Microchip Capillary Electrophoresis Assays and Reagents

Abstract

Microchip capillary electrophoresis (MCE) assays and reagents are provided for assessing the purity of and identifying impurities in protein drug product samples. A method is provided for analyzing analytes in a protein drug sample.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. application Ser. No. 17 / 335,756, filed June 1, 2021, which is a continuation-in-part of U.S. application Ser. No. 16 / 355,050, filed March 15, 2019, which claims the benefit of and priority to U.S. Provisional Application No. 62 / 644,933, filed March 19, 2018. This application is also a continuation-in-part of U.S. application Ser. No. 16 / 355,050, filed March 15, 2019, which claims the benefit of and priority to U.S. Provisional Application No. 62 / 644,933, filed March 19, 2018. Each of these applications is incorporated by reference in its entirety.

[0002] Aspects of the present invention are directed generally to the field of capillary electrophoresis, and in particular to microchip capillary electrophoresis. [Background technology]

[0003] To meet the testing demands of biological products in current quality control (QC) laboratories, the implementation of robust, reproducible, and user-friendly technologies is critical. Technology upgrades are necessary to facilitate increased production volumes while continually generating high-quality analytical data and minimizing the number of invalid test results and equipment-related investigations. Electrophoresis has historically been used in QC for product purity and fragmentation analysis, but the technique has transitioned from gel-based to capillary-based and more recently to microchips. Microchip capillary electrophoresis (MCE) allows for dramatic reductions in 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)).

[0004] 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 it can be prone to assay interference.

[0005] It is therefore an object of the present invention to provide improved MCE assays and compositions that reduce assay interference.

[0006] Another object of the present invention is to provide MCE assays and compositions for improved detection of impurities in protein drug products. Summary of the Invention

[0007] MCE assays and reagents are provided for assessing purity and identifying impurities in protein drug product samples. Methods are provided for analyzing analytes in protein drug samples. Preferred protein drugs include, but are not limited to, recombinant proteins such as antibodies and their antigen-binding fragments, and fusion proteins. The assay uses 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. In some embodiments, the MCE assays and reagents provided herein can be used in the analysis and purification testing of anti-SARS-CoV-2 products, such as therapeutic protein products, including REGEN-COV™ (casirivimab and imdevimab).

[0008] One embodiment provides a non-reducing aqueous electrophoresis sample buffer containing an alkylating agent such as 2-iodoacetamide (IAM), iodoacetic acid (IAA), or N-ethylmaleimide (NEM). In one embodiment, the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, 155-175 mM 2-iodoacetamide, 0.50-1.5% lithium dodecyl sulfate, and 65-95 mM sodium phosphate, and the aqueous electrophoresis sample buffer has a pH less than 7. In one embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer contains 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0009] In another embodiment, the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, for example, 50-250 or 155-200 or 155-250 mM 2-iodoacetamide, 0.50-1.5% lithium dodecyl sulfate, and 40-80 mM or 50-70 mM sodium phosphate, and the aqueous electrophoresis sample buffer has a pH of 8 or less. In one embodiment, the pH of the buffer is 8. In yet another embodiment, the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0010] In another embodiment, the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, such as 155-200 mM 2-iodoacetamide, 0.50-1.5% lithium dodecyl sulfate, and 50-70 mM sodium phosphate, and the aqueous electrophoresis sample buffer has a pH of 8 or less. In one embodiment, the pH of the buffer is 6. In yet another embodiment, the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0011] A reducing buffer is also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5-1.5% lithium dodecyl sulfate, 55-85 mM sodium phosphate, and a reducing agent, the aqueous electrophoresis sample buffer having a pH greater than 8. In one embodiment, the pH of the buffer is 9. In one embodiment, the reducing buffer contains 135-155 mM dithiothreitol. Yet another embodiment provides a reducing buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

[0012] In another embodiment, the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5-1.5% lithium dodecyl sulfate, 45-85 mM sodium phosphate, and a reducing agent, and the aqueous electrophoresis sample buffer has a pH of 8 or greater. In one embodiment, the pH of the buffer is 8. In one embodiment, the reducing buffer contains 80-155 mM dithiothreitol. Yet another embodiment provides a reducing buffer containing 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol.

[0013] HEPES-based buffers may also be used with the disclosed methods. One embodiment provides a non-reducing HEPES-based aqueous electrophoresis sample buffer containing an alkylating agent, such as 55-75 mM 2-iodoacetamide, 0.1-1.0% lithium dodecyl sulfate, 5-85 mM HEPES, and 5-115 mM sodium chloride, the aqueous electrophoresis sample buffer having a pH less than 9. In another embodiment, the pH of the buffer is 8. In yet another embodiment, the aqueous buffer contains 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.

