Microchip capillary electrophoresis assays and reagents

Abstract

To provide improved MCE assays and compositions that reduce assay interferences, and MCE assays and compositions for improving detection of impurities in a protein drug product.SOLUTION: MCE assays and reagents to assess purity and to identify impurities in protein drug product samples are provided. Methods for analyzing analytes in a protein drug sample are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical field of the invention Aspects of the present invention are directed generally to the field of capillary electrophoresis, and particularly to microchip capillary electrophoresis. [Background technology]

[0002] Background of the Invention To meet the demands of today's quality control (QC) laboratories for biological product testing, robust, reproducible, and user-friendly technologies are essential. Technology upgrades are necessary to facilitate increased output while continuing to generate high-quality analytical data and minimizing the number of invalid test results and instrument-related investigations. While electrophoresis has historically been used for product purity QC and fragmentation analysis, methods have transitioned from gel-based to capillary-based and more recently to microchips. Microchip capillary electrophoresis (MCE) dramatically reduces sample analysis time while maintaining the performance and reproducibility standards required for QC analysis (Ouimet, C., et al., Expert Opin Drug Discov., 12(2):213-224 (2017)).

[0003] MCE has emerged as a promising technique that is increasingly being used in the pharmaceutical industry for biopharmaceutical characterization, quality control, and drug discovery, but is prone to assay interference.

[0004] It is therefore an object of the present invention to provide improved MCE assays and compositions that reduce assay interference.

[0005] Another object of the present invention is to provide MCE assays and compositions that improve the detection of impurities in protein drug products. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Ouimet, C., et al., Expert Opin Drug Discov.,12(2):213-224(2017) Summary of the Invention

[0007] MCE assays and reagents are provided for assessing purity and identifying impurities in protein drug product samples. Methods for analyzing analytes in protein drug samples are also provided. Preferred protein drugs include, but are not limited to, antibodies and their antigen-binding fragments, fusion proteins, and recombinant proteins. The assay employs MCE technology to separate, identify, and quantify protein products and impurities in protein products. Impurities include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing and non-reducing buffers are also provided.

[0008] In one embodiment, a non-reducing aqueous electrophoresis sample buffer is provided that includes an alkylating agent, such as 155-175 mM 2-iodoacetamide; 0.50-1.5% lithium dodecyl sulfate; and 65-95 mM sodium phosphate, where the aqueous electrophoresis sample buffer has a pH of less than 7. In a preferred embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer includes 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0009] Reducing buffers are also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer comprising 0.5-1.5% lithium dodecyl sulfate, 55-85 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 8. In a preferred embodiment, the buffer has a pH of 9. In one embodiment, the reducing buffer comprises 135-155 mM dithiothreitol. In yet another embodiment, a reducing buffer is provided comprising 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

[0010] HEPES-based buffers can also be used with the disclosed methods. In one embodiment, a non-reducing HEPES-based aqueous electrophoresis sample buffer is provided, comprising an alkylating agent, e.g., 55-75 mM 2-iodoacetamide; 0.1-1.0% lithium dodecyl sulfate; 5-85 mM HEPES; and 5-115 mM sodium chloride, wherein the aqueous electrophoresis sample buffer has a pH of less than 9. In another embodiment, the pH of the buffer is 8. In yet another embodiment, the aqueous buffer comprises 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.

[0011] In another embodiment, a reducing HEPES-based aqueous electrophoresis sample buffer is provided, comprising 0.05-0.75% lithium dodecyl sulfate, 5 mM-115 mM sodium chloride, 5 mM-115 mM HEPES, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 7. In a preferred embodiment, the pH of the buffer is 8. In one embodiment, the reducing buffer comprises 35-50 mM dithiothreitol. In yet another embodiment, a reducing buffer is provided, comprising 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.

[0012] In one embodiment, a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample is provided, comprising adding the protein sample to the non-reducing buffer described above to form a buffered protein drug sample. The buffered protein drug sample is heated to between 65°C and 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample. In a preferred embodiment, the buffered protein drug sample is heated to 70°C for 10 minutes.

