Microchip capillary electrophoresis assay and reagent

Abstract

MCE assays and reagents for assessing the purity of a sample of a proteinaceous drug product and identifying impurities in the sample of the proteinaceous drug product are provided. Methods for analyzing an analyte in a protein drug sample are provided.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201980020003.2, application date March 15, 2019, and invention name “Microchip capillary electrophoresis assay and reagent”, and the original application is a national phase application with international application number PCT / US2019 / 022525. The international application claims priority to the U.S. provisional patent application with application number 62 / 644,933, filed on March 19, 2018, the entire contents of which are incorporated by reference into this application. Technical Field

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

[0003] The implementation of robust, reproducible, user-friendly technologies is essential to meet the testing needs of today's quality control (QC) laboratories for biological products. Technology upgrades must be made to facilitate increased output while continuing to generate high-quality analytical data and attempting to minimize the number of invalid test results and instrument-related investigations. Although electrophoresis has historically been used for QC of product purity and fragmentation analysis, the method has shifted from gel-based to capillary-based and, more recently, to microchip-based. Microchip capillary electrophoresis (MCE) can significantly reduce 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] Although MCE has become a promising technique that is increasingly used in the pharmaceutical industry for characterization of biopharmaceuticals, quality control, and for drug discovery, it can be susceptible to assay perturbations.

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

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

[0007] MCE assays and reagents for assessing the purity of a protein drug product sample and identifying impurities in the protein drug product sample are provided. Methods for analyzing analytes in a protein drug sample are provided. Preferred protein drugs include, but are not limited to, antibodies and antigen-binding fragments thereof, 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 buffers and non-reducing buffers are also provided.

[0008] One embodiment provides a non-reducing aqueous electrophoresis sample buffer comprising an alkylating agent, such as 155 mM to 175 mM 2-iodoacetamide; 0.50% to 1.5% lithium dodecyl sulfate; and 65 mM to 95 mM sodium phosphate, wherein 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 comprises 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0009] A reducing buffer is also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5% to 1.5% lithium dodecyl sulfate, 55mM to 85mM 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. In one embodiment, the reducing buffer contains 135mM to 155mM dithiothreitol. Another embodiment provides a reducing buffer containing 0.69% lithium dodecyl sulfate, 69mM sodium phosphate and 142mM dithiothreitol.

[0010] HEPES-based buffers can 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 55mM to 75mM 2-iodoacetamide; 0.1% to 1.0% lithium dodecyl sulfate; 5mM to 85mM HEPES, and 5mM to 115mM 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 another embodiment, the aqueous buffer contains 66.4mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2mM HEPES, and 48.6mM sodium chloride.

[0011] Another embodiment provides a reducing HEPES-based aqueous electrophoresis sample buffer containing 0.05% to 0.75% lithium dodecyl sulfate, 5mM to 115mM sodium chloride, 5mM to 115mM 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 contains 35mM to 50mM dithiothreitol. Another embodiment provides a reducing buffer containing 0.28% lithium dodecyl sulfate, 41.5mM sodium chloride, 13.8mM HEPES and 42.5mM dithiothreitol.

[0012] One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample, comprising the step of 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 65° C. to 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°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample. Preferred detectable labels include, but are not limited to, Dyomics DY-631NHS ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., as well as those disclosed in U.S. Patent No. 6,924,372, which is incorporated herein by reference in its entirety. In a preferred embodiment, the protein drug sample with the added label is heated to 35°C for 30 minutes. Optionally, excess label is removed from the sample, for example by using a spin filter.

[0014] The denatured labeled protein drug product is diluted and subjected to MCE on a microchip capillary electrophoresis system to separate the diluted protein drug sample, thereby generating an electropherogram. In one embodiment, the final concentration of the sample starting from 0.5 mg / ml and then injected on the microchip is 9 μg / ml to MCE. The electropherogram contains peaks corresponding to the protein drug product and impurities. The method draws a conclusion by identifying the peaks corresponding to the contaminants or impurities in the electropherogram.

