of 2000 U / ml.

[0296] VII. Patient selection for treatment Patients can be selected for therapies according to the present invention using the detection of HER2 expression or amplification. Several FDA-approved commercial assays are available to identify patients with HER2-positive, HER2-expressing, HER2-overexpressing, or HER2-amplifying cancers. These methods include HERCEPTEST® (Dako) and PATHWAY® HER2 (immunohistochemical (IHC) assays), as well as PathVysion® and HER2 FISH pharmDx® (FISH assays). Users should refer to the documentation provided with the specific assay kit for information on the validity and performance of each assay.

[0297] For example, HER2 expression or overexpression can be analyzed by IHC using, for example, HERCEPTEST® (Dako). Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC assays and matched to the following HER2 protein staining intensity criteria: Score 0: No staining is observed, or membrane staining is observed in less than 10% of tumor cells. Score 1+: Faint / almost imperceptible membrane staining is detected in more than 10% of tumor cells. Only a portion of these cells' membranes are stained. Score 2+: Weak to moderate complete membrane staining is observed in more than 10% of tumor cells. Score 3+: Moderate to intense complete membrane staining is observed in more than 10% of tumor cells.

[0298] These tumors with a HER2 overexpression assessment score of 0 or 1+ can be characterized as HER2-negative, and those with a score of 2+ or 3+ can be characterized as HER2-positive.

[0299] Tumors that overexpress HER2 may also be assessed by an immunohistochemical score corresponding to the copy number of HER2 molecules expressed per cell, and can be biochemically determined: 0 = 0 to 10,000 copies / cell, 1+=at least approximately 200,000 copies / cell, 2+ = at least approximately 500,000 copies / cell, 3+ = at least approximately 2,000,000 copies / cell.

[0300] Overexpression of HER2 at the 3+ level leads to ligand-independent activation of tyrosine kinase (Hudziak et al., Proc. Natl. Acad. Sci. USA, Vol. 84: pp. 7159-7163 (1987)), occurring in approximately 30% of breast cancers, and these patients experience reduced recurrence-free survival and overall survival (Slamon et al., Science, Vol. 244: pp. 707-712 (1989); Slamon et al., Science, Vol. 235: pp. 177-182 (1987)).

[0301] The presence of HER2 protein overexpression and gene amplification is highly correlated, and therefore, alternatively or additionally, the use of in situ hybridization (ISH), such as fluorescence in situ hybridization (FISH), an assay for detecting gene amplification, can be used for the selection of patients suitable for treatment according to the present invention. A FISH assay, such as INFORM® (marketed by Ventana, Arizona) or PathVysion® (Vysi, Illinois), can be performed on formalin-fixed paraffin-embedded tumor tissue to determine the degree of HER2 amplification (if present) in the tumor.

[0302] Most commonly, HER2 positivity is confirmed using one of the methods described above, along with archival paraffin-embedded tumor tissue.

[0303] Preferably, HER2-positive patients having an IHC score of 2+ or 3+ and / or being FISH or ISH positive are selected for treatment according to the present invention. Patients having an IHC score of 3+ and being FISH / ISH positive are particularly suitable for treatment according to the present invention.

[0304] HER2 mutations associated with responsiveness to HER2-targeted therapy have also been identified. Such mutations include, but are not limited to, insertions into exon 20 of HER2, deletions around amino acid residues 755-759 of HER2, mutations of G309A, G309E, S310F, D769H, D769Y, V777L, P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov 2013; vol. 3: p. 114), and identical non-synonymous putative activating mutations (or indels) previously reported in the COSMIC database found in two or more independent specimens.

[0305] For alternative assays for screening patients for pertuzumab therapy, see also U.S. Patent No. 7,981,418 and its examples. TIFF2026108626000001.tif241170TIFF2026108626000002.tif232170 [Examples]

[0306] Example 1 Pertuzumab / Trastuzumab FDC Pertuzumab and trastuzumab, the two active ingredients in FDC Pharmaceuticals LD and MD, are recombinant humanized monoclonal antibodies of the IgG1 subclass against the extracellular domain of HER2. The third active ingredient in FDC Pharmaceuticals, rHuPH20, is a transient reactive enzyme (recombinant human hyaluronidase) that functions as a local permeation enhancer, enabling subcutaneous delivery of therapeutic drugs that are traditionally delivered intravenously.

[0307] FDC drugs are provided as a colorless to slightly brownish sterile solution for subcutaneous injection. They do not contain preservatives. The following two formulations are available:

[0308] Loading amount FDC chemical LD Each 20 mL single-dose vial contains 1200 mg (nominal) of pertuzumab, 600 mg (nominal) of trastuzumab, and 2000 U / mL of hyaluronidase (rHuPH20, bor hyaluronidase alfa) at a target pH of 5.5. The drug is formulated with 80 mg / mL of pertuzumab and 40 mg / mL of trastuzumab. The excipients used in the formulation are L-histidine, L-histidine hydrochloride monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0309] Maintenance dose FDC drug MD Each 15 mL single-dose vial contains 600 mg (nominal) of pertuzumab, 600 mg (nominal) of trastuzumab, and 2000 U / mL of hyaluronidase (rHuPH20, bor hyaluronidase alfa) at a target pH of 5.5. The drug is formulated with 60 mg / mL of pertuzumab and 60 mg / mL of trastuzumab. The excipients used in the formulation are L-histidine, L-histidine hydrochloride monohydrate, L-methionine, α,α-trehalose dihydrate, sucrose, and polysorbate 20.

[0310] Example 2 Pertuzumab / trastuzumab FDC titers by cell-based assays This method determines the titers of pertuzumab and trastuzumab by measuring their ability to inhibit the proliferation of either MDA-MB-175-VII cells or BT-474 cells, respectively. In a typical assay, MDA-MB-175-VII cells or BT-474 cells are seeded in a 96-well microtiter plate and incubated overnight at 37°C with 5% carbon dioxide in a humidified incubator. After incubation, the medium is removed and reference standards, assay controls, and samples of various concentrations are added to the plate. The plate is then incubated for 3 days, and the relative number of viable cells is quantified indirectly using the redox dye alamarBlue.

[0311] Fluorescence is measured using excitation at 530 nm and emission at 590 nm.

[0312] The alamarBlue dye is blue and non-fluorescent in its oxidized state, but is reduced to a pink form in the intracellular environment of cells, which is highly fluorescent. The changes in color and fluorescence are proportional to the number of living cells. The results, expressed in RFU, are plotted against antibody concentration, and the antiproliferative activity of the FDC sample relative to a reference standard is estimated using a parallel line analysis program.

[0313] Cell-based assays show selective sensitivity to one or the other antibody of the FDC drug, but not to both antibodies, as shown in Figures 7A and B. Individual analyses reveal that trastuzumab has antiproliferative activity in BT-474 cells but not in MDA-MD-175-VII cells, and pertuzumab has antiproliferative activity in MDA-MB-175-VII cells, but its activity against BT-474 cells is strongly shifted to higher concentrations. The difference in sensitivity between the two cell lines may be based on different HER2 expression levels (high and moderate for BT-474 and MDA-MB-175-VII, respectively), rather than differences in affinity for HER2. Additionally, HER3 expression levels and other potential parameters involved in overall antiproliferative activity (e.g., presence or absence of the HER3 endogenous ligand heregulin) may contribute to the difference in sensitivity. In addition, in somatic cell-based assays, the presence of one antibody influences the response of the other, masking potential quality changes that may occur with one or the other antibody. Pertuzumab and trastuzumab have complementary mechanisms of action that disrupt HER2 signaling, resulting in high antiproliferative activity when both are present (Figures 8A and B). Trastuzumab alone cannot inhibit the proliferation of MDA-MB-175-VII cells in the pertuzumab antiproliferative assay (Figures 7A and B), but the addition of pertuzumab shifts the trastuzumab dose-response curve to a lower EC50 value, reflecting the high titer when trastuzumab and pertuzumab are combined (Figure 8A). Therefore, slight quality changes in pertuzumab in FDC drugs are not detected by the MDA-MB-175-VII antiproliferative assay. Similar observations, though less prominent, were made with pertuzumab in the BT-474 antiproliferative assay (Figure 8B). Furthermore, slight changes in antibody quality in the reverse direction can result in 100% titer.

[0314] To demonstrate that substantial quality changes in either antibody of the FDC drugs cannot be detected by antiproliferative assays, pertuzumab and trastuzumab HER2 affinity mutants with directional mutations in the CDR (HC S55A mutation and LC H91A mutation, respectively) were tested in pertuzumab and trastuzumab antiproliferative assays (Figure 9A and B). The significantly reduced affinity of the mutants for HER2 correlated with reduced antiproliferative activity in the corresponding cell-based assays. Addition of pertuzumab to the trastuzumab mutant (or trastuzumab to the pertuzumab mutant) partially restored the shape of the dose-response curve and thus restored antiproliferative activity.

[0315] In summary, based on the selective sensitivity, complementarity mechanisms, and shielding effects observed in antiproliferation assays, these assays are considered unsuitable for detecting changes related to the activity of either antibody in the combination drug. These limitations make antiproliferation assays unsuitable for use in determining and controlling the biological activity of FDC drugs. Therefore, we design two selective titer ELISAs unaffected by such cross-interference to control relevant changes in the binding activity of the two antibodies in the FDC drug. The selectivity of the ELISA is ensured by using different epitopes of the HER2 receptor as primary binding targets.

[0316] Example 3 Pertuzumab titer in FDC by ELISA The titer of the FDC drug is controlled using two separate ELISAs. Here, we describe ELISAs that control the biological activity of the pertuzumab component of the FDC drug. Pertuzumab is a monoclonal IgG1 antibody against HER2 that is specific to the extracellular subdomain II of HER2. Upon binding, pertuzumab blocks HER2 activation by preventing heterodimerization of HER2 at the ligand-activating member of the HER receptor family. This results in inhibition of downstream signaling pathways in HER2-overexpressing cells. The ELISA for pertuzumab determines specific biological activity as the ability of pertuzumab to specifically bind to the recombinant HER2 epitope (i.e., subdomain II). Figure 6 depicts schematic diagrams of the capture reagents used in the ELISA for pertuzumab and trastuzumab (see Example 6 for details).