[0013] The protein drug sample is mixed with a detectable label and heated at 30-40°C for 20-40 minutes to form a denatured, labeled protein drug sample. Preferred detectable labels include, but are not limited to, DY-631 NHS ester from Dyomics. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, and the like, as well as those disclosed in U.S. Patent No. 6,924,372, which is incorporated by reference in its entirety. In a preferred embodiment, the labeled protein drug sample is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter.

[0014] The denatured, labeled protein drug product is diluted and subjected to MCE, and the diluted protein drug sample is separated on a microchip capillary electrophoresis system to generate an electropherogram. In one embodiment, the sample, initially injected onto the microchip at 0.5 mg / ml, has a final concentration of 9 μg / ml for MCE. The electropherogram contains peaks corresponding to the protein drug product and impurities. The method is concluded by identifying peaks in the electropherogram that correspond to contaminants or impurities.

[0015] In another embodiment, a reducing MCE method for identifying contaminants or impurities in a protein drug sample is provided. This method begins by adding a protein sample to any of the reducing buffers described above to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample to 65-85°C, preferably 70°C, for 10 minutes to form a denatured protein drug sample. The protein drug sample is mixed with a detectable label and heated at 30-40°C for 20-40 minutes to form a denatured, labeled protein drug sample. In a preferred embodiment, the labeled protein drug sample is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter. Preferred detectable labels include, but are not limited to, DY-631 NHS ester (Dynomics). Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, and the like, and such as those disclosed in US Pat. No. 6,924,372, which is incorporated by reference in its entirety.

[0016] In one embodiment, the established assay range for sample concentration is 0.4 mg / ml to 0.6 mg / ml, corresponding to a final concentration being analyzed of approximately 7 μg / ml to 11 μg / ml, which is subjected to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram. The method is concluded by identifying peaks in the electropherogram that correspond to contaminants or impurities. [The present invention 1001] 155-175 mM 2-iodoacetamide, 0.50 to 1.5% lithium dodecyl sulfate; 75-95mM sodium phosphate Including, having a pH of less than 7, Aqueous electrophoresis sample buffer. [The present invention 1002] 1001. An aqueous buffer solution of the present invention, having a pH of 6. [The present invention 1003] An aqueous buffer solution of invention 1001 or 1002 comprising 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate. [The present invention 1004] 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate; 81 mM sodium phosphate and It consists of having a pH of 6.0, Aqueous electrophoresis sample buffer. [The present invention 1005] 0.50 to 1.5% lithium dodecyl sulfate; 45-75 mM sodium phosphate; Reducing agent and Including, having a pH greater than 8, Aqueous electrophoresis sample buffer. [The present invention 1006] 1005. An aqueous buffer solution of the present invention, having a pH of 9. [The present invention 1007] The aqueous buffer solution of the present invention 1005 or 1006, comprising 135 to 155 mM dithiothreitol. [The present invention 1008] The aqueous buffer solution of any one of 1005 to 1007 of the present invention, comprising 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol. [The present invention 1009] 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate, 142 mM dithiothreitol and It consists of having a pH of 9.0, Aqueous electrophoresis sample buffer. [The present invention 1010] 1. A method for identifying contaminants or impurities in a protein drug sample, comprising: Adding the protein drug sample to any one of the buffer solutions of the present invention 1001 to 1004 to form a buffered protein drug sample; heating the buffered protein drug sample to 65-85°C for 5-15 minutes to form a denatured buffered protein drug sample; adding a detectable label to the denatured, buffered protein drug sample and heating at 30-40°C for 20-40 minutes to form a denatured, labeled protein drug sample; diluting the denatured, labeled protein drug sample and subjecting it to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system and generate an electropherogram; identifying peaks in the electropherogram that correspond to contaminants or impurities; A method comprising: [The present invention 1011] 10. The method of claim 10, wherein the buffered protein drug sample is heated at 70° C. for 10 minutes. [The present invention 1012] 1012. The method of claim 1010 or 1011, wherein the labeled protein drug sample is heated at 35° C. for 30 minutes. [The present invention 1013] 13. The method of any one of claims 1010 to 1012, wherein the diluted protein drug sample is 9 mg / ml. [The present invention 1014] 1. A method for identifying contaminants or impurities in a protein drug sample, comprising: adding the protein sample to any one of the buffers of the present invention 1005 to 1009 to form a buffered protein drug sample; heating the buffered protein drug sample to 65-85°C for 5-15 minutes to form a denatured protein drug sample; adding a detectable label to the denatured protein drug sample and heating at 30-40°C for 20-40 minutes to form a denatured labeled protein drug sample; diluting the denatured labeled protein drug sample and subjecting it to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram; identifying peaks in the electropherogram that correspond to contaminants or impurities; A method comprising: [The present invention 1015] 1015. The method of claim 1014, wherein the buffered protein drug sample is heated at 70° C. for 10 minutes. [The present invention 1016] 1016. The method of claim 1014 or 1015, wherein the sample is heated at 35° C. for 30 minutes. [The present invention 1017] 17. The method of any one of claims 1014 to 1016, wherein the diluted protein drug sample is 9 μg / ml. [The present invention 1018] 1018. The method of any one of claims 1010 to 1017, wherein said detectable label is DY-631 N-hydroxysuccinimidyl ester. [The present invention 1019] A kit comprising the buffer solution of any one of claims 1001 to 1009 and written instructions for preparing a sample for electrophoresis in said buffer solution. [The present invention 1020] 55-75 mM 2-iodoacetamide, 0.1 to 1.0% lithium dodecyl sulfate; 5 to 115 mM sodium chloride, 5 to 85 mM HEPES and Including, having a pH of less than 9, Aqueous electrophoresis sample buffer. [The present invention 1021] 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate; 48.6 mM NaCl, 16.2 mM HEPES and Including, having a pH of less than 9, Aqueous electrophoresis sample buffer. [The present invention 1022] 1020 or 1021, an aqueous buffer solution of the present invention, having a pH of 8. [The present invention 1023] 0.05 to 0.75% lithium dodecyl sulfate; 5mM to 115mM NaCl, 5mM to 85mM HEPES, 35-50mM dithiothreitol Including, having a pH greater than 7, Aqueous electrophoresis sample buffer. [The present invention 1024] 0.28% lithium dodecyl sulfate; 41.5 mM NaCl, 13.8 mM HEPES, 42.5 mM dithiothreitol and Including, having a pH greater than 7, Aqueous electrophoresis sample buffer. [The present invention 1025] 1023 or 1024, an aqueous buffer solution of the present invention, having a pH of 8. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1A shows an electropherogram of a typical non-reduced sample run. Figure 1B shows an electropherogram of a typical reduced sample run. The X-axis represents time in minutes, and the Y-axis represents relative fluorescence units (RFU). Increasing migration time corresponds to increasing protein size. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention I. Definition Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the claimed invention (particularly in the context of the claims), the use of "a," "an," "the," and similar referents should be construed to cover both the singular and the plural.