[0014] Another embodiment provides a reducing HEPES-based aqueous electrophoresis sample buffer containing 0.05-0.75% lithium dodecyl sulfate, 5 mM-115 mM sodium chloride, 5 mM-115 mM HEPES, and a reducing agent, the aqueous electrophoresis sample buffer having a pH greater than 7. In one embodiment, the pH of the buffer is 8. In one embodiment, the reducing buffer contains 35-50 mM dithiothreitol. Yet another embodiment provides a reducing buffer containing 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.

[0015] One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample, comprising adding the protein sample to a non-reducing buffer as described above to form a buffered protein drug sample. The buffered protein drug sample is heated at 65-85°C for 5-15 minutes to form a denatured buffered protein drug sample. In one embodiment, the buffered protein drug sample is heated at 70°C for 10 minutes.

[0016] In another embodiment, the non-reducing MCE method for identifying contaminants or impurities in a protein drug sample includes adding the protein sample to a non-reducing buffer as described above to form a buffered protein drug sample. The buffered protein drug sample is heated at 50-72°C for 5-15 minutes to form a denatured buffered protein drug sample. Alternatively, the buffered protein drug sample is heated between 45-75°C for 5-15 minutes to form a denatured buffered protein drug sample. In one embodiment, the buffered protein drug sample is heated at 60°C-65°C for 10 minutes. In another embodiment, the buffered protein drug sample is heated at 63°C for 10 minutes. In another embodiment, the buffered protein drug sample is heated at 60°C for 10 minutes.

[0017] In some embodiments, the protein drug sample is mixed with a detectable label and heated at 30-40°C for 20-40 minutes or 10-40 minutes to form a denatured labeled protein drug sample. In other embodiments, the protein drug sample is mixed with a detectable label and heated at 30-40°C for 15 minutes to form a denatured labeled protein drug sample. Detectable labels include, but are not limited to, Dyomics DY-631 NHS ester. In some embodiments, the label is diluted with MilliQ purified water, and in other embodiments, the label is diluted in sodium phosphate buffer. In some examples, the detectable label is reconstituted with dimethyl sulfoxide (DMSO) and subsequently diluted with 200 mM sodium phosphate pH 7.2 buffer. Other detectable labels can be used, including other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., and those disclosed in U.S. Patent No. 6,924,372. In one embodiment, the protein drug sample to which the label has been added is heated at 35°C for 30 minutes. In another embodiment, the protein drug sample with added label is heated at 35° C. for 15 minutes. Excess label is removed from the sample, if necessary, for example, by using a spin filter. In yet another embodiment, excess label is not quenched. In yet another embodiment, excess label is not removed.

[0018] The denatured labeled protein drug product is diluted and subjected to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system to obtain an electropherogram. In one embodiment, the final concentration of the sample, starting at 0.5 mg / ml, then injected onto the microchip is 9 μg / ml for MCE. In another embodiment, the final concentration of the sample, starting at 0.2 mg / ml, then injected onto the microchip is 3.6 μg / ml for MCE. The electropherogram includes peaks corresponding to the protein drug product and impurities. The method ends by identifying peaks in the electropherogram that correspond to the protein drug product, contaminants, and / or impurities.

[0019] Another embodiment provides a reduction MCE method for identifying contaminants or impurities in a protein drug sample. The method begins by adding a protein sample to any one of the reduction buffers described above to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample at 65-85°C, preferably 75°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 one embodiment, the protein drug sample with the added label is heated at 35°C for 30 minutes.

[0020] In another embodiment, the buffered protein drug sample is denatured by heating the buffered protein drug sample at 50-72°C, preferably 60°C, for 10 minutes to form a denatured protein drug sample. For example, the protein drug sample is mixed with a detectable label and heated at 30-40°C for 15 minutes to form a denatured, labeled protein drug sample. In another embodiment, the protein drug sample with the added label is heated at 35°C for 15 minutes.

[0021] Excess label is removed from the sample as needed, for example, by using a spin filter. In yet another embodiment, excess label is not quenched. In yet another embodiment, excess label is not removed. Exemplary detectable labels include, but are not limited to, Dyomics DY-631 NHS ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., and those disclosed in U.S. Patent No. 6,924,372.

[0022] In one embodiment, the established assay range for the sample concentration is 0.4 mg / ml to 0.6 mg / ml, which corresponds to a final analyzed concentration of about 7 μg / ml to 11 μg / ml, which is subjected to MCE analysis in a microchip capillary electrophoresis system to generate an electropherogram. In another embodiment, the established assay range for the sample concentration is 0.2 mg / ml to 0.6 mg / ml, which corresponds to a final analyzed concentration of about 3.6 μg / ml to 11 μg / ml. The method concludes by identifying peaks in the electropherogram that correspond to the protein drug product, contaminants, and / or impurities. [Brief description of the drawings]

[0023] [Figure 1A] Electropherograms of a typical non-reduced sample analysis are shown.