[0317] Binding is measured using a peroxidase-conjugated secondary antibody. Dose-response curves generated with samples and standards provide the basis for quantification. In ELISA, the actual protein content of pertuzumab (not the actual total protein content of the FDC drug) is considered in the dilution preparation. ELISA for pertuzumab is used for both LD and MD of the FDC drug.

[0318] Equipment and materials 96-well immunosorbent plate (e.g., Maxisorp ELISA) Absorption plate reader A computer equipped with 4-parameter data organization software and parallel processing analysis software (e.g., SoftMaxPro). Microplate washing machine

[0319] reagent ● Pertuzumab-coated reagent: Recombinant HER2 extracellular domains I, II, and III are fused to mouse Fc; domain IV (contained in the trastuzumab epitope) is deleted (SEQ ID NO: 27). ●Detected antibody: HRP conjugate goat anti-human antibody ( (Specific to the F(ab')2 portion of human IgG) (e.g., Jackson ImmunoResearch) ● 1×DPBS without calcium and magnesium ● Purified water, for example, Milli-Q ●BSA fraction V ●Tween20 ●ABTS substrate solution ● Phosphate concentrate (85%)

[0320] solution Note: The prescription is for the nominal quantity of reagents and can be adjusted to meet assay requirements.

[0321] Washing buffer: 1 x DPBS, 0.05% Tween20 Assay diluent: 1 × DPBS, 0.05% Tween20, 0.5% BSA fraction V Coating solution: Pertuzumab coating reagent (1 μg / mL) in 1 × DPBS Detected antibody: 0.8 mg / mL HRP-conjugated goat anti-human antibody Detection solution: Prepare the detection solution by diluting the detection antibody (0.8 mg / mL) with the assay diluent to a concentration of 16 ng / mL. Prepare a fresh solution before use. Stopping solution: 1M phosphoric acid Reference Standard: FDC's MD Reference Standard

[0322] Plate coating Transfer 100 μL of the coating solution to each well of the microtiter plate. - Incubate the coated plates at 2°C to 8°C for 30 to 60 minutes.

[0323] Plate blocking - Wash all coated plates three times with 300 μL / well of washing buffer to remove any excess coating solution. Add 100 μL of assay diluent to each well and block all plates. - Incubate the plate at room temperature for 60-90 minutes while gently shaking it. - Wash the plate three times with 300 μL / well of washing buffer.

[0324] Sample transfer Transfer 100 μL / well of FDC reference standard, product control, and sample diluent to the wells of the immunoplate. - Incubate the plate at room temperature for 60-90 minutes while gently shaking it.

[0325] detection Transfer 100 μL of the detection solution (16 ng / mL) to each well of the plate. - Incubate the plate at room temperature for 30-90 minutes while gently shaking it. - Wash the plate three times with 300 μL / well of washing buffer.

[0326] Substrate transfer and measurement Transfer 100 μL / well of ABTS substrate solution to each well of the plate. - Incubate the plate at room temperature for 20-35 minutes while gently shaking it. - To stop the reaction, transfer 100 μL / well of stop solution to each well of the plate. - Gently stir the plate for at least 1 minute to mix. Within 30 minutes, measure the 0D value at a wavelength of 405 nm (reference wavelength 490 nm) using an absorbance plate reader.

[0327] evaluation -Calculate the OD value of each well as follows: OD(405nm)-OD(490nm). In the formula, OD(405nm): detected absorbance at 405nm, and OD(490nm): reference absorbance at 490nm. - The average OD values ​​of the replicas are averaged to determine the average OD. - The mean OD(y) is plotted against the pertuzumab antibody concentration ng / mL(x) to generate dose-response curves for the standard, product control, and sample. - Apply nonlinear regression using the following four-parameter equation: TIFF2026108626000003.tif32170 formula: A: Lower asymptote B: Hill Slope C:EC 50 value D: Upper asymptote -Calculate the R² value of the standard, product control, and sample curves. -Calculate the standard delta OD as follows: Standardized delta OD = (standardized average maximum OD) - (standardized average minimum OD) - Determine the maximum OD as follows: The maximum OD value is the maximum OD value at 405 nm obtained within all copies of the dose-response curve.

[0328] Calculation of potency - Use 4-parameter parallel line analysis to calculate the common set of Hill slopes, upper asymptotes, and lower asymptotes for the standard and sample (or product control) curves. - The resulting curve equations for the standard and sample (or product control) are as follows: TIFF2026108626000004.tif29170 formula: A = Common downward asymptote B = Hill slope C 標準 =Standard EC 50 value D = Common upper asymptote ρ = Titers of sample and product control relative to the reference standard -Calculate the relative titer as follows: Relative titer = ρ × activity of the reference standard

[0329] Reference standard potency allocation The pertuzumab titer of FDC drugs is based on the protein content of pertuzumab, not the total protein content of the FDC drug. Therefore, titration is independent of the ratio of the two molecules in the FDC drug, and the titers of MD and LD samples of FDC drugs can be determined using a single-molecule reference standard. The MD reference standard of DC was selected as the reference standard for titer.

[0330] Details of the FDC's MD reference criterion potency allocation are provided as follows: - The titer is 1.00 × 10⁻⁶ 4 I set it to U / mg. - Pertuzumab titer determination by ELISA was performed against the commercially available pertuzumab IV reference standard anti2C4907-2. - Trastuzumab titer was determined by ELISA against the commercially available trastuzumab SC reference standard G005.03EP1.

[0331] Results: Pertuzumab binding to a fixed-dose pertuzumab / trastuzumab combination was analyzed using a pertuzumab ELISA assay. A representative dose-response curve is shown in Figure 10.

[0332] Example 4 Specificity of pertuzumab ELISA To assess the specificity of the pertuzumab ELISA in Example 3, the formulation buffer and structurally relevant molecules were dual-tested at the highest assay concentration on a single plate. If interference by structurally relevant molecules was observed (mean replication was 3 times higher than the lower asymptote mean of the reference standard dose-response curve), an augmentation of the complete dose-response curve with one reportable decision (n=1) was performed.

[0333] The results demonstrate that the pertuzumab ELISA is specific to pertuzumab: ●Both the LD and MD formulation buffers of rHuPH20-containing FDCs did not interfere with the assay, demonstrating their suitability for the assay to analyze the use of FDC formulations in these matrices. Structurally related molecules, including trastuzumab (excluding pertuzumab; see below), did not interfere with the pertuzumab ELISA. This is indicated by the mean OD value of the replicas, which is three times lower than the mean OD value of the lower asymptote of the reference standard dose-response curve. ●As expected, the pertuzumab SC active ingredient formulated as FDC and pertuzumab IV both bind to the same HER2 domain II, and therefore showed interference with the pertuzumab ELISA.

[0334] The results are shown in Table 3. TIFF2026108626000005.tif195170

[0335] Example 5 Robustness of pertuzumab ELISA The robustness of the pertuzumab ELISA was assessed by the arbitrary variability of assay parameters, a potential source of variability in practice. Robustness results were evaluated by comparing the obtained dose-response curve parameters, system suitability, and similarity criteria with the procedural conditions of the method. The overall robustness results are summarized in Table 4. TIFF2026108626000006.tif118170

[0336] Example 6 Trastuzumab potency in FDCs by ELISA The titer of FDC drugs is controlled using two similar ELISAs. This section describes ELISAs that control the biological activity of the trastuzumab component of FDC drugs. Trastuzumab is a monoclonal IgG1 antibody against HER2 that is specific to the extracellular subdomain IV of HER2. Upon binding, trastuzumab blocks HER2 activation by preventing homodimerization and shedding of the HER2 extracellular domain.

[0337] This results in the inhibition of downstream signaling pathways in HER2-overexpressing cells. Trastuzumab ELISA determines specific biological activity as trastuzumab's ability to specifically bind to the recombinant HER2 epitope (i.e., subdomain IV). Figure 6 depicts a schematic diagram of the capture reagent used in trastuzumab ELISA.

[0338] Binding is measured using a peroxidase-conjugated secondary antibody. Dose-response curves generated with samples and standards provide the basis for quantification. In ELISA, the actual protein content of trastuzumab (not the actual total protein content of the FDC drug) is considered in the dilution preparation. ELISA for trastuzumab is used for both LD and MD of the FDC drug.

[0339] The reagents, buffers, and procedures are as outlined in Example 3, with the exception of the coating reagent and coating solution. ● Trastuzumab-coated reagent: Recombinant HER2 extracellular domains I, II, III, and IV fused to mouse Fc; Domain II is replaced with a structurally related EGFR domain II (SEQ ID NO: 32) that cannot bind to pertuzumab. ●Coating solution: Trastuzumab coating reagent (1 μg / mL) in 1× DPBS

[0340] evaluation -Calculate the OD value of each well as follows: OD(405nm)-OD(490nm). In the formula, OD(405nm): detected absorbance at 405nm, and OD(490nm): reference absorbance at 490nm. - The average OD values ​​of the replicas are averaged to determine the average OD. - Plot the mean OD(y) against the trastuzumab antibody concentration ng / mL(x) to generate dose-response curves for the standard, product control, and sample. - Apply nonlinear regression using the following four-parameter equation: TIFF2026108626000007.tif32170 formula: A: Lower asymptote B: Hill Slope C:EC 50 value D: Upper asymptote -Calculate the R² value of the standard, product control, and sample curves. -Calculate the standard delta OD as follows: Standardized delta OD = (standardized average maximum OD) - (standardized average minimum OD) - Determine the maximum OD as follows: The maximum OD value is the maximum OD value at 405 nm obtained within all copies of the dose-response curve.

[0341] Calculation of potency - Use 4-parameter parallel line analysis to calculate the common set of Hill slopes, upper asymptotes, and lower asymptotes for the standard and sample (or product control) curves. - The resulting curve equations for the standard and sample (or product control) are as follows: TIFF2026108626000008.tif29170 formula: A = Common downward asymptote B = Common hill slope C 標準 =Standard EC 50 value D = Common upper asymptote ρ = Titers of sample and product control relative to the reference standard -Calculate the relative titer as follows: Relative titer = ρ × activity of the reference standard

[0342] Reference standard potency allocation The trastuzumab titer of FDC drugs is based on the protein content of trastuzumab, not the total protein content of the FDC drug. Therefore, titration is independent of the ratio of the two molecules in the FDC drug, and the titers of MD and LD samples of FDC drugs can be determined using a single-molecule reference standard. The MD reference standard of FDC was selected as the reference standard for titer.