[0019] Unless otherwise stated herein, the recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein.

[0020] When the term "about" is used, it is intended to describe a value that is either about + / -10% above or below the stated value. In other embodiments, values ​​may range from about + / -5% above or below the stated value. In other embodiments, values ​​may range from about + / -2% above or below the stated value. In other embodiments, values ​​may range from about + / -1% above or below the stated value. The preceding ranges are intended to be clear by context, and no further limitations are implied. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended solely to clearly illustrate the invention and do not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as suggesting that any non-claimed element is essential to the practice of the invention.

[0021] "Protein" refers to a molecule containing two or more amino acid residues linked together via peptide bonds. Proteins include polypeptides and peptides and may further include modifications such as glycosylation, lipidation, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs. Representative proteins include enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins. Proteins are produced by various types of recombinant cells using well-known cell culture methods and are typically introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-less sequences, etc.), either episomally or integrated into the cell's genome.

[0022] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, linked together via disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions are further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" also includes antibody molecules prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from a host cell transfected for antibody expression. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to multiple, distinct epitopes. Bispecific antibodies are generally described in U.S. Pat. No. 8,586,713, which is incorporated herein by reference.

[0023] An "Fc fusion protein" comprises part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not otherwise found together in nature. Preparation of fusion proteins comprising specific heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, in Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88:10535 (1991); Byrn et al., Nature 344:677 (1990); and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11 (1992). A "receptor-Fc fusion protein" comprises one or more extracellular domains of a receptor linked to an Fc portion, which in some embodiments comprises the hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein comprises two or more different receptor chains that bind to one or more ligands, e.g., the Fc fusion protein is a trap, such as an IL-1 trap or a VEGF trap.