[0024] [Figure 1B] Electropherograms of a typical reduced sample analysis are shown. The X-axis represents time (min) and the Y-axis represents relative fluorescence units (RFU). Increasing migration time corresponds to increasing protein size. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] I. Definition In the context of describing the present invention (particularly in the context of the claims), the use of the terms "a," "an," "the," and similar referents are to be construed to encompass both the singular and the plural, unless otherwise indicated in the specification or clearly contradicted by context.

[0026] The recitation of ranges of values ​​herein, unless otherwise indicated 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.

[0027] Use of the term "about" is intended to describe values ​​above or below the stated value by about + / -10%, in other embodiments, values ​​may range anywhere above or below the stated value by about + / -5%, in other embodiments, values ​​may range anywhere above or below the stated value by about + / -2%, and in other embodiments, values ​​may range anywhere above or below the stated value by about + / -1%. The foregoing ranges are intended to be made clear by the context, and no further limitations are implied. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "to etc.") provided herein is intended merely to better clarify the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0028] "Protein" refers to a molecule that contains two or more amino acid residues linked together by peptide bonds. Proteins include polypeptides and peptides, and may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs, and include enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins, among others. Proteins are produced by various types of recombinant cells using well-known cell culture methods, and are generally introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-free sequences, etc.), where the protein may be present as an episome or integrated into the genome of the cell.

[0029] "Antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by 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 can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus 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" includes antibody molecules prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes bispecific antibodies, including heterotetrameric immunoglobulins capable of binding to two or more different epitopes. Bispecific antibodies are generally described in U.S. Patent No. 8,586,713.

[0030] 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. The preparation of fusion proteins comprising certain 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, and in some embodiments, the hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein comprises two or more distinct receptor chains that bind to one or more ligands, for example, the Fc fusion protein is a trap, such as, for example, an IL-1 trap or a VEGF trap.

[0031] The term "MCE" or "microchip capillary electrophoresis" refers to microchip-based capillary electrophoresis (CE) separation of analytes.

[0032] II. MCE Assay and Buffers A method is provided for analyzing analytes in protein drug samples. Protein drugs include, but are not limited to, antibodies and their antigen-binding fragments, fusion proteins, and recombinant proteins. The assay uses 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.

[0033] Microchip capillary electrophoresis (MCE) provides analysis of protein purity and impurities suitable for in-process testing, lot release, and stability indication. Protein samples such as casirivimab and imdevimab were prepared by denaturing the sample by heating and adding lithium dodecyl sulfate (LDS) with either an alkylating reagent (non-reducing) or a reducing reagent (reducing). Without intending to be bound by theory, this produces a linear, negatively charged protein-LDS polypeptide chain, which is then incubated with an amine ester dye to covalently label any free amine groups. The labeled sample can be injected onto a microchip, where application of an electric current separates the sample components according to mass-to-charge ratio. Detection can be done by laser-induced fluorescence, which produces an electropherogram. Analysis of the electropherogram provides the relative purity of the sample compared to the impurities present.

[0034] A. Buffer 1. Non-reducing buffer One embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155-200 mM of an alkylating agent, such as 2-iodoacetamide, 0.50-1.5% lithium dodecyl sulfate, and 60-95 mM sodium phosphate, the aqueous electrophoresis sample buffer having a pH less than 7. In one embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer contains 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0035] Another embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155-200 mM alkylating agent, 0.50-1.5% lithium dodecyl sulfate, and 60-95 mM sodium phosphate, and having a pH of less than or equal to 8. In another embodiment, the pH of the buffer is 8. In another embodiment, the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0036] Another embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155-200 mM alkylating agent, 0.50-1.5% lithium dodecyl sulfate, and 60-95 mM sodium phosphate, and having a pH less than 8. In another embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0037] 2. Reducing Buffer A reducing buffer is also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5-1.5% lithium dodecyl sulfate, 65-95 mM sodium phosphate, and a reducing agent, the aqueous electrophoresis sample buffer having a pH greater than 8. In another embodiment, the reducing buffer contains 0.5-1.5% lithium dodecyl sulfate, 45-95 mM sodium phosphate, and a reducing agent, the aqueous electrophoresis sample buffer having a pH greater than or equal to 8. In one embodiment, the pH of the buffer is 9. In another embodiment, the pH of the buffer is 8.

[0038] 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, 2 BME, 2-ME, b-mer, CAS 60-24-2), 2-aminoethanethiol (2-MEA-HCl, also called 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 known in the art, such as immobilized reducing agent columns, which contain resins on which thiol-based reducing agents are immobilized to allow for solid-phase reduction of peptide and protein disulfide bonds. Additional reducing agents suitable for cleaving disulfide bonds are also contemplated.

[0039] In one embodiment, the reduction buffer contains 135 to 155 mM dithiothreitol. In another embodiment, the reduction buffer contains 80 to 155 mM dithiothreitol.