[0343] Details of the FDC's MD reference criterion potency allocation are provided as follows: - The titer is 1.00 × 10⁻⁶ 4 I set it to U / mg. - Pertuzumab titer determination by ELISA was performed against the commercially available pertuzumab reference standard anti2C4907-2. - Trastuzumab titer was determined by ELISA against the commercially available trastuzumab SC reference standard G005.03EP1.

[0344] Results: Trastuzumab binding to a fixed-dose pertuzumab / trastuzumab combination drug was analyzed using a trastuzumab ELISA assay. A representative dose-response curve is shown in Figure 11.

[0345] Example 7 Specificity of trastuzumab ELISA To assess the specificity of the trastuzumab ELISA, the formulation buffer and structurally relevant molecules were dual-tested on a single plate at the highest assay concentration. If interference by structurally relevant molecules was observed (mean replication was 3 times higher than the lower asymptote mean of the reference standard dose-response curve), an augmentation of the complete dose-response curve with one reportable decision (n=1) was performed.

[0346] The results demonstrate that the trastuzumab ELISA is specific to trastuzumab: ●Both the LD and MD formulation buffers of rHuPH20-containing FDCs did not interfere with the assay, demonstrating their suitability for the assay to analyze the use of FDC formulations in these matrices. ● Structurally related molecules, including pertuzumab (excluding trastuzumab; see below), did not interfere with the trastuzumab ELISA. This is indicated by the mean OD value of the replicas, which is three times lower than the mean OD of the lower asymptote of the reference standard dose-response curve. ●As expected, trastuzumab (IV and SC), as well as trastuzumab emtansine, show interference in the trastuzumab ELISA because they both bind to the same HER2 domain IV.

[0347] The results are shown in Table 5. TIFF2026108626000009.tif215170

[0348] Example 8 Robustness of trastuzumab ELISA The robustness of the trastuzumab ELISA was assessed by arbitrary variability in assay parameters, a potential source of variability in practice. Robustness results were evaluated by comparing the obtained dose-response curve parameters, system suitability, and similarity criteria with the method's procedural conditions. The overall robustness results are summarized in Table 6. TIFF2026108626000010.tif128170

[0349] Example 9 Development of IEC for analyzing FDC charge variants Various ion exchange chromatography protocols were tested to degrade FDC charge variants. The following parameters were tested: column type, buffer type and concentration, salt concentration, flow rate, injection volume, pH value, column temperature, and gradient profile.

[0350] Develop a test method to separate and determine the relative abundance (percentage of total peak area) of the following peaks / peak groups. - Total of Peaks 1-3 -Peak 4 (the main peak of pertuzumab) - Total of Peaks 5-6 -Peak 7 (the main peak of trastuzumab) - Peak 8 - Total of peaks 9-10

[0351] We developed an IE-HPLC method for FDC and optimized it to achieve the best separation of pertuzumab and trastuzumab charge variants. Trastuzumab SC and pertuzumab SC can be analyzed separately to infer predicted charge variants. Experiments were conducted using Perjeta SC (lot GB0005, c=120 mg / mL) and Herceptin SC (lot P0003, c=120 mg / mL) individually and as a co-mixture.

[0352] In the first step, the registration IE-HPLC method for individual molecules of Perjeta IV and Herceptin IV / SC was tested. These methods are published, 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, Ver. 3, pp. 794-812, March 1, 2013, DOI: https: / / doi.org / 10.1002 / jps.23403. These methods utilize a weak cation exchange column (WCX-10) (see Table 7, Methods 1 and 2). In the next step, the ProPac WCX-10 column was tested under operating conditions that had been successful with other mAb products carrying similar pI values ​​of pertuzumab / trastuzumab (see Table 7, Method 3). In the next step, a strong cation exchange column was used to test different buffers and pH values. The parameters used and the results are summarized in Table 7 below.

[0353] In the next step, different columns were screened. The best resolution was achieved with a strong cation exchange column (Mab PAC SCX-10). Different buffers and pH values ​​were tested (Methods 4-6). The parameters used and results are summarized in Table 7. A further series of processes were performed based on Method 6 to obtain the best results (see Table 8).

[0354] result Method 1: When pertuzumab-trastuzumab FDC was analyzed under the conditions of Method 1, the peak resolution did not meet the requirements of the product release assay. The resolution of peak 7 (trastuzumab main peak) and peak 8 (trastuzumab IsoAsp102) was insufficient, and the basic region of pertuzumab overlapped with the main peak of trastuzumab. Method 2: When pertuzumab-trastuzumab FDC was analyzed under the conditions of Method 2, the peak resolution did not meet the requirements of the product release assay. The basic region of pertuzumab completely overlapped with the trastuzumab main peak (peak 7) and peak 8, and was therefore unacceptable. Method 3: The main peaks of both trastuzumab and pertuzumab could be separated, with only slight overlap in the basic region of pertuzumab observed. However, the acidic region of trastuzumab overlapped with the main peak of pertuzumab. Method 4: The basic region of pertuzumab is overlapped with the main peak of trastuzumab and the IsoAsp102 peak of trastuzumab (peak 8). Method 5:1: pH 7.5: Both the main peak and peak 8 showed good separation, with only slight overlap between the basic region of pertuzumab and the main peak of trastuzumab. Method 52: pH 8.0: There is good separation at peak 8, but compared to Method 5 at pH 7.5, there is strong overlap between the basic region of pertuzumab and the main peak of trastuzumab. Method 6: Good isolation is obtained for all target species. TIFF2026108626000011.tif129170JPEG2026108626000012.jpg107170

[0355] Based on the HPLC parameters listed in Table 8 above, several experimental designs (Design of Experiment, DoE) were performed. The following parameters were tested: ● Gradient Profile ●Flow rate (0.5~1.0mL / min) ● pH values ​​of mobile phases A and B (7.4-7.6 and 6.8) ●NaCl concentration in transport B (100-300 mM) ● Column temperature (25~40℃)

[0356] Analyzing the data obtained within the framework of the experimental design, the following test parameters showed the best results. ● Eluting agent A: 20 mM ACES, pH 7.5 ● Eluting agent B: 20 ​​mM ACES, 200 mM NaCl, pH 7.5 ● Column: MabPac SCX-10, BioLC, 4 x 250 mm ● Column temperature: 40℃ ●Flow rate 0.8mL / min ●Injection volume: 10 μL (100 μg of protein) ●Gradient: 1-47% in 40 minutes (B)

[0357] To analyze the robustness of this developed method, an experimental factorial design based on Doe was conducted. Therefore, the following parameters varied depending on the matrix. ●ACES concentration: 18~22mM ●NaCl concentration: 180~220mM ●Column temperature: 36~44℃ ●Flow rate: 0.7~0.9mL / min ● pH: 7.4~7.6 ●Injection volume: 8~12μL (80~120μg)

[0358] The results of these experiments demonstrated that the test method was robust within the test range for the primary peaks (peak 7) and peak 8 of trastuzumab. However, high variability was observed in the purity values ​​obtained for the primary peak (peak 4) of pertuzumab and in the intermediate region (the region between the primary peaks of pertuzumab and trastuzumab). This variability was strongly dependent on pH and column temperature. Statistical evaluation of this experiment led to the development of the final method. ● Eluting agent A: 20 mM ACES, pH 7.6 ● Eluting agent B: 20 ​​mM ACES, 200 mM NaCl, p7.6 ● Column temperature: 36℃ ●Flow rate 0.8mL / min ●Injection volume: 10 μL (100 μg of protein) ●Gradient: 1-47% in 40 minutes (B)

[0359] We evaluated alternative methods for determining the charge heterogeneity of pertuzumab-trastuzumab FDC variants. Among these alternatives, we assessed the suitability of different column types and pH gradient methods.

[0360] We also attempted several separations using a ProPac WAX-10 bio LC anion exchange column, 4×250mm, under the following chromatographic conditions. ●The following diluents A and B were prepared and tested. 1. A = 20 mM CAPSO4 pH 10.0, B = 20 mM CAPSO4 + 250 mM NaCl, pH 10.0 2. A = 20 mM piperazine, pH 10.0; B = 20 mM piperazine + 250 mM NaCl, pH 10.0 3. A = 20 mM trisma, pH 10.5; B = 20 mM trisma + 250 mM NaCl, pH 10.5 4. A = 20 mM Trizma, pH 8.0; B = 20 mM Trizma + 250 mM NaCl, pH 8.0 5. A = 20 mM phosphat pH 11.0, B = 20 mM phosphat + 250 mM NaCl, pH 11.0 ● Column temperature: 30℃ ●Flow rate 0.8mL / min; 1.0mL / min ●Injection volume: 5 μL (50 μg of protein) ●Gradient 1: 0-100% B in 60 minutes ●Gradient 2: 0-100% B in 40 minutes

[0361] Under all conditions tested with a weak anion exchange column, the target species was not retained in the column but eluted along with the injection peak, thus indicating that these experimental conditions are all unsuitable for the isolation of charge variants of pertuzumab-trastuzumab FDC.

[0362] Experiments in pH gradient separation mode The suitability of the IEC method based on pH gradients was assessed as a possible alternative to the salt gradient method. A strong cation exchange column (MabPac SCX-10 column) was used with the following HPLC test parameters. ● Eluting agent A: 10 mM Tris, 10 mM Phosphat, 10 mM Piperazine, pH 6.0 ● Eluting agent B: 10 mM Tris, 10 mM Phosphat, 10 mM Piperazine, pH 11.0 ● Eluting agent C: 100 mM NaCl ● Eluting agent D: Pure water ● Column: MabPac SCX-10, BioLC, 4 x 250 mm ● Column temperature: 35℃ ●Flow rate 0.5mL / min ●Injection volume: 10 μL (10 μg of protein) ● Gradient: 10-50% in 45 minutes (see below for details) ●Equipment Waters Alliance

[0363] By combining eluents C and D, NaCl concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM were provided. Therefore, the ratio of eluent C to eluent D varied from 0% eluent C / 50% eluent D (0 mM NaCl) to 50% eluent C / 0% eluent D (50 mM NaCl). Experiments were conducted using Perjeta SC (lot GB0005, c=120 mg / mL) and Herceptin SC (lot P0003, c=120 mg / mL) individually and as a co-mixture. Test samples were diluted with 90% eluent A / 10% eluent B to a final concentration of 1 mg / mL.