[0024] The term "MCE" or "microchip capillary electrophoresis" refers to the separation of analytes by capillary electrophoresis (CE) using a microchip.

[0025] II. MCE Assay and Buffers A method for analyzing analytes in protein drug samples is provided. Preferred protein drugs include, but are not limited to, antibodies and their antigen-binding fragments, fusion proteins, and recombinant proteins. The assay employs MCE technology to separate, identify, and quantify protein products and impurities in protein products. Impurities include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing and non-reducing buffers are also provided.

[0026] A. Buffer solution 1. Non-reducing buffer In one embodiment, a non-reducing aqueous electrophoresis sample buffer is provided that includes 155-175 mM alkylating agent, such as 2-iodoacetamide or NEM; 0.50-1.5% lithium dodecyl sulfate; and 75-95 mM sodium phosphate, where the aqueous electrophoresis sample buffer has a pH of less than 7. In a preferred embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer includes 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0027] 2. Reducing buffer A reducing buffer is also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer comprising 0.5-1.5% lithium dodecyl sulfate, 65-95 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 8. In a preferred embodiment, the pH of the buffer is 9.

[0028] Reducing agents are known in the art. Exemplary reducing agents include, but are not limited to, dithiothreitol (DTT, CAS 3483-12-3), β-mercaptoethanol (BME, 2BME, 2-ME, b-mer, CAS 60-24-2), 2-aminoethanethiol (2-MEA-HCl, also known as cysteamine-HCl, CAS 156-57-0), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, CAS 5961-85-3), cysteine ​​hydrochloride (Cys-HCl, CAS 52-89-1), or 2-mercaptoethanesulfonic acid sodium salt (MESNA). Other methods for reducing protein bonds are also known in the art, such as immobilized reduction columns, which contain a resin to which a thiol-based reducing agent is immobilized, allowing for solid-phase reduction of disulfide bonds in peptides and proteins. It is contemplated that reducing agents, including oxidizing agents, are suitable for reducing chemical interactions between polypeptides.

[0029] In one embodiment, the reducing buffer contains 135 to 155 mM dithiothreitol.

[0030] In yet another embodiment, a reducing buffer is provided that comprises 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

[0031] 3. Non-reducing buffer using HEPES HEPES-based buffers can also be used with the disclosed methods. In one embodiment, a non-reducing HEPES-based aqueous electrophoresis sample buffer is provided, comprising an alkylating agent, e.g., 55-75 mM 2-iodoacetamide; 0.1-1.0% lithium dodecyl sulfate; 5-85 mM HEPES; and 5-115 mM sodium chloride, wherein the aqueous electrophoresis sample buffer has a pH of less than 9. In a preferred embodiment, the buffer has a pH of 8. In another embodiment, the aqueous buffer comprises 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.

[0032] 4. Reducing buffer using HEPES In another embodiment, a reducing HEPES-based aqueous electrophoresis sample buffer is provided, comprising 0.05-0.75% lithium dodecyl sulfate, 5 mM-115 mM sodium chloride, 5 mM-115 mM HEPES, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 7. In a preferred embodiment, the pH of the buffer is 8. In one embodiment, the reducing buffer comprises 35-50 mM dithiothreitol. In yet another embodiment, a reducing buffer is provided, comprising 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.

[0033] B. Assay 1. Non-reducing Assay In one embodiment, a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample is provided, the method comprising adding the protein sample to a non-reducing buffer as discussed above to form a buffered protein drug sample. The buffered protein drug sample is heated to between 65°C and 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample. In a preferred embodiment, the buffered protein drug sample is heated to 70°C for 10 minutes. A detectable label is then added to the denatured buffered protein drug sample and heated at 30 to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample. In a preferred embodiment, the denatured protein drug sample with added label is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter.

[0034] Preferred detectable labels include, but are not limited to, DY-631 NHS ester from Dynomics, Inc. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, and the like, and those disclosed in U.S. Patent No. 6,924,372, which is incorporated by reference in its entirety.