[0040] Yet another embodiment provides a reducing buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol, or 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol.

[0041] 3. HEPES-based non-reducing buffer HEPES-based buffers may also be used with the disclosed methods. One embodiment provides a non-reducing HEPES-based aqueous electrophoresis sample buffer containing an alkylating agent, such as 55-75 mM 2-iodoacetamide, 0.1-1.0% lithium dodecyl sulfate, 5-85 mM HEPES, and 5-115 mM sodium chloride, and having a pH of less than 9. In one embodiment, the pH of the buffer is 8. In another embodiment, the aqueous buffer contains 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.

[0042] 4. HEPES-based reducing buffer Another embodiment provides a reducing HEPES-based aqueous electrophoresis sample buffer containing 0.05-0.75% lithium dodecyl sulfate, 5 mM-115 mM sodium chloride, 5 mM-115 mM HEPES, and a reducing agent, the aqueous electrophoresis sample buffer having a pH greater than 7. In one embodiment, the pH of the buffer is 8. In one embodiment, the reducing buffer contains 35-50 mM dithiothreitol. Yet another embodiment provides a reducing buffer containing 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.

[0043] B. Assay 1. Non-reduced Assay One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample, the method comprising adding a protein sample to a non-reducing buffer as described above to form a buffered protein drug sample. The buffered protein drug sample is heated at 50-85°C for 5-15 minutes to form a denatured buffered protein drug sample. In one embodiment, the buffered protein drug sample is heated at 75°C for 10 minutes. In another embodiment, the buffered protein drug sample is heated between 45-75°C for 5-15 minutes to form a denatured buffered protein drug sample. In another preferred embodiment, the buffered protein drug sample is heated at 60°C for 10 minutes. In yet another preferred embodiment, the buffered protein drug sample is heated at 63°C for 10 minutes.

[0044] The detectable label is prepared in dimethyl sulfoxide (DMSO) according to the supplier's recommendations. Subsequent dilutions are prepared in MilliQ according to the supplier's recommendations. In other embodiments, the label is prepared by dilution in 200 mM sodium phosphate pH 7.2. In some embodiments, the label is diluted to a 5 μM solution. In other embodiments, the label is diluted to a 16 μM solution. The diluted label is then added to the denatured buffered protein drug sample and heated at 30-40° C. for 10-40 minutes to form the denatured labeled protein drug sample. In another embodiment, the detectable label is then added to the denatured buffered protein drug sample and heated at 30-40° C. for 15 minutes to form the denatured labeled protein drug sample. In one embodiment, the denatured protein drug sample with added label is heated at 35° C. for 30 minutes. In another preferred embodiment, the denatured protein drug sample with added label is heated at 35° C. for 15 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter. In yet another embodiment, the excess label is not quenched, hi yet another embodiment, the excess label is not removed.

[0045] Preferred detectable labels include, but are not limited to, Dyomics DY-631 NHS ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., and those disclosed in U.S. Patent No. 6,924,372.

[0046] The denatured labeled protein drug product is diluted and subjected to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system to obtain an electropherogram. In one embodiment, the final concentration of the sample, starting at 0.5 mg / ml, then injected onto the microchip is 9 μg / ml for MCE. In another embodiment, the sample starting concentration is 0.2 mg / ml. The electropherogram contains peaks corresponding to the protein drug product and impurities. The method ends by identifying peaks in the electropherogram that correspond to contaminants or impurities.

[0047] 2. Reduction Assay Another embodiment provides a reduction MCE method for identifying contaminants or impurities in a protein drug sample. The method begins by adding the protein drug sample to any one of the reduction buffers described above to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample at 65-85°C, preferably 75°C, for 10 minutes to form a denatured protein drug sample. In another embodiment, the buffered protein drug sample is denatured by heating the buffered protein drug sample at 50-72°C, or 45-75°C, or preferably 60°C, for 10 minutes to form a denatured protein drug sample. In yet another preferred embodiment, the buffered protein drug sample is heated at 63°C for 10 minutes. The temperature required to denature the target protein drug sample can vary depending on the structure of the target protein drug sample.

[0048] The label-added protein drug sample is then heated at 30-40°C for 20-40 minutes or 10-20 minutes or 10-40 minutes to form a denatured, labeled protein drug sample. In one embodiment, the label-added protein drug product sample is heated at 35°C for 30 minutes. In another preferred embodiment, the label-added protein drug product sample is heated at 35°C for 15 minutes. Excess label is optionally removed from the sample, for example, by using a spin filter. In yet another embodiment, excess label is not quenched. In yet another embodiment, excess label is not removed. Preferred detectable labels include, but are not limited to, Dyomics DY-631 NHS ester. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., and those disclosed in U.S. Patent No. 6,924,372.