[0364] The best separation was obtained using 40 mM NaCl, but even then, the peaks eluted very early under these conditions, and the two main peaks presented a broad shape and reduced height.

[0365] The design of experiments (DoE) was performed with a salt concentration of 40 mM NaCl, varying the gradient profile and flow rate. Nevertheless, a design model could be established, demonstrating that even slight changes in the test conditions compromised the robustness of the method of the present invention.

[0366] From the experiments conducted above, it appears that the most important parameters for the IEC method are 1. pH value, 2. column type, 3. column temperature, and 4. gradient profile. These parameters have a significant effect on resolution. On the other hand, buffer type and concentration, salt concentration, flow rate, and injection volume have only a small effect on resolution.

[0367] Example 10 IEC analysis of FDC charge variants Purpose and Principles IE-HPLC separates proteins present in a chemical according to their charge properties in solubility. This separation is based on the interaction between the surface charge of the protein and the charged groups present on the surface of the column packing. In the cation exchange HPLC used in this analytical procedure, acidic species elute first in the salt gradient, followed by more basic species. The same method is applied to the LD and MD of FDC chemicals. The FDC MD reference standard is used for both the LD and MD tests of FDC chemicals.

[0368] Equipment and materials ●HPLC system equipped with a UV detector (Waters Alliance 2695 / e2695 with a 2487 / 2489 detector or equivalent) ●HPLC column (Thermo Scientific MAbPac SCX-10, 4mm~250mm, particle size: 10μm or equivalent to 10μm)

[0369] solution ●Medicinal dilution buffer: 20 mM L-histidine / L-histidine monohydrochloride, 105 mM trehalose, 100 mM sucrose, 10 mM methionine, 0.04% [w / v] polysorbate 20, pH 5.5 ± 0.2 ●Mobile phase A: 20mM ACES, pH 7.60±0.05 ●Mobile phase B: 20 ​​mM ACES, 200 mM sodium chloride, pH 7.60 ± 0.05 ●CpB solution: 1 mg / mL of CpB (in mobile phase A)

[0370] Preparation of sample solution: The FDC reagent is diluted with mobile phase A to prepare a sample solution containing a total protein concentration of approximately 10 mg / mL and a CpB concentration of approximately 0.08 mg / mL.

[0371] Preparation of blank solution: Dilute the chemical dilution buffer using the same method as the sample.

[0372] CpB digestion Incubate the reference standard, sample, and blank solution at 37°C ± 2°C for 20 ± 5 minutes. Store the sample at 10°C ± 4°C until analysis, and complete the HPLC analysis within 24 hours.

[0373] procedure Before injecting the first sample, rinse 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 demonstrates a consistent profile over at least two consecutive injections.

[0374] Operating parameters ●Detection wavelength: 280nm ●Injection volume: 10μL ●Flow rate: 0.8mL / min ●Column temperature: 36℃±2℃ ● Autosampler temperature: 10℃±4℃ ●Driving time: 60 minutes

[0375] TIFF2026108626000013.tif49170

[0376] Injection protocol The injections will be performed in the following order: 1. Mobile phase A 2. Blank solution 3.Reference standard 4. Samples (up to 10 samples) 5.Reference standard 6. Blank solution Note: If more than 10 samples are to be analyzed, handle them in batches of 10 samples by injecting the reference standard.

[0377] result: An IE-HPLC method for FDC reagents was developed and optimized to achieve the best separation of pertuzumab and trastuzumab charge variants. Due to the similar isoelectric points of pertuzumab (pI 8.7) and trastuzumab (pI 8.4), IE-HPLC cannot completely separate the charge variants of the two antibody molecules (see Figure 13). All important charge variants of individual molecules can be controlled by FDC reagents, as all relevant peaks are degraded. The reported assay parameters for FDC reagents are the sum of peaks 1-3, peak 4 (main peak of pertuzumab), the sum of peaks 5-6, peak 7 (main peak of trastuzumab), peak 8, and the sum of peaks 9-10. An exemplary chromatogram is shown in Figure 12.

[0378] Example 11 Robustness and repeatability studies of HPLC Various experiments were conducted to evaluate the robustness of the analytical procedure of Example 10 for different input variables. These input variables are, in particular, as follows: ● Column temperature (32°C, 36°C, 40°C) ●Flow rate (0.7mL / min, 0.8mL / min, 0.9mL / min) ● pH of mobile phases A and B (pH 7.5~7.7) ●Sodium chloride concentration in mobile phase B (180 mM to 220 mM)

[0379] The profiles and results obtained from analyses after changing the target parameters were compared with the profiles and results from analyses performed according to the target parameters. The relative peak regions (region %) of Peak 4, Peak 7, the sum of Peaks 1-3, and Peak 8 were used to calculate the relative differences in the results. The results met the acceptance criteria, thus demonstrating that the procedure is suitably robust for its intended purpose.

[0380] The repeatability of the analysis procedure was demonstrated within the following ranges at peak 4, peak 7, the sum of peaks 1-3, and peak 8: -The LD of FDC drugs is 50 μg to 149 μg of injected protein, covering 50% to 149% of the nominal processing amount (100 μg of protein). -FDC Pharmaceuticals' MD (Medical Dosage) involved injecting 51 μg to 153 μg of protein, covering 51% to 153% of the nominal processing volume (100 μg of protein).

[0381] Example 12 Stability index Non-stressed and stressed MD and LD samples of FDC drugs were tested using the method of Example 10, demonstrating the procedure's ability to separate, identify, and determine the purity of antibodies 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 non-stressed samples (Figures 12 and 13), the chromatographs of stressed samples showed increased amounts of peaks 1-3 and peak 8 (data not shown). In conclusion, the procedure is an indicator of stability.

[0382] Example 13 ELISA-based titers of trastuzumab and pertuzumab charge variants and CDR affinity mutants in FDCs The ability of ELISA to reflect antiproliferative activity was demonstrated in charge mutants and CDR affinity mutants. The pertuzumab and trastuzumab HER2 affinity mutants described above were tested using antiproliferation assays and ELISA (Figures 9 and 14, respectively). The absence or shift to high concentrations of the dose-response curves observed in the HER2 affinity mutants, along with the failure to meet the similarity criteria (parallel processing of antiproliferation assays and ELISA and high asymptotic deviation, respectively), similarly demonstrate a significant reduction in titer. For the evaluation of charge variants, cell-based assays were used to exclude cross-interference of the aforementioned FDC drugs by using supportive technology batches containing either trastuzumab or pertuzumab in the MD formulation buffer of the FDC drugs.

[0383] All IE-HPLC fractions showed similar titers in both cell-based assays and ELISAs (considering the precision of the corresponding methods), with the exception of peak 9 (trastuzumab with increased oxidation of Fc Met261). Although this Fc oxidation at Met261 should not affect the target binding activity of CDR, this variant showed a reduced titer in trastuzumab ELISA (73% vs. 91% in cell-based assays).

[0384] We were unable to fully resolve whether the fragmentation process of these isoforms, which exist only in very small amounts, contributes to this finding, and whether the titers of both assays are truly different. However, the trastuzumab ELISA is considered conservative in this respect, as it shows a decrease in titer that is not reflected in cell-based antiproliferative assays.

[0385] ELISA is equivalent to antiproliferative assays in its ability to control the biological activity of product variants known to affect biological activity, and is detailed below. ●The trastuzumab deamidation product mutants HC Asn55 / isoAsp55 and LC Asn30 / Asp30 at peak 1 showed reduced activity in both assays. ●A trastuzumab product variant having succinimide at the Asp102 position of one of the heavy chains and increased FcMet oxidation at peak 10 showed reduced activity in both assays. Other IE-HPLC fractions, including peaks 4 and 7 corresponding to the main peaks of pertuzumab and trastuzumab, respectively, all showed 80%–120% invariant activity in both assays, as expected.

[0386] Similar titers at peak 8 were obtained in both cell-based assays and ELISAs, but the known negative effect of HC IsoAsp102 on the titer of trastuzumab IV was not observed in this study. Isomerization of HC Asp102 to ISoAsp in one of the heavy chains of trastuzumab, eluted at peak 4 in IE-HPLC, corresponds to peak 8 in IE-HPLC of the FDC drug. Additional studies conducted during the development of trastuzumab SC on the effect of HC Asp102 / isoAsp102 morphology on antiproliferative activity did not show a significant effect on trastuzumab SC compared to trastuzumab IV.

[0387] This may be due to formulation optimization (e.g., pH changes) and increased stability of trastuzumab SC. Finally, it should be noted that control of this variant was maintained in the FDC drug via a confirmed acceptance criterion determined by IE-HPLC. TIFF2026108626000014.tif131170

[0388] Example 14 Characterization of charge mutants Charge variants of FDC drugs, separated and isolated by IE-HPLC, were characterized (Figure 13). In addition, charge variants of individual antibodies in the released FDC formulations were isolated and characterized using the same IE-HPLC method (Figure 13).

[0389] A comprehensive peak characterization study using the following methods was conducted to confirm the charge variants of FDC drugs. ●LC-MS / MS of trypsin antibody peptides for assessment of chemical degradation sites ● Boronic acid affinity chromatography for evaluating lysine glycosylation content ● 2-AB labeling combined with HILIC for analysis of Fc glycosylation

[0390] LC-MS peptide mapping: LC-MS / MS peptide mapping and quantification of relevant amino acid modifications were performed as described by Schmid et al., 2018 (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; Vol. 1: p. 28). Briefly, all samples were denatured with 8 mol / L guanidine hydrochloride (pH 6.0) and reduced with dithiothreitol at 50°C for 1 hour.

[0391] The sample was buffered (0.02 mol / L, histidine hydrochloride, pH 6.0) and further digested with trypsin at 37°C for 18 hours. Peptide separation on a BEH C18 column was performed using an ACQUITY UPLC system. Online mass spectrometry detection was achieved using a Synapt G2 HDMS Q-ToF mass spectrometer. Relative quantification of modified peptides was performed using GRAMS AI software.