[0035] The denatured, labeled protein drug product is diluted and subjected to MCE, and the diluted protein drug sample is separated on a microchip capillary electrophoresis system to generate an electropherogram. In one embodiment, the sample, initially injected onto the microchip at 0.5 mg / ml, has a final concentration of 9 μg / ml for MCE. In another embodiment, the starting sample concentration is 0.2 mg / ml. The electropherogram contains peaks corresponding to the protein drug product and impurities. The method is concluded by identifying peaks in the electropherogram that correspond to contaminants or impurities.

[0036] 2. Reducibility Assay In another embodiment, a reducing MCE method for identifying contaminants or impurities in a protein drug sample is provided. This method begins by adding the protein drug sample to one of the reducing buffers described above to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample to 65-85°C, preferably 70°C, for 10 minutes to form a denatured protein drug sample. The protein drug sample to which a label has been added is then heated at 30-40°C for 20-40 minutes to form a denatured, labeled protein drug sample. In a preferred embodiment, the labeled protein drug product sample is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter. Preferred detectable labels include, but are not limited to, DY-631 NHS ester (Dynomics). Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, and the like, and such as those disclosed in US Pat. No. 6,924,372, which is incorporated by reference in its entirety.

[0037] In one embodiment, the established assay range for sample concentration is 0.4 mg / ml to 0.6 mg / ml, corresponding to a final concentration being analyzed of approximately 7 μg / ml to 11 μg / ml, which is subjected to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram. The method is concluded by identifying peaks in the electropherogram that correspond to contaminants or impurities.

[0038] C. Measuring equipment Instrumentation for performing the MCE assay of the present disclosure is commercially available. In a preferred embodiment, the MCE assay of the present disclosure is performed using a LabChip GXII or LabChip GXII Touch HT, and a LabChip® HT Protein Express Chip.

[0039] III. Protein of Interest The proteins of interest, e.g., protein drug products, assayed using the disclosed MCE assays and reagents can be any protein of interest suitable for expression in prokaryotic or eukaryotic cells and can be used in the provided artificial host cell systems. For example, proteins of interest include, but are not limited to, antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, ScFvs or fragments thereof, Fc fusion proteins or fragments thereof, growth factors or fragments thereof, cytokines or fragments thereof, or the extracellular domain of cell surface receptors or fragments thereof. Proteins of interest can be simple polypeptides consisting of a single subunit or complex multi-subunit proteins containing two or more subunits. Proteins of interest can be biopharmaceuticals, food additives, or preservatives, or any protein product subject to purification and quality standards.

[0040] In some embodiments, the protein drug product (protein of interest) is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or a tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0041] In some embodiments, the antibody is an anti-programmed cell death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,018,356), an anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies described in U.S. Pat. No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies described in U.S. Pat. No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies described in U.S. Pat. No. 9,132,192 or anti-EGFRvIII antibodies described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-proprotein convertase antibodies, butyrilin kexin-9 antibodies (e.g., anti-PCSK9 antibodies described in U.S. Patent No. 8,062,640 or U.S. Patent No. 9,540,449), anti-growth differentiation factor 8 antibodies (e.g., anti-GDF8 antibodies (also known as anti-myostatin antibodies) described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibodies described in U.S. Patent Application Publication No. 2015 / 0337045A1 or U.S. Patent Application Publication No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., For example, anti-IL4R antibodies described in U.S. Patent Application Publication No. 2014 / 0271681A1, or U.S. Patent No. 8,735,095 or U.S. Patent No. 8,945,559), anti-interleukin 6 receptor antibodies (e.g., anti-IL6R antibodies described in U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., U.S. Patent Nos. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies described in U.S. Patent Application Publication No. 9,447,173), anti-cluster of differentiation 3 (e.g., anti-CD3 antibodies described in U.S. Patent Nos. 9,447,173 and 9,447,173, and U.S. Patent No. 62 / 222,605), anti-cluster of differentiation 20 (e.g., anti-CD20 antibodies described in U.S. Patent Nos. 9,657,102 and 20150266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of differentiation 48 (e.g., anti-CD48 antibodies described in U.S. Patent No. 9,228,014), anti-Fel d1 antibodies (e.g., antibodies described in U.S. Patent Application Publication No. 9,079,948), anti-Middle East respiratory syndrome virus (e.g., anti-MERS antibodies described in U.S. Patent Application Publication No. 2015 / 0337029A1), anti-Ebola virus antibodies (e.g., antibodies described in U.S. Patent Application Publication No. 2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibodies or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., antibodies described in U.S. Patent Application Publication No. 2016 / 0017029, and U.S. Patent Nos. 8,309,088 and 9,353,176), and anti-protein Y antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3x anti-CD20 bispecific antibody (as described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 20150266966A1), an anti-CD3x anti-mucin 16 bispecific antibody (e.g., an anti-CD3x anti-Muc16 bispecific antibody), and an anti-CD3x anti-prostate specific membrane antigen bispecific antibody (e.g., an anti-CD3x anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-ato, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodatumab, canakinumab, capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, entansinellirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselk ... mab, ibritumomab tiuxetan, idarucizumab, infliximab-abda, infliximab-dive, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesbacumab, nivolumab, obilutoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramuciltunab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevoglumab, ustekinumab, and vedolizumab. ,