[0049] In one embodiment, the established assay range for the sample concentration is 0.4 mg / ml to 0.6 mg / ml, which corresponds to a final analyzed concentration of about 7 μg / ml to 11 μg / ml, which is subjected to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram. In another embodiment, the established assay range for the sample concentration is 0.2 mg / ml to 0.6 mg / ml, which corresponds to a final analyzed concentration of about 3.6 μg / ml to 11 μg / ml, which is subjected to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram. The method concludes by identifying peaks in the electropherogram that correspond to the protein drug product, contaminants, and / or impurities.

[0050] C. Instruments Instrumentation for performing the disclosed MCE assay is commercially available. In one embodiment, the disclosed MCE assay is performed using a LabChip GXII or LabChip GXII Touch HT and a LabChip® HT Protein Express Chip.

[0051] III. Protein of Interest The protein of interest, e.g., a protein drug product, 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 engineered host cell system provided. For example, the protein of interest can include, but is not limited to, an antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, an ScFv or a fragment thereof, an Fc fusion protein or a fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, or an extracellular domain of a cell surface receptor or a fragment thereof. The protein of interest can be a simple polypeptide consisting of a single subunit, or a complex multi-subunit protein containing two or more subunits. The protein of interest can be a biopharmaceutical product, a food additive or preservative, or any protein drug product that is subject to purification and quality standards.

[0052] 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.

[0053] Embodiments can be used with any known therapeutic antibody therapeutic. 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-hemophilic antibody (e.g., the anti-angiopoietin-2 antibody described in U.S. Patent No. 9,018,356), an anti-angiopoietin-like antibody (e.g., the anti-angptl3 antibody described in U.S. Patent No. 9,018,356), an anti-hemophilic antibody (e.g., the anti-hemophilic ... No. 9,302,015), anti-complement 5 antibodies (e.g., the anti-C5 antibody described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., the anti-EGFR antibody described in U.S. Patent Application Publication No. 9,132,192 or the anti-EGFR antibody described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-EGFRvIII antibodies), anti-proprotein convertase subtilisin kexin-9 antibodies (e.g., anti-PCSK9 antibodies described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449), anti-growth differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Pat. No. 8,871,209 or U.S. Pat. No. 9,260,515), anti-glucagon receptor (e.g., anti-GCG antibodies described in U.S. Patent Application Publication No. 2015 / 0337045A1 or U.S. Patent Application Publication No. 2016 / 0075778A1), anti-proliferation differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Patent Application Publication No. 2015 / 0337045A1 or U.S. Patent Application Publication No. 2016 / 0075778A1), anti-proliferation differentiation factor-8 antibodies (e.g., anti-GCG antibodies, also known as anti-GCG ...GDF8 antibodies, also known as anti-myostatin antibodies, as described R antibody), anti-VEGF antibody, anti-IL1R antibody, interleukin 4 receptor antibody (e.g., anti-IL4R antibody 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 antibody (e.g., anti-IL6R antibody described in U.S. Patent No. 7,582,298, U.S. Patent No. 8,043,617 or U.S. Patent No. 9,173,880), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody,Anti-interleukin 33 (e.g., anti-IL33 antibodies described in U.S. Pat. No. 9,453,072 or U.S. Pat. No. 9,637,535), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies described in U.S. Pat. App. No. 9,447,173), anti-group 3 (e.g., anti-CD3 antibodies described in U.S. Pat. Nos. 9,447,173 and 9,447,173, and U.S. Patent Application No. 62 / 222,605), anti-group 20 (e.g., anti-CD20 antibodies described in U.S. Pat. Nos. 9,657,102 and US2015 / 0266966A1, and U.S. Pat. No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-group 48 (e.g., anti-CD48 antibodies described in U.S. Pat. No. 9,228,014), anti-Fel The antibody may be selected from the group consisting of a d1 antibody (e.g., as described in U.S. Pat. No. 9,079,948), an anti-Middle East Respiratory Syndrome virus antibody (e.g., an anti-MERS antibody described in U.S. Pat. App. Pub. No. 2015 / 0337029A1), an anti-Ebola virus antibody (e.g., as described in U.S. Pat. App. Pub. No. 2016 / 0215040), an anti-Zika virus antibody, an anti-lymphocyte activation gene 3 antibody (e.g., an anti-LAG3 antibody, or an anti-CD223 antibody), an anti-nerve growth factor antibody (e.g., an anti-NGF antibody described in U.S. Pat. App. Pub. No. 2016 / 0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176), and an anti-protein Y antibody. In some embodiments, the bispecific antibody may be selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1), an anti-CD3 x anti-mucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Muc16 bispecific antibody), and an anti-CD3 x anti-prostate specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of casirivimab, imdevimab, ravulizumab-cwvz, abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab,belimumab、benralizumab、bevacizumab、bezlotoxumab、blinatumomab、brentuximab vedotin、brodalumab、canakinumab、capromab pendetide、certolizumab pegol、cemiplimab、cetuximab、denosumab、dinutuximab、dupilumab、durvalumab、eculizumab、elotuzumab、emic izumab-kxwh、emtansinealirocumab、evinacumab、fasinumab、golimumab、guselkumab、ibritumab tiuxetan、idarucizumab、infliximab、infliximab-abda、infliximab-dyyb、ipilimumab、xekizumab、mepolizumab、necitumum ab、nesvacumab、nivolumab、obiltoxaximab、obinutuzumab、ocrelizumab、ofatumumab、omalizumab、panitumumab、 pembrolizumab、pertuzumab、ramucirumab、ranibizumab、raxibacumab、reslizumab、rinucumab、rituximab、sarilumab、secuki namab、siltuximab、tocilizumab、tocilizumab、trastuzumab、t revogrumab, or vedolizumab, have been reported in the literature.