[0392] Boronic acid affinity chromatography: Boronic acid affinity chromatography was performed using a TSKgel Boronate-5PW affinity column. 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) was used for chromatographic separation using an HPLC system with 280 nm UV detection. Peak integration and glycation quantification were performed as described (Fischer S, Hoernschemeyer J, Mahler HC. Glycation during storage and administration of monoclonal antibody formulations. Eur J Pharm Biopharm. 2008; vol. 70: pp. 42-50).

[0393] Analysis of glycans: To assess Fc glycosylation, the sample was buffered with ammonium formate buffer (pH 8.6) and incubated with PNGase F at 45°C for 1 hour. Glycan 2-AB labeling was performed at 65°C for 2 hours. The labeled glycan structure was separated by HILIC, and subsequent peak integration and glycan quantification were detected by fluorescence detection as described (Reusch D, Haberger M, Maier B et al., Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1:separation-based methods. MAbs. 2015; Vol. 7: pp. 167-169).

[0394] Results and conclusions: All charge variants (relative abundance ≥1%) found in individual pertuzumab and trastuzumab molecules of the FDC formulation were also detected in the FDC drug. No new charge variants were detected in the FDC drug compared to the individual antibodies of the FDC formulation at release and after storage. Table 10 summarizes the findings. TIFF2026108626000015.tif105170TIFF2026108626000016.tif129170

[0395] The sum of peaks 1-3 contains acidic variants of pertuzumab (deamidation of HC-Asn-391, FC sialic acid and lysine glycosylation), as well as acidic variants of trastuzumab (deamidation of LC-Asn-30 and HC-Asn-55).

[0396] Peak 4 contains the major charge variant of pertuzumab (i.e., the native antibody), as well as low levels of trastuzumab acidic variants (deamidation of LC-Asn-30 and isomerization of HC-Asp-102).

[0397] The combined peaks 5-6 contain basic variants of pertuzumab (N-terminal VHS in the heavy and light chains, and C-terminal lysine in the heavy chain), as well as acidic variants of trastuzumab (deamidation of HC-Asn-392, lysine glycosylation, and increased Fc sialic acid content).

[0398] Peak 7 contains the major charge variant of trastuzumab (i.e., the native antibody) and does not overlap with the pertuzumab variant.

[0399] Peak 8 contains trastuzumab with isolated isomerization of HC-Asp-102 to isoaspartic acid and does not overlap with the pertuzumab charge variant.

[0400] -The sum of peaks 9-10 contains trastuzumab-charged mutants with increased FC oxidation (at HC-Met-255 and -431), and isomerization of HC-Asp-102 does not show overlap with pertuzumab mutants.

[0401] All the abundant charge variants found in pertuzumab SC and trastuzumab SC were also detected in the FDC (Full Decompression Cell) formulations. No new charge variants were detected in the FDC formulation materials at release or after storage. All important charge variants of individual molecules can be controlled in the FDC formulations.

[0402] The absence of additional co-elution or increased existing peak co-elution is expected or anticipated during stability, as stress-induced charge variants of pertuzumab and trastuzumab shift to early and late elution times, respectively. The peak pattern of stressed pertuzumab shifts to the acidic region of the chromatogram, while the peak pattern of stressed trastuzumab shifts to the basic region.

[0403] Example 15 FDC composition Total of peaks 1-3 of FDC chemicals measured by IE-HPLC The sum of peaks 1-3 of the FDC drug consists of the following variants: ● Acidic variant of pertuzumab (deamidation of HC Asn391, Fc sialic acid and lysine glycosylation). ● Deamidation of Asn327 in pertuzumab was observed only at trace levels in IE-HPLC characterization studies of FDC drugs. ● Acidic mutants of trastuzumab (mainly deamidation of LC Asn30 and HC Asn55). Compared to LC Asn30, the lower degradation sensitivity of HC Asn55 was verified under bioprocess 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; Vol. 1: p. 28).

[0404] Acceptable criteria for the end of shelf life of FDC drugs are justified based on clinical experience and predicted impacts on the PK / bioactivity and safety / immunogenicity profiles. Proposed acceptable criteria are suitable for controlling product quality and for covering potential impacts on the active ingredients and drug processing and storage.

[0405] For FDC drugs, the following acceptable end-of-shelf-life criteria were established: total of peaks 1-3: ≤23.0% (LD) / ≤21.0% (MD). These acceptable criteria were established based on clinical experience and the anticipated impacts on bioactivity / PK and safety / immunogenicity profiles. Expansion beyond current clinical experience is considered justified by low impact on bioactivity and PK, and no risk to immunogenicity / safety.

[0406] Safety and Immunogenicity Considerations: Since the acidic variants found in the pertuzumab and trastuzumab materials are modifications commonly found in IgG antibodies, any increase in the level of acidic variants within acceptable limits is not expected to represent new forms and therefore is not predicted to increase the risk of toxicity and ADA occurrence. This is supported by the low incidence of ADA in FDC drug clinical studies and favorable safety profiles. Aging clinical study materials up to 18.7 percent of FDC drug LD and up to 16.0 percent of FDC drug MD were administered to patients during the core study. No new acidic variants are generated during storage and handling. Furthermore, it has been published for trastuzumab (Schmid et al., 2018) that the degradation of solvent-exposed residues located at storage Fc (deamidation of HC Asn387, Asn392, and Asn393) and also at CDR (mainly deamidation of LC Asn30 and isomerization of HC Asn102) generally occurs significantly faster in vivo (within a few days) compared to bioprocess and real-time storage conditions. The degradation levels of the same Fc Asn deamidation sites in endogenous human antibodies were significantly higher than those observed in liquid drug formulations stored at 5°C. Therefore, it has been concluded that these degradations do not impose an increased risk of safety / immunogenicity on patients (Liu YD, van Enk JZ, Flynn GC. Human antibody Fc deamidation in vivo. Biologicals 2009; vol. 37: pp. 313-322). This conclusion can also be applied to pertuzumab deamidation, since it is detected in this peak region where the only deamidation site is located at the Fc portion (HC Asn391).

[0407] In summary, the potential 4.3% increase in the total of Peaks 1–3, which exceeds the level of patient exposure, is not expected to alter the product's immunogenicity and safety profile.

[0408] Considerations regarding biological activity: Compared to the maximum clinical experience at 18.7% (LD) and 16.5% (MD) regions for acidic variants of pertuzumab and trastuzumab (total of peaks 1-3), the specification limits of 23.0% (LD) and 21.0% (MD) could lead to a reduction of approximately 4% (according to the ELISA titers listed in Table 8) in pertuzumab and trastuzumab binding activity. A 4% change in biological activity is not considered to have a significant impact. Therefore, efficacy is expected to be maintained even if the total of peaks 1-3 is at the specification limit.

[0409] Considerations regarding PK: Antibodies of Fc are involved in clearance (Jefferis R. Antibody therapeutics: isotype and glycoform selection. Expert Opin Biol Ther 2007; Vol. 7: pp. 1401-13), and therefore, deamidation in CDR is not expected to affect PK. While it is known that charge properties affect the PK behavior of antibodies, the single negative charge introduced by deamidation does not affect PK (Khawli et al., 2010). Notably, only low levels of modification in Fc deamidation (IE-HPLC peak 3: pertuzumab HC Asn391; IE-HPLC peak 6: trastuzumab HC Asn392) were observed during FDC drug stability. Therefore, even if the sum of peaks 1-3 is at the specification limit, PK is not expected to be affected.

[0410] Peak 4 of FDC drugs by IE-HPLC Peak 4 of the FDC drug is part of the reported assay parameters for the IE-HPLC method and constitutes the desired primary charge isoform of pertuzumab. Including it in the specification ensures consistent purity of the product.

[0411] Acceptable criteria for release and stability testing of active ingredients and chemicals were established in relation to other reported assay parameters by IE-HPLC, along with considerations of manufacturing experience and stability effects. FDC chemical acceptance criteria of ≥38% (LD) and ≥28% (MD) at the end of shelf life ensure product purity and appropriate control of the manufacturing process.

[0412] Total of peaks 5-6 of FDC drugs measured by IE-HPLC The combined peaks 5-6 of FDC drugs consist of the following variants. ● Basic amino acids of pertuzumab (N-terminal VHS of the heavy and light chains, N-terminal pyroglutamate, and C-terminal lysine and prolineamide in the heavy chain) ● Acidic variant of trastuzumab (deamidation of HC Asn392, lysine glycosylation, and increased FC sialic acid content)

[0413] The sum of peaks 5-6 is not controlled by FDC drug release or stability testing because the basic variant of pertuzumab and the acidic variant of trastuzumab remain unchanged during drug manufacturing and storage and are therefore not considered stability indicator parameters.

[0414] Peak 7 of FDC drugs by IE-HPLC Peak 7 of the FDC drug is part of the output of the IE-HPLC method and constitutes the desired primary charge isoform of trastuzumab. This standard ensures consistent purity of the product. Acceptable criteria for drug release and stability testing were established in relation to other reported assay parameters by IE-HPLC, along with considerations of manufacturing experience and stability effects. The FDC drug acceptance criteria of ≥16.0% (LD) and ≥23.0% (MD) at the end of shelf life ensure product quality and appropriate control of the manufacturing process.

[0415] Peak 8 of FDC drugs by IE-HPLC Peak 8 of the FDC drug contains trastuzumab with a sole isomerization of HC Asp102 to isoaspartic acid (on one of the heavy chains) and does not show co-elution with the pertuzumab charge variant. The peak is controlled in release and stability tests of the FDC drug.

[0416] The acceptable thresholds of ≤9.0% (LD) / ≤12.0% (MD) for the end of shelf life of FDC drugs are justified based on clinical experience and the expected impacts on the PK / bioactivity and safety / immunogenicity profiles. The proposed acceptable thresholds are suitable for controlling product quality and for covering potential impacts on the active ingredients and drug processing and storage.