[0042] In some embodiments, the protein of interest is a recombinant protein (such as an Fc fusion protein) comprising an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor-Fc fusion protein comprising one or more extracellular domains of a receptor linked to the Fc portion. In some embodiments, the Fc portion comprises a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor-Fc fusion protein comprises two or more separate receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g., rilonacept, which comprises an IL-1RacP ligand binding domain fused to the IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, the entire contents of which are incorporated herein by reference), or a VEGF trap (e.g., aflibercept or div-aflibercept, which comprises the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159). In other embodiments, the Fc fusion protein is an ScFv-Fc fusion protein, which comprises one or more antigen binding domains (such as the variable heavy and light chain fragments of an antibody bound to the Fc portion).

[0043] IV. Cell culture The protein drug product assayed using the disclosed MCE assay and reagents is a cell culture. The cell culture can be a "fed-batch cell culture" or "fed-batch culture," which refers to a batch culture. In this batch culture, cells and medium are first supplied to the culture vessel. Additional culture nutrients are slowly supplied to the culture in discrete increments during the culture. This nutrient supply is with or without periodic cell and / or product harvesting before the end of the culture. Fed-batch culture includes "semi-continuous fed-batch culture," in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple "batch culture," in which all components for cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the beginning of the culture process in the batch culture. Fed-batch culture can differ from "perfusion culture" insofar as the supernatant is not removed from the culture vessel during the standard fed-batch process. In perfusion culture, cells are confined in culture, for example by filtration, and culture medium is continuously or intermittently introduced and removed from the culture vessel. In contrast, during fed-batch cell culture, it is contemplated that samples may be removed for testing purposes. The fed-batch process is continued until a maximum working volume and / or protein production is determined to have been reached, and the protein is subsequently harvested.

[0044] Cell culture is sometimes referred to as "continuous cell culture." This cell culture is a technique used to continuously grow cells, usually at a particular growth stage. For example, maintaining a cell culture at a particular growth stage may be necessary when a constant supply of cells is required or when a particular protein of interest is to be produced. Therefore, maintaining cells at that particular stage requires that conditions be continuously monitored and adjusted accordingly.

[0045] Cells are cultured in cell culture media. The terms "cell culture medium" and "culture medium" refer to a nutrient solution used to grow mammalian cells. This nutrient solution typically provides the nutrients necessary to promote cell growth, such as carbohydrate energy sources, essential amino acids (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, histidine), non-essential amino acids (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine), trace elements, energy sources, lipids, vitamins, etc. Cell culture media may contain extracts, such as serum or peptone (hydrolysates), that provide raw materials to support cell growth. Instead of animal-derived extracts, media may contain yeast-derived or soybean extracts. Chemically defined media refer to cell culture media in which all chemical components are known (i.e., have known chemical structures). A chemically defined medium does not contain any animal-derived components, such as serum or animal-derived peptones. In one embodiment, the medium is a chemically defined medium.

[0046] The solution may also contain components (such as hormones and growth factors) that enhance growth and / or survival rates above minimum levels. The solution may be formulated to provide an optimal pH and salt concentration for the survival and proliferation of the particular cells being cultured.