[0054] In some embodiments, the protein of interest is a recombinant protein (e.g., an Fc fusion protein) containing an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein that contains one or more extracellular domains of a receptor coupled to an 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 contains two or more different receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein such as IL-1 TRAP (e.g., rilonacept, which contains the IL-1RAcP ligand binding domain fused to the Il-1R1 extracellular domain fused to the Fc of hIgG1, see U.S. Patent No. 6,927,044), or VEGF TRAP (e.g., aflibercept or ziv-aflibercept, which contains 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 a scFv-Fc fusion protein, which contains one or more of the antigen binding domains (e.g., variable heavy and variable light fragments) of an antibody bound to the Fc portion.

[0055] IV. Cell culture The protein drug product assayed using the disclosed MCE assays and reagents is the cell culture produced. The cell culture may be a "fed-batch cell culture" or "fed-batch culture", which refers to a batch culture, in which the cells and culture medium are initially fed into the culture vessel, and additional culture nutrients are slowly fed in discrete increments to the culture during the culture, 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 by fresh medium. Fed-batch culture is distinct from simple "batch culture", in which all components for cell culture (including animal cells and all culture nutrients) are fed into the culture vessel at the beginning of the culture process in batch culture. Fed-batch culture may differ from "perfusion culture" insofar as the supernatant is not removed from the culture vessel during the standard fed-batch process, in which the cells are confined in the culture, for example, by filtration, and the culture medium is continuously or intermittently introduced and removed from the culture vessel. However, removal of samples for testing during fed-batch cell culture is contemplated. The fed-batch process continues until it is determined that maximum working volume and / or protein production has been reached, after which the protein is harvested.

[0056] The cell culture may be a "continuous cell culture", which is a technique used to continuously grow cells, usually in a particular growth phase. For example, a cell culture may require maintenance in a particular growth phase when a constant supply of cells is required or when production of a particular protein of interest is required. Therefore, to maintain the cells in that particular phase, conditions must be continuously monitored and adjusted accordingly.

[0057] 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, typically providing nutrients necessary to enhance cell growth, such as carbohydrate energy sources, essential amino acids (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and non-essential amino acids (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine), trace elements, energy sources, lipids, vitamins, and the like. Cell culture media may contain extracts, such as serum or peptones (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 refers to cell culture media in which all chemical components are known (i.e., have known chemical structures). A chemically defined medium is completely free of animal-derived components such as serum or animal-derived peptones, hi one embodiment the medium is a chemically defined medium.

[0058] The solution may also contain components that enhance growth and / or survival beyond a minimum rate, including hormones and growth factors. The solution may be formulated to an optimal pH and salt concentration for survival and growth of the particular cells being cultured.

[0059] "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."

[0060] The term "cell" includes 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 or quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell may be 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 aforementioned cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) that expresses a viral gene. In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0061] V.Kit One embodiment provides a kit that includes one or more of the disclosed buffers or components for making the disclosed buffers. The kit can include a container for the buffer or components. The buffer can be in solution or lyophilized form. The kit also optionally includes a second container that includes a diluent or reconstitution solution for lyophilized formulations, and optionally includes instructions for use or reconstitution of the solution and / or use of the lyophilized buffer or powdered components.

[0062] The kit may further include additional reagents required to perform the disclosed MCE assay, including one or more of buffers, diluents, and filters. The buffers and reagents may be present in bottles, vials, or test tubes.

[0063] The following examples are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0064] Example 1: MCE assay for therapeutic protein purity and impurity analysis. Methods and Materials: material: LabChip GXII or LabChip GXII Touch HT and LabChip® HT Protein Express Chip were used for capillary electrophoresis separation and data collection (Perkin Elmer). The non-reducing and reducing denaturing buffers disclosed above were used for the MCE assay.