[0417] Safety and Immunogenicity Considerations: Since the acidic variants found in the trastuzumab material are modifications commonly found in IgG antibodies, any increase in the level of acidic variants within acceptable limits is not expected to represent a new form and therefore not expected to increase the risk of toxicity and ADA occurrence. The FDC drug was generally safe and showed good tolerability. The acidic profile was comparable to the safety profile of pertuzumab IV + trastuzumab IV (P+H IV). The occurrence of ADA was low (≤5%), and there were no clinical outcomes regarding PK, efficacy, or safety. Aging clinical study material up to 6.4 percent of peak 8 for FDC drug LD and up to 9.4 percent of peak 8 for FDC drug MD was administered to patients during the central study. No new charge variants were generated during storage and handling. Furthermore, it has been published for trastuzumab (Schmid et al., 2018) that degradation of solvent-exposed residues located at storage Fc (deamidation of HC Asn387, Asn392, and Asn393) and also at CDR (mainly deamidation of LC Asn30 and isomerization of HC Asn102) generally occurs significantly faster in vivo (within a few days) compared to bioprocess and real-time storage conditions. Degradation levels at the same Fc Asn deamidation sites in endogenous human antibodies were significantly higher than those observed in liquid drug formulations stored at 5°C. Therefore, it was concluded that these degradations do not pose an increased safety / immunogenicity risk to patients (Liu et al., 2009). The potential 2.5% increase in peak 8 (isomerization of HC Asp102 to isoaspartic acid), which exceeds the level to which patients are exposed, was not expected to alter the immunogenicity profile of the product.

[0418] Considerations regarding biological activity: The enriched peak 8 (peak purity 92%, mainly containing the isomerization of HC Asp102 to isoaspartic acid in one of the heavy chains) exhibits similar trastuzumab activity (100% binding activity) compared to the reference standard. Therefore, even if peak 8 is present at the specification limit, the efficacy of the FDC drug is expected to be maintained.

[0419] Considerations regarding PK: Aspartate isomerization to isoaspartate in the CDRs of pertuzumab and trastuzumab does not change the charge and is not expected to affect PK. Therefore, monoaspartate isomerization of trastuzumab HC Asp102 does not affect PK.

[0420] Total of peaks 9-10 of FDC drugs measured by E-HPLC The sum of peaks 9-10 for the FDC drug consists of trastuzumab with a single isomerization of HC Asp102 to succinimide (on one of the heavy chains) and does not overlap with the pertuzumab charge variant. In addition, low levels of trastuzumab Fc oxidation are detected in these peaks. Due to the low levels, no impact is expected. Succinimide (sum of peaks 9-10) is in equilibrium with peak 8 (iso-Asp) and peak 7 (Asp), and is therefore indirectly controlled by the tolerance criteria of peaks 8 and 7. Consequently, no tolerance criteria are required for the sum of peaks 9-10 in the control system.

[0421] Example 16 Manufacturing of FDC compositions The pertuzumab SC active ingredient is transferred from the drug storage container to a steam-sterilized stainless steel container. Multiple batches of pertuzumab SC active ingredient can be combined for drug manufacturing.

[0422] Based on the amount of pertuzumab added to the compounding container (determined by the mass, density, and pertuzumab content of the transferred pertuzumab SC active ingredient), the target dose of trastuzumab is defined (i.e., a 1:1 API ratio for the maintenance dose). Multiple batches of trastuzumab SC active ingredient to be added to the compounding container (based on density and trastuzumab content) can then be combined for FDC drug manufacturing.

[0423] Based on the volume (determined by mass and density) of pertuzumab SC and trastuzumab SC active ingredients to be added to the compounding container, the required amount of thawed rHuPH20 (based on the rHuPH20 solution content and activity) is added to the compounding container. Multiple rHuPH20 batches can be combined for drug manufacturing.

[0424] After transferring all the ingredients to the mixing container, the solution is homogenized by mixing.

[0425] Example 17 Development of an RP-UPHLC assay to determine FDC content device The same equipment and appropriate operating conditions can be used. HPLC system: HPLC system equipped with data acquisition software (with a series-connected vacuum degasser). Detector: UV / visible absorptometer or photodiode array detector Membrane filter: 0.2 μm filter (e.g., Corning catalog number 430049) Column: TSK-Gel G3000SWXL, 7.8×300mm, 5μm (Bosoh Bioscience catalog number 08541) or BioSuite 250, 7.8×300mm, 5μm (Waters catalog number 186002165)

[0426] reagent ● Purified water (water treated with Milli-Q) ● Trifluoroacetic acid (TFA) (Fluka catalog number 40967) ● Acetonitrile (Merck catalog number 1.00030.2500) ● L-histidine anhydrous (Sigma catalog number H8000) ● Sucrose (Merck catalog number 1.07687) ● L-methionine (Sigma catalog number 64319) ● Glacial acetic acid (Merck catalog number 1.00063.1000) ●Polysorbate 20 (Sigma catalog number 93773)

[0427] Solvent A: 0.1% TFA in Milli-Q water Solvent B: 0.1% TFA in acetonitrile Formulation buffer: 20 mM histidine acetate, 240 mM sucrose, 10 mM methionine, and polysorbate 20, 0.02% [w / v], pH 5.7 ± 0.2 Dilution buffer: 20 mM histidine acetate, pH 5.5 Column storage solution: 60% acetonitrile (v / v)

[0428] Sample solution: Dilute the sample with formulation buffer to approximately 10 mg / mL. Dilute the 10 mg / mL test sample solution with dilution buffer to approximately 1 mg / mL. Blank: Inject the formulation buffer and dilution buffer undiluted.

[0429] Flow rate: 0.4mL / min Maximum pressure: 400 bar / 6000 psi Wavelength: 280nm Driving time: 29 minutes Column temperature setting: 60℃ Autosampler temperature setting: ≤10℃ Injection volume: Sample and reference standard: 25 μg of protein (nominal) Blank and mobile phase: Same injection volume as the reference standard.

[0430] TIFF2026108626000017.tif46170

[0431] While the pertuzumab and trastuzumab peaks were clearly separated by this method, a major carryover problem with the above method became apparent. After five injections of blank sample (formulation buffer), trace amounts of Herceptin / Perjeta were still detectable. Therefore, further method development was 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 in relation to the accuracy and repeatability of the method.

[0432] Effect of column type on separation Different types of columns were tested with pertuzumab / trastuzumab FDC. JPEG2026108626000018.jpg127170

[0433] Several potential columns were identified for pertuzumab / trastuzumab FDC. For example, BEH300 C4 showed good separation but required a high column temperature (90°C). Agilent AdvanceBio RP mAb had similar separation to Agilent Zorbax RRHD 300-Diphenyl but had lower overall resolution. The most preferred column was determined to be Agilent Zorbax RRHD 300-Diphenyl, 2.1 × 100 mm column, which exhibited low carryover and improved separation of the two antibodies compared to the initial method.

[0434] Design of Experiment (DoE) for Agilent Zorbax RRHD 300-Diphenyl columns Mobile phase, flow rate, gradient, and column compartment temperature were tested using an Agilent Zorbax RRHD 300-Diphenyl, 2.1 × 100 mm column. The DoE for the development of the reverse-phase protein content method was set using MODDE®. A summary of the factors tested within the DoE range is listed in Table 12. JPEG2026108626000019.jpg39170

[0435] Evaluation of "Resolution of trastuzumab / pertuzumab" Overall, the resolution of reversed-phase chromatography methods for determining protein content is strongly influenced by flow rate and gradient length. Lower flow rates and longer gradient lengths resulted in improved resolution. Column compartment temperature and starting conditions had a weak but significant effect on the method. A temperature of 70°C and relatively high starting conditions of 30% B proved to produce the best results. Adding additional retention time did not affect the resolution.

[0436] Evaluation of "Total amount of trace forms" The total amount of trace forms was strongly dependent on the starting conditions (high) and column temperature (low). Flow rate and gradient time had only a slight effect. Retention time was negligible on its own, but showed an effect when combined with flow rate and column temperature.

[0437] Evaluation of "high trastuzumab ratio" To achieve a high ratio, i.e., to avoid shouldering the trastuzumab main peak, the temperature needed to be low. Flow analysis was unclear. Gradient time and starting conditions should ideally be within the high range. Here again, additional retention time had no effect.

[0438] Evaluation of "Pertuzumab USP Tailing" To reduce tailing of the pertuzumab main peak, the flow rate should be increased, and the gradient and starting conditions should be decreased. Again, additional holding time did not have an effect.

[0439] Based on the DoE results, the following parameters were selected. Flow rate 0.8mL / min Wavelength 280nm Column temperature 70°C Autosampler temperature 10℃ Driving time: 20 minutes TIFF2026108626000020.tif40170

[0440] Based on these results, the column temperature, gradient, and flow rate were further optimized.

[0441] Effect of column temperature on separation Increased temperature in reverse-phase chromatography can have a significant impact on peak separation, tailing effects, and system pressure. Three different temperatures were selected and tested on an Agilent Zorbax RRHD 300 Diphenyl column. Temperature testing was performed within the DoE range (results not shown). Overall, retention times shifted to earlier elution as the column compartment temperature increased. This was expected because the viscosity of the eluent and secondary column interactions decrease with increasing temperature. However, increasing the column compartment temperature also decreased the overall resolution. Therefore, the most suitable column compartment temperature within the experimental range was 70°C.

[0442] Influence of gradient profiles on separation The gradient has a dramatic impact on the separation of the analyte. Four major gradients were tested on an Agilent Zorbax RRHD 300 Diphenyl column for protein content determination by reverse-phase chromatography (see Table 14). For direct gradient comparison, the column compartment temperature was kept constant at 70°C and the flow rate was set to 0.6 mL / min. Once the final flow rate was set, the gradient needed to be re-evaluated. The final gradients for the RP protein content method are listed as gradient 5 in Table 14. The initial DoE gradient (Table 13) was modified for optimal separation and for equilibrium time with the new flow rate (0.3 mL / min). JPEG2026108626000021.jpg63170

[0443] observation: All five gradients tested showed sufficient protein retention and selected starting conditions in the 20-30% B range. Any starting condition within this range is suitable for the separation of pertuzumab / trastuzumab FDCs. However, to reduce gradient time and run time, a 30% starting condition was selected. Considering the gradient time, the tested range (10-20 minutes) was selected. Since the gradient time is also greatly affected by the flow rate, a 15-minute isolation time at a flow rate of 0.3 mL / min was ultimately selected.

[0444] With a 10-minute isolation time, both antibodies eluted within a time frame of just 1-2 minutes, especially with a steep gradient of 30%B. However, a 20-minute isolation time and a steep gradient of 20%B resulted in a broader elution profile and a less strong detection signal.