[0047] A "cell line" refers to a cell or cells derived from a particular lineage through serial passage or subculture of the cells. The term "cell" is used interchangeably with "cell population."

[0048] The term "cell" encompasses any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells, such as bacterial cells, mammalian cells, human cells, non-human animal cells, avian cells, insect cells, yeast cells, or cell fusions, e.g., hybridomas and quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is selected from the following cells: Chinese hamster ovary (CHO) (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocytes such as Jurkat (T lymphocytes) or Daudi (B lymphocytes), A431 (epidermal), U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, one or more viral genes are contained within the cell, e.g., retinal cells (e.g., PER.C6® cells) expressing viral genes. In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0049] V. Kit In one embodiment, a kit for producing a buffer solution of the present disclosure is provided, comprising one or more of the buffer solutions or components of the present disclosure. The kit can include a container for the buffer solution or component. The buffer solution can be in solution or lyophilized form. The kit also optionally includes a second container containing a diluent or reconstitution solution for the lyophilized formulation; and optionally includes instructions for using the solution or reconstitution solution and / or the lyophilized buffer solution or powdered component.

[0050] The kit may further include reagents necessary to perform the MCE assay of the present disclosure, including one or more of buffers, diluents, and filters. The buffers and reagents may be in bottles, vials, or test tubes. [Example]

[0051] Example 1: MCE assay for therapeutic protein purity and impurity analysis Methods and Materials: material: For capillary electrophoresis separation and data collection, a LabChip GXII or LabChip GXII Touch HT and a LabChip® HT Protein Express Chip were used (Perkin Elmer). The non-reducing and reducing denaturing buffers disclosed above were used for the MCE assay.

[0052] method: Table 1 shows the workflow steps for preparing samples for the MCE assay. Briefly, protein samples were diluted to 0.5 mg / ml. 1 μl of non-reducing (NR) or reducing (R) denaturing buffer and 4 μl of diluted sample were added to a 96-well plate. The samples were mixed, centrifuged, and heated to the temperature specified in the manufacturer's instructions, typically 75°C for 10 minutes. The samples were then labeled with 5 μM of a commercially available dye (e.g., Dynomics DY-631 NHS ester). The samples were mixed, centrifuged, and then heated at 35°C for 30 minutes. The labeled samples were then diluted with 105 μl of stop solution. The samples were separated using a LabChip GXII or LabChip GXII Touch HT.

[0053] buffer solution Stock solutions of 200 mM sodium phosphate monobasic monohydrate, 200 mM sodium phosphate dibasic heptahydrate, and 10% lithium dodecyl sulfate (LDS) were prepared. Using this stock solution and Milli-Q® water, solutions of 100 mM sodium phosphate, 1% LDS, pH 6, and 100 mM sodium phosphate, 1% LDS, pH 9 were prepared.

[0054] A non-reducing buffer was prepared by adding 34 μL of 1 M iodoacetamide (IAM) (freshly prepared in Milli-Q® water) + 166 μL of 100 mM sodium phosphate, 1% LDS, pH 6 + 5 μL of Milli-Q® water. The final concentrations were 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0055] A reducing buffer was prepared by adding 68 μL of 10x reducing agent (500 mM dithiothreitol (DTT) + 166 μL of 100 mM sodium phosphate, pH 9 with 1% LDS + 6 μL of Milli-Q® water). The final concentrations were 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate; and 142 mM dithiothreitol.

[0056] Table 1. Sample preparation method for the MCE assay TIFF2025156526000001.tif70154

[0057] result Microchip capillary electrophoresis (MCE) allows for dramatic reductions in sample analysis time while maintaining the performance and reproducibility standards required for QC analysis. MCE assays were developed using the non-reduced and reduced denaturing buffers disclosed herein. Figures 1A-1B show representative electropherograms illustrating the analysis of proteins in non-reduced and reduced samples.

[0058] While the foregoing specification has described the invention in connection with certain specific embodiments thereof, and has set forth numerous details for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that the specific details described herein can be varied considerably without departing from the underlying principles of the invention.

[0059] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes. Accordingly, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention.