[0065] method: Microchip capillary electrophoresis (MCE) provides analysis of protein purity and impurities suitable for in-process testing, lot release, and stability indication. Protein samples of casirivimab and imdevimab were prepared by denaturing the samples by heating and adding lithium dodecyl sulfate (LDS) with either an alkylating agent (non-reducing) or a reducing agent (reducing). The resulting samples were then incubated with amine ester dyes to covalently label any free amine groups. The labeled samples were injected onto a microchip where a current was applied to separate the sample components according to size (low to high molecular weight). Detection was performed by laser-induced fluorescence and electropherograms were generated. Analysis of the electropherograms provided the relative purity of the samples compared to the impurities present.

[0066] Table 1 shows the workflow steps for preparing samples for the first MCE assay. Briefly, protein samples were diluted to 0.5 mg / ml. 1 μl of either non-reducing (NR) or reducing (R) denaturation buffer and 4 μl of diluted sample were added to a 96-well plate. Samples were mixed, centrifuged, and heated to the temperature specified for the product, typically 75° C. for 10 minutes. Samples were then labeled with 5 μM of a commercially available dye (e.g., Dyomics DY-631 NHS Ester, also referred to as PICO dye). Samples were mixed, centrifuged, and then heated to 35° C. for 30 minutes. The labeled samples were then diluted with 105 μl of stop solution. Samples were separated using a LabChip GXII or LabChip GXII Touch HT. [Table 1]

[0067] Table 2 shows the workflow steps for preparing samples for the second MCE assay. Briefly, protein samples were diluted to 0.5 or 2 mg / ml. 10 μl of either non-reducing (NR) or reducing (R) denaturing buffer and 40 μl of diluted sample were added to a 96-well plate. Samples were mixed, centrifuged, and heated to the temperature specified for the product, typically 60° C., for 10 minutes. Samples were then labeled with 16 μM of a commercially available dye (e.g., Dyomics DY-631 NHS ester). Samples were mixed, centrifuged, and then heated to 35° C. for 15 minutes. The labeled samples were then diluted with 105 μl of stop solution or diluent solution. Samples were separated using a LabChip GXII or LabChip GXII Touch HT.

[0068] Example 1A: Non-reducing buffer The sample preparation step followed the general preparation shown in Table 1 using a non-reducing aqueous electrophoresis sample buffer containing 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate, pH 6. Samples were heated at 75°C for 10 minutes. The sample labeling step used 5 μM dye solution. The final dilution step used 105 μL of stop solution.

[0069] Example 1B: Reduction Buffer The sample preparation step followed the general preparation shown in Table 1 using reducing aqueous electrophoresis sample buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, 142 mM dithiothreitol, pH 9. Samples were heated at 75° C. for 10 minutes. The sample labeling step used 5 μM dye solution. The final dilution step used 105 μL of stop solution. [Table 2]

[0070] 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. Alternatively, commercially prepared pH 8 sodium phosphate buffer was used.

[0071] Solutions of 100 mM sodium phosphate 1% LDS pH 6 and 100 mM sodium phosphate 1% LDS pH 9 were prepared using stock solutions and Milli-Q® water.

[0072] Alternate buffers were made using stock solutions and Milli-Q water including 100 mM sodium phosphate 2% LDS pH 6 and 100 mM sodium phosphate 2% LDS pH 8 solutions.

[0073] 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.

[0074] An alternative non-reducing buffer was prepared by adding 800 μL of 1 M iodoacetamide (IAM) (freshly prepared in Milli-Q® water) + 1200 μL of 100 mM sodium phosphate 2% LDS pH 8. The final concentrations were 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0075] Yet another non-reducing buffer was prepared by adding 800 μL of 1 M iodoacetamide (IAM) (freshly prepared in Milli-Q® water) + 1200 μL of 100 mM sodium phosphate 2% LDS pH 6. The final concentrations were 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

[0076] The reducing buffer was prepared by adding 68 μL of 10× reducing agent (500 mM dithiothreitol (DTT) + 166 μL of 100 mM sodium phosphate 1% LDS pH 9 + 6 μL of Milli-Q® water). The final concentrations were 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

[0077] An alternative reducing buffer was prepared by adding 320 μL of 10× reducing agent (500 mM dithiothreitol (DTT) + 1200 μL of 100 mM sodium phosphate 2% LDS pH 8 + 480 μL of Milli-Q® water). The final concentrations were 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol.

[0078] Example 2A: Non-reducing buffer The sample preparation step followed the general preparation shown in Table 2 using a non-reducing aqueous electrophoresis sample buffer containing 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate at pH 8. Samples were heated at 60° C. for 10 minutes. The sample labeling step used 16 μM dye solution. The final dilution step used 105 μL of stop solution.

[0079] Example 2B: Reduction Buffer The sample preparation step followed the general preparation shown in Table 2 using reducing aqueous electrophoresis sample buffer containing 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, 80 mM dithiothreitol, pH 8. Samples were heated at 60° C. for 10 minutes. The sample labeling step used 16 μM dye solution. The final dilution step used 105 μL of stop solution.