[0445] Finally, a 15-minute separation time was combined with a steep gradient of 15%B. When combined with a slow flow rate (0.3 mL / min), it showed good baseline separation of both antibodies without significant signal intensity loss.

[0446] Effect of flow rate on separation Ultimately, it was necessary to determine the most suitable flow rate. Faster flow rates usually mean earlier elution, but can lead to degradation loss. Initial experiments were performed at flow rates of 0.6 or 0.8 mL / min. It was later found that lower flow rates were more beneficial for this particular RP protein content method. Four different flow rates were tested using single mAb-containing samples on an Agilent Zorbax RRHD 300 Diphenyl column (0.3 mL / min to 0.6 mL / min). For direct comparisons, the column compartment temperature was set constant at 70°C, and the gradients listed in Table 13 were used for all separations.

[0447] Reducing the flow rate resulted in a narrower peak shape and higher signal intensity. Retention time shifted to late elution. Side peak resolution, in particular, improved with slower flow rates. Therefore, a flow rate of 0.3 mL / min is ideal for this method. A gradient run time of 30 minutes, with a flow rate of 0.3 mL / min, demonstrated sufficient column reequilibrium.

[0448] Based on these experiments, the most important parameters for this method were found to be column type, column temperature, and flow rate. The use of a phenyl-based column resulted in improved resolution and no carryover issues. Temperatures of 64°C–76°C and 66°C–74°C were tested and did not significantly affect the performance of the method. Within the robustness limits of Phase III experiments and the validity of the BLA / MAA method, flow rates of 0.4 and 0.2 mL / min were tested and found not to significantly affect the performance of the method.

[0449] Example 18 RP-UPHLC assay to determine FDC content Note: Equivalent equipment, appropriate operating conditions, and solvents, chemicals, and reagents of equivalent quality can be used.

[0450] The pertuzumab and trastuzumab content in FDC drugs is determined by RP-UHPLC with UV detection. Pertuzumab and trastuzumab are separated based on their hydrophobicity differences. The corresponding pertuzumab and trastuzumab content is calculated using an external calibration curve generated by each of a series of analyses injecting various doses of FDC reference standards. The same method is applied to the LD and MD of FDC drugs. Each dosage form is measured against the corresponding reference standard.

[0451] Equipment and materials ● UHPLC system with UV detector (Thermo Ultimate 3000 RS or equivalent) ● UHPLC column (Agilent Zorbax RRHD 300-Diphenyl, 2.1 × 100 mm, particle size: 1.8 μm or equivalent to 1.8 μm)

[0452] reagent ●2-propanol ● Acetonitrile ●TFA ● L-histidine anhydrous ● L-histidine monohydrochloride monohydrate ● Sucrose ● Trehalose ● L-methionine ●Polysorbate 20 ●Sodium hydroxide ● Hydrochloric acid ● Purified water (e.g., Milli-Q)

[0453] solution Chemical dilution buffer 20 mM L-histidine / L-histidine monohydrochloride, 105 mM trehalose, 100 mM sucrose, 10 mM methionine, 0.04% (w / v) polysorbate 20, pH 5.5 ± 0.2

[0454] Mobile phase A 2% (v / v) 2-propanol, 0.1% (v / v) TFA in water

[0455] Mobile phase B 70% (v / v) 2-propanol, 20% (v / v) acetonitrile, 10% (v / v) mobile phase A

[0456] Preparation of reference standard solutions Note: For the measurement of LD and MD samples of FDC drugs, FDC LD reference standards and FDC MD reference standards must be prepared, respectively. The corresponding reference solutions must be prepared in duplicate (reference solution A and reference solution B). Dilute the corresponding reference standards to a total protein concentration of 1 mg / mL using drug dilution buffer.

[0457] Preparation of sample solution Dilute the FDC reagent with dilution buffer to prepare a sample solution containing a total protein concentration of approximately 1 mg / m².

[0458] procedure Before injecting the first sample, rinse 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 demonstrates a consistent profile over at least two consecutive injections.

[0459] Operating parameters ●Detection wavelength: 280nm ●Injection volume: Refer to the injection protocol below. ●Flow rate: 0.3mL / min ●Column temperature: 70℃±2℃ ● Autosampler temperature: 10℃±4℃ • Driving time: 30 minutes

[0460] TIFF2026108626000022.tif48170

[0461] Injection protocol Each dosage form requires a different sequence using the corresponding reference standard. Sample injections should be performed in the order shown in Table 16. TIFF2026108626000023.tif80170

[0462] result Typical chromatographic profiles are shown in Figure 15 for FDC reagent LD and in Figure 16 for FDC reagent MD.

[0463] The final method (Example 18) provides a substantial improvement over the initial protein content method, including improved overall resolution / peak analysis and elimination of sample carryover, i.e., carryover does not exceed 0.2% in subsequent analysis. Furthermore, the final method enables quantitative determination of the protein content of maintenance and loading doses of pertuzumab and trastuzumab. Different phenyl-based RP columns exhibit improved specificity for the two antibodies, allowing for detection of only trace amounts of sample carryover and enabling accurate protein content determination. The final reversed-phase U-HPLC method for determining the protein content in pertuzumab / trastuzumab FDC involves separating the two molecules on a phenyl-based reversed-phase column (Agilent Zorbax RRHD 300-Diphenyl) at 70°C using a water-2-propanol / acetonitrile gradient and 0.1% TFA.

[0464] Figure 15 depicts an example of an RP-UHPLC chromatogram analyzing the protein content of an FDC LD reference standard, and Figure 16 depicts an example of an RP-UHPLC chromatogram analyzing the protein content of an FDC MD reference standard.

[0465] Data Analysis Integrate the pertuzumab and trastuzumab peaks in the chromatograms of reference solutions A and B, and in the sample solution. The integration is defined with the help of representative chromatograms in Figure 15 for FDC drug LD and Figure 16 for FDC drug MD. Generate standard curves for each antibody by plotting the peak region for each standard level injection volume (μg). Fit the standard curve data using linear regression. Do not force the curve through zero.

[0466] By using the standard curve equation, the amounts of pertuzumab and trastuzumab are calculated using the corresponding peak regions for each sample shaker and the injection of reference B. TIFF2026108626000024.tif18170

[0467] Slope calibration curve To calculate the pertuzumab and trastuzumab content, divide the amount by the corresponding injection volume and multiply by the dilution factor. TIFF2026108626000025.tif18170

[0468] Example 19 HIHPLC to determine FDC content Hydrophobic interaction chromatography (HI-HPLC) was evaluated. HI-HPLC is a common antibody analysis method, particularly for identifying molecular variants, such as post-translational modifications or antibody-drug conjugate species. In addition, because HI-HPLC is a non-denaturing chromatography method, it is possible to identify misfolded proteins or conformational changes.

[0469] Compared to RP-UHPLC, the main differences with HI-HPLC are as follows: ●HI chromatography is non-destructive, and proteins remain folded. ● Due to natural protein folding, protein column interactions occur only at amino acids located on the protein surface. ● Elution is not promoted by increasing the organic solvent concentration, but by reducing the amount of ammonium sulfate, for example, the hydrophobic-hydrophobic interaction between the protein and the stationary phase is weakened. Therefore, reducing the number of hydrophobic species leads to earlier elution.

[0470] Two columns were tested using HIC-HPLC. - TSKgel ether column, 75mm x 7.5mm, particle size 10μm - TSKgel butyl column, 35 mm × 4.6 mm, particle size 2.5 μm

[0471] The mobile phase was tested. - Eluting agent A: 50 mM sodium phosphate, pH 7.0 ± 0.05, 5% (v / v) ethanol - Eluting agent B: 50 mM sodium phosphate, 2 M ammonium sulfate, pH 7.0 ± 0.05

[0472] result: HI-HPLC can separate pertuzumab / trastuzumab FDC molecules with any column type. Butyl columns have significantly better resolution than ether columns in combination samples (data not shown). Regarding the comparison between RP-UHPLC and HI-HPLC, RPUHPLC was preferred over HI-HPLC, particularly in protein content analysis. HI chromatography separated the two antibodies but lacked sufficient resolution and exhibited a noticeable tailing effect. Reverse-phase chromatography shows improved resolution for pertuzumab and trastuzumab compared to HI-HPLC. In particular, the shoulder peaks of pertuzumab and trastuzumab are better degraded with RPC than with HIC. Furthermore, RPC results in a horizontal baseline, which is preferable to the slanted baseline of HIC. In addition, the use of a water-organic solvent gradient is less stressful on the HPLC system than a high-low salt gradient. TIFF2026108626000026.tif93170

[0473] While certain embodiments of the present invention have been shown and described herein, it will be understood by those skilled in the art that such embodiments are provided only as examples. Herein, those skilled in the art will anticipate numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternative forms to the embodiments described herein may be used in carrying out the present invention. The following claims define the scope of the present invention and are intended to encompass the methods and structures within the claims, as well as their equivalents.

[0474] SEQUENCE LISTING <110> GENENTECH, INC. F. HOFFMANN-LA ROCHE AG <120> ASSAYS FOR 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: Synthetic 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: Synthetic 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: Synthetic 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: Synthetic 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: Synthetic 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: Synthetic 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: Synthetic 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> / note="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: Synthetic 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: Synthetic 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: Synthetic peptide" <220> <221> VARIANT <222> (10)..(10) <223> / replace="Ser" <220> <221> MISC_FEATURE <222> (10)..(10) <223> / note=" Residue given in the sequence has no preference with respect to that in the annotations for said position " <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: Synthetic 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: Synthetic 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: Synthetic peptide" <400> 20 Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (5)..(5) <223> / replace="Leu" <220> <221> VARIANT <222> (6)..(6) <223> / replace="Glu" <220> <221> VARIANT <222> (7)..(7) <223> / replace="Ser" <220> <221> MISC_FEATURE <222> (5)..(7) <223> / note="Residue given in the sequence has no preference with respect to that in the annotations for said position " <400> 21 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 22 Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5 <210> 23 <211> 119 <212> PRT <213> Homo sapiens <400> 23 Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40 45 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 50 55 60 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 65 70 75 80 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 85 90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys 115 <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> Capture reagent for anti-HER2 antibody <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> capturing 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> Capturing 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> Artificial Sequence <220> <223> Capturing 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> Capturing 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> Capturing 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 ...