Claims

1. 155-175 mM 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; 75-95 mM sodium phosphate and Including, having a pH of less than 7; Aqueous electrophoresis sample buffer.

2. 2. The aqueous buffer solution of claim 1, wherein the pH is 6.

3. 3. The aqueous buffer solution of claim 1, comprising 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

4. 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate; 81 mM sodium phosphate and It consists of having a pH of 6.0, Aqueous electrophoresis sample buffer.

5. 0.50 to 1.5% lithium dodecyl sulfate; 45-75 mM sodium phosphate; Reducing agent and Including, having a pH greater than 8; Aqueous electrophoresis sample buffer.

6. 6. The aqueous buffer solution of claim 5, having a pH of 9.

7. 7. The aqueous buffer solution according to claim 5, comprising 135 to 155 mM dithiothreitol.

8. 8. An aqueous buffer solution according to any one of claims 5 to 7, comprising 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

9. 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate; 142 mM dithiothreitol and It consists of having a pH of 9.0, Aqueous electrophoresis sample buffer.

10. 1. A method for identifying contaminants or impurities in a protein drug sample, comprising: adding the protein drug sample to a buffer solution according to any one of claims 1 to 4 to form a buffered protein drug sample; heating the buffered protein drug sample to 65-85°C for 5-15 minutes to form a denatured buffered protein drug sample; adding a detectable label to the denatured, buffered protein drug sample and heating at 30-40°C for 20-40 minutes to form a denatured, labeled protein drug sample; diluting the denatured labeled protein drug sample and subjecting it to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system and generate an electropherogram; identifying peaks in the electropherogram that correspond to contaminants or impurities; A method comprising:

11. 11. The method of claim 10, wherein the buffered protein drug sample is heated at 70°C for 10 minutes.

12. 12. The method of claim 10 or 11, wherein the labeled protein drug sample is heated at 35°C for 30 minutes.

13. The method of any one of claims 10 to 12, wherein the diluted protein drug sample is 9 mg / ml.

14. 1. A method for identifying contaminants or impurities in a protein drug sample, comprising: adding the protein sample to a buffer solution according to any one of claims 5 to 9 to form a buffered protein drug sample; heating the buffered protein drug sample to 65-85°C for 5-15 minutes to form a denatured protein drug sample; adding a detectable label to the denatured protein drug sample and heating at 30-40°C for 20-40 minutes to form a denatured labeled protein drug sample; diluting the denatured labeled protein drug sample and subjecting it to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram; identifying peaks in the electropherogram that correspond to contaminants or impurities; A method comprising:

15. 15. The method of claim 14, wherein the buffered protein drug sample is heated at 70°C for 10 minutes.

16. 16. The method of claim 14 or 15, wherein the sample is heated at 35°C for 30 minutes.

17. The method of any one of claims 14 to 16, wherein the diluted protein drug sample is 9 μg / ml.

18. The method of any one of claims 10 to 17, wherein the detectable label is DY-631 N-hydroxysuccinimidyl ester.

19. A kit comprising a buffer solution according to any one of claims 1 to 9 and written instructions for preparing samples for electrophoresis in said buffer solution.

20. 55-75 mM 2-iodoacetamide; 0.1 to 1.0% lithium dodecyl sulfate; 5 to 115 mM sodium chloride; 5 to 85 mM HEPES and Including, having a pH of less than 9; Aqueous electrophoresis sample buffer.

21. 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate; 48.6 mM NaCl, 16.2 mM HEPES and Including, having a pH of less than 9; Aqueous electrophoresis sample buffer.

22. 22. An aqueous buffer solution according to claim 20 or 21, having a pH of 8.

23. 0.05 to 0.75% lithium dodecyl sulfate; 5 mM to 115 mM NaCl; 5 mM to 85 mM HEPES; 35-50 mM dithiothreitol and Including, having a pH greater than 7; Aqueous electrophoresis sample buffer.

24. 0.28% lithium dodecyl sulfate; 41.5 mM NaCl, 13.8 mM HEPES, 42.5 mM dithiothreitol and Including, having a pH greater than 7; Aqueous electrophoresis sample buffer.

25. 25. An aqueous buffer solution according to claim 23 or 24, having a pH of 8.

Images