[0080] Example 2C: Non-reducing buffer, non-quenching The sample preparation step followed the general preparation shown in Table 2 using a non-reducing aqueous electrophoresis sample buffer containing 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate, pH 6. Samples were heated at 63° C. for 10 minutes. The sample labeling step used 16 μM dye solution. The final dilution step used 105 μL of dilution solution.

[0081] Example 2D: Reducing Buffer, Non-Quenching The sample preparation step followed the general preparation shown in Table 2 using reducing aqueous electrophoresis sample buffer containing 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, 80 mM dithiothreitol, pH 8. Samples were heated at 63° C. for 10 minutes. The sample labeling step used 16 μM dye solution. The final dilution step used 105 μL of diluent solution.

[0082] result: Microchip capillary electrophoresis (MCE) allows for dramatically shortened sample analysis times while maintaining the performance and reproducibility standards required for QC analysis. MCE assays were developed using non-reducing and reducing denaturing buffers as disclosed herein. Figures 1A-1B show representative electropherograms of proteins in non-reduced and reduced samples from a first MCE assay. Figure 1A shows the non-reduced analytical electropherogram. The main peak (MP) at 0.426 corresponds to the main antibody and the labeled low molecular weight (LMW) peaks correspond to antibody fragments. In some cases, peaks corresponding to one or more high molecular weight fragments are present, although not present in this example. Figure IB shows the reduced analytical electropherogram. Peaks corresponding to light chain (LC), heavy chain, and non-glycosylated heavy chain (NGHC), low molecular weight (LMW), and high molecular weight (HMW) antibody fragments are labeled accordingly. It should be noted that different products may have variations in the electropherograms generated and therefore may have different labeling practices.

[0083] In the foregoing specification, the invention has been described in relation to specific embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to further embodiments, and that the specific details described herein may be modified considerably without departing from the underlying principles of the invention.

[0084] The present invention may be embodied in other specific forms without departing from its spirit or essential attributes, and therefore, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention.

Claims

1. 1. An aqueous electrophoresis sample buffer comprising: 155-200 mM 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; 60-95 mM sodium phosphate; The aqueous electrophoresis sample buffer has a pH of 8 or less.

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

3. 2. The aqueous buffer of claim 1 comprising 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.

4. The aqueous electrophoresis sample buffer of claim 1, comprising: 165.9 mM 2-iodoacetamide; 0.81% lithium dodecyl sulfate; 81 mM sodium phosphate; has a pH of 6.0, or 200 mM 2-iodoacetamide; 1.2% lithium dodecyl sulfate; 60 mM sodium phosphate; has a pH of 6.0, or 200 mM 2-iodoacetamide; 1.2% lithium dodecyl sulfate; 60 mM sodium phosphate; having a pH of 8.0; Aqueous electrophoresis sample buffer.

5. The aqueous electrophoresis sample buffer of claim 1, comprising: 0.50 to 1.5% lithium dodecyl sulfate; 45-75 mM sodium phosphate; 80-155 mM dithiothreitol; The aqueous electrophoresis sample buffer has a pH of 8 or greater, preferably pH 8 or pH 9.

6. 6. The aqueous buffer of claim 5, comprising 80 mM dithiothreitol or 142 mM dithiothreitol.

7. 6. The aqueous buffer solution of claim 5 comprising 1.2% lithium dodecyl sulfate and 60 mM sodium phosphate.

8. 1. An aqueous electrophoresis sample buffer comprising: 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 80-155 mM dithiothreitol; has a pH of 9.0, or 1.2% lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol, having a pH of 8.0; Aqueous electrophoresis sample buffer.

9. 1. A method for identifying contaminants or impurities in a protein drug sample, the method comprising: adding said protein drug sample to a buffer according to any one of claims 1 to 7 to form a buffered protein drug sample; heating the buffered protein drug sample to 45-75° 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 it at 30-40° C. for 10-40 minutes to form a denatured, labeled protein drug sample; diluting the denatured, labeled protein drug sample and (a) subjecting it to MCE on a microchip capillary electrophoresis system to separate the diluted protein drug sample and generate an electropherogram, or (b) subjecting it to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram; and identifying peaks in the electropherogram that correspond to the protein drug, contaminants, and / or impurities.

10. 10. The method of claim 9, wherein the buffered protein drug sample is heated at 60°C for 10 minutes and / or the labeled protein drug sample is heated at 35°C for 15 minutes.

11. 10. The method of claim 9, wherein the diluted protein drug sample is 3.6 μg / ml.

12. 10. The method of claim 9, wherein the detectable label is DY-631 N-hydroxysuccinimidyl ester.

13. 10. The method of claim 9, wherein the detectable label is reconstituted in dimethylsulfoxide (DMSO) and subsequently diluted in 200 mM sodium phosphate pH 7.2 buffer.

14. A buffer solution according to any one of claims 1 to 8, and instructions for preparing a sample for electrophoresis in said buffer.

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