Claims

1. A binding assay for a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Contacting FDC with a capture reagent containing a modified HER2 ECD subdomain; b. Contacting the sample with a detectable antibody; c. Quantifying the level of antibodies bound to the capture reagent using a detection method for detectable antibodies, A binding assay including the following.

2. The binding assay according to claim 1, wherein the fixed-dose formulation comprises an antibody that binds to HER2 extracellular subdomain II and an antibody that binds to HER2 extracellular subdomain IV.

3. The binding assay according to claim 1 or 2, wherein the binding of an antibody that binds to the extracellular subdomain II of HER2 is quantified.

4. The binding assay according to any one of claims 1 to 3, wherein the capture reagent comprises recombinant HER2 extracellular domain II.

5. The binding assay according to claim 4, wherein the capture reagent comprises SEQ ID NO: 2 or SEQ ID NO:

23.

6. The binding assay according to any one of claims 1 to 5, wherein the capture reagent comprises recombinant HER2 extracellular domains I, II, and III.

7. The binding assay according to claim 6, wherein the capture reagent comprises SEQ ID NO:

24.

8. The binding assay according to any one of claims 3 to 6, wherein the capture reagent does not contain the HER2 subdomain IV.

9. The binding assay according to claim 1 or 2, wherein the binding of an antibody bound to the HER2 subdomain IV is quantified.

10. The binding assay according to claim 9, wherein the capture reagent comprises recombinant HER2 extracellular domain IV.

11. The binding assay according to claim 10, wherein the capture reagent comprises SEQ ID NO: 4 or SEQ ID NO:

28.

12. The binding assay according to any one of claims 9 to 11, wherein the capture reagent does not contain HER2 subdomain II.

13. The binding assay according to any one of claims 9 to 12, wherein the capture reagent comprises recombinant HER2 extracellular domains I, III, IV and EGFR domain II.

14. The binding assay according to any one of claims 9 to 13, wherein the capture reagent comprises SEQ ID NO:

29.

15. A binding assay according to any one of claims 1 to 14 for analyzing the titer of one of the anti-HER2 antibodies.

16. The binding assay according to claim 15, wherein the titer is measured in a cell-based assay and quantified by correlating the level of antibody bound to the capture reagent with the biological activity of the isolated antibody.

17. The binding assay according to any one of claims 1 to 16, wherein the capture reagent is coated onto a microtiter plate.

18. The conjugation assay according to any one of claims 1 to 17, wherein the detectable antibody targets the F(ab')2 portion of the anti-HER2 antibody.

19. The binding assay according to any one of claims 1 to 18, wherein the fixed-dose formulation additionally contains hyaluronidase.

20. Isolated protein containing SEQ ID NO:

24.

21. Isolated protein containing SEQ ID NO:

29.

22. A kit for specifically quantifying the binding of an antibody to the HER2 extracellular subdomain II in a fixed-dose combination (FDC) of a first antibody and a second anti-HER2 antibody that bind to the HER2 extracellular subdomain II, a. A container containing proteins including SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 34 as capture reagents, b. Instructions for quantifying the binding of antibodies to the extracellular subdomain II of HER2, A kit that includes this.

23. A kit for specifically quantifying the binding of an antibody to the HER2 extracellular subdomain IV in a fixed-dose combination drug (FDC) of an antibody that binds to the HER2 extracellular subdomain IV and a second anti-HER2 antibody, a. A container containing proteins including SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 3 and SEQ ID NO: 4 as capture reagents, b. Instructions for quantifying the binding of antibodies to the HER2 extracellular subdomain IV, A kit that includes this.

24. A method for evaluating a fixed-dose composition containing pertuzumab and trastuzumab, a. Using a loading buffer with a pH of approximately 7.5 to approximately 7.65, the antibody is bound to the ion exchange material. b. Elute the antibody with an elution buffer with a pH of approximately 7.5 to approximately 7.7, Methods that include...

25. The method according to claim 24, wherein the ion exchange material is a cation exchange material.

26. The method according to claim 25, wherein the cation exchange chromatography material is a strong cation exchange material.

27. The method according to claim 25 or 26, wherein the cation exchange material comprises a sulfonate group.

28. The method according to any one of claims 24 to 27, wherein step b is carried out on a salt gradient.

29. The method according to any one of claims 24 to 28, wherein the elution buffer contains sodium.

30. The method according to any one of claims 24 to 29, wherein the elution buffer contains sodium chloride.

31. c. A step of selectively detecting charge variants of pertuzumab and trastuzumab in the composition. The method according to any one of claims 24 to 30, further comprising:

32. The method according to any one of claims 24 to 31, carried out at a temperature of 32 to 40°C.

33. The method according to any one of claims 24 to 32, wherein a combination of pertuzumab and trastuzumab in a fixed dose further comprises hyaluronidase.

34. A method for preparing a composition, comprising: (1) producing a fixed-dose combination containing pertuzumab, trastuzumab, and one or more variants thereof; and (2) subjecting the composition thus produced to an analytical assay to evaluate the amount of variants therein, wherein the variants include (i) pertuzumab deamidated with HC-Asn-391, pertuzumab FC sialic acid variant, and pertuzumabridin glycated variant; (ii) pertuzumab native antibody; (iii) trastuzumab native antibody; and (vi) trastuzumab having isomerization of one heavy chain with HC-Asp-102 to isoaspartic acid.

35. The method according to claim 35, wherein the analytical assay is the assay described in any one of claims 24 to 33.

36. The method according to claim 34 or 35, wherein the fixed-dose formulation additionally contains hyaluronidase.

37. The method according to any one of claims 34 to 36, wherein the composition comprises 40 to 60 mg / mL of trastuzumab and 60 to 80 mg / mL of pertuzumab.

38. A composition comprising pertuzumab and trastuzumab, wherein the composition comprises less than 23% of pertuzumab acidic variants selected from deamidation of HC-Asn-391, Fc sialic acid and lysine glycosylation, trastuzumab variants deamidated with LC-Asn-30, and trastuzumab variants deamidated with HC-Asn-55, at least 28% of pertuzumab native antibody, at least 16% of trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

39. The composition according to claim 38, comprising less than 23% of a pertuzumab acidic variant selected from deamidation of HC-Asn-391, Fc sialic acid, and lysine glycosylation, a trastuzumab variant deamidated with LC-Asn-30, and a trastuzumab variant deamidated with HC-Asn-55, at least 38% of a pertuzumab native antibody, at least 16% of a trastuzumab native antibody, and less than 9% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

40. The composition according to claim 38, comprising less than 21% of a pertuzumab acidic variant selected from deamidation of HC-Asn-391, Fc sialic acid, and lysine glycosylation, a trastuzumab variant deamidated with LC-Asn-30, and a trastuzumab variant deamidated with HC-Asn-55, at least 28% of a pertuzumab native antibody, at least 23% of a trastuzumab native antibody, and less than 12% of trastuzumab having isomerization of HC-Asp-102 to isoaspartic acid in one of its heavy chains.

41. A composition comprising pertuzumab and trastuzumab, wherein, as determined by the method of any one of claims 24 to 33, the composition comprises a peak region of less than 23% of the sum of peaks 1 to 3, a peak region of at least 28% of peak 4 (pertuzumab natural antibody), a peak region of at least 16% of peak 7 (trastuzumab natural antibody), and a peak region of less than 12% of peak 8.

42. The composition according to claim 41, comprising, as determined by the method of any one of claims 24 to 33, a peak region of less than 23% of the sum of peaks 1 to 3, a peak region of at least 38% of peak 4 (pertuzumab natural antibody), a peak region of at least 16% of peak 7 (trastuzumab natural antibody), and a peak region of less than 9% of peak 8.

43. The composition according to claim 41, comprising, as determined by the method of any one of claims 24 to 33, a peak region of less than 21% of the sum of peaks 1 to 3, a peak region of at least 28% of peak 4 (pertuzumab natural antibody), a peak region of at least 23% of peak 7 (trastuzumab natural antibody), and a peak region of less than 12% of peak 8.

44. The composition according to any one of claims 38 to 43, further comprising rHuPH20.

45. The composition according to any one of claims 38 to 44, comprising 40 to 60 mg / mL of trastuzumab and 60 to 80 mg / mL of pertuzumab.

46. a. Add a predetermined amount of pertuzumab to the mixing container, b. Adding trastuzumab in a 1:1 ratio of trastuzumab to pertuzumab or a 1:2 ratio of trastuzumab to pertuzumab, c. Add rHuPH20. A composition according to any one of claims 38 to 45, obtained by the means of the method described herein.

47. A method for analyzing the protein content of a fixed-dose combination (FCD) of two anti-HER2 antibodies, a. Prepare an RP-HPLC phenyl column, b. Loading an RP-HPLC column with a fixed-dose combination of two anti-HER2 antibodies (FCD), c. Separating two anti-HER2 antibodies at a flow rate of 0.2–0.4 mL / min, with a column temperature of 64°C–76°C, Methods that include...

48. The method according to claim 47, wherein the fixed-dose combination agent comprises pertuzumab and trastuzumab.

49. The method according to claim 47 or 48, wherein a combination of pertuzumab and trastuzumab in a fixed dose further comprises hyaluronidase.

50. The method according to any one of claims 47 to 49, wherein the separation is achieved by a water-2-propanol / acetonitrile gradient.

51. The method according to any one of claims 47 to 50, wherein the flow rate is approximately 0.3 mL / min.

52. The method according to any one of claims 47 to 51, wherein the antibody is separated over a period of 10 to 20 minutes.

53. The method according to claim 52, wherein the antibody is separated over a period of 15 minutes.

54. The method according to claim 52 or 53, wherein the antibody is separated over a period of 15 minutes at a flow rate of approximately 0.3 mL / min.

55. The method according to any one of claims 47 to 54, wherein the column temperature is 70°C ± 2°C.

56. The method according to any one of claims 47 to 55, wherein the phenyl column is a column selected from the group consisting of Acclaim Phenyl-1 (Dionex), Pursuit® XRs Diphenyl, Pinnacle® Biphenyl, Zorbax® Eclipse® Plus Hexyl Phenyl, Ascentis Phenyl, and Agilent AdvanceBio RP mAb Diphenyl and Agilent Zorbax RRHD 300-Diphenyl columns.

57. Essentially, the methods, kits, and compositions described herein.

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