[0015] Another embodiment provides a reductive MCE method for identifying contaminants or impurities in a protein drug sample. The method begins by adding the protein sample to any of the above-mentioned reducing buffers to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample to 65°C to 85°C, preferably to 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°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample. In a preferred embodiment, the protein drug sample with the added label is heated to 35°C for 30 minutes. Optionally, for example, excess labels are removed from the sample by using a spin filter. Preferred detectable labels include, but are not limited to, Dyomics DY-631NHS ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., as well as those disclosed in U.S. Patent No. 6,924,372, which is incorporated herein 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 analytical 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. The method draws conclusions by identifying peaks in the electropherogram corresponding to contaminants or impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A A typical electropherogram of a non-reduced sample analysis is shown. Figure 1B A typical electropherogram of a reduced sample analysis is shown. The X-axis represents time in minutes and the Y-axis represents relative fluorescence units (RFU). Increased migration time corresponds to increased protein size. DETAILED DESCRIPTION

[0018] I. Definitions

[0019] The use of the terms "a," "an," "the" and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0020] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0021] The use of the term "about" is intended to describe values ​​that are within a range of approximately + / -10% above or below the stated value; in other embodiments, the numerical range of values ​​may be within a range of approximately + / -5% above or below the stated value; in other embodiments, the numerical range of values ​​may be within a range of approximately + / -2% above or below the stated value; in other embodiments, the numerical range of values ​​may be within a range of approximately + / -1% above or below the stated value. The preceding ranges are intended to be explained clearly by the context and no further limitation is implied. Unless otherwise stated herein or the context is clearly contradictory, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (such as "such as") provided herein is intended only to better illustrate the present invention, rather than to limit the scope of the present invention. No language in the specification should be interpreted as indicating that any element not claimed for protection is essential to the implementation of the present invention.

[0022] "Protein" refers to a molecule comprising two or more amino acid residues connected to each other by peptide bonds. Proteins include polypeptides and peptides, and may also include modifications such as glycosylation, lipid linkage, sulfation, gamma carboxylation, alkylation, hydroxylation and ADP-ribosylation of glutamic acid residues. Proteins may have scientific or commercial value, including protein-based drugs, and proteins particularly include enzymes, ligands, receptors, antibodies and chimeric proteins 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., such as sequences encoding chimeric proteins, or codon-optimized sequences, intron-free sequences, etc.), where they may exist as episomes or be integrated into the cell genome.

[0023] "Antibody" refers to an immunoglobulin molecule composed of four polypeptide chains (two heavy chains (H) and two light chains (L)) 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, namely 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 region and VL region can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes glycosylated and non-glycosylated immunoglobulins of any isotype or subtype. The term "antibody" includes antibody molecules prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins that can bind to more than one different epitope. Bispecific antibodies are generally described in U.S. Patent No. 8,586,713, which is incorporated herein by reference.

[0024] "Fc fusion proteins" contain part or all of two or more proteins that are not found together in nature, one of which is the Fc portion of an immunoglobulin molecule. The preparation of fusion proteins containing certain heterologous polypeptides fused to various portions of antibody-derived polypeptides, including the Fc domain, has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins," Current Protocols in Immunology, Supplement 4, pp. 10.19.1-10.19.11, 1992. A "receptor Fc fusion protein" comprises one or more extracellular domains of a receptor coupled to an Fc portion, which in some embodiments comprises a hinge region of an immunoglobulin followed by a CH2 and CH3 domain. In some embodiments, an Fc fusion protein comprises two or more unique receptor chains that can bind to one or more ligands. For example, an Fc fusion protein is a trap, such as an IL-1 trap or a VEGF trap.

[0025] The term "MCE" or "microchip capillary electrophoresis" refers to microchip-based capillary electrophoresis (CE) separation of analytes.

[0026] II. MCE Assay and Buffers

[0027] Methods for analyzing analytes in protein drug samples are provided. Preferred protein drugs include, but are not limited to, antibodies and antigen-binding fragments thereof, fusion proteins, and recombinant proteins. The assay uses MCE technology to separate, identify, and quantify impurities in the protein product and the protein product. Impurities include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing buffers and non-reducing buffers are also provided.

[0028] A. Buffer

[0029] 1. Non-reducing buffer

[0030] One embodiment provides a non-reducing aqueous electrophoresis sample buffer comprising 155 mM to 175 mM alkylating agent, such as 2-iodoacetamide or NEM; 0.50% to 1.5% lithium dodecyl sulfate; 75 mM to 95 mM sodium phosphate, wherein 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 comprises 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0031] 2. Reducing buffer

[0032] A reducing buffer is also provided. In one embodiment, the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5% to 1.5% lithium dodecyl sulfate, 65mM to 95mM 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.

[0033] 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 known in the art, such as immobilized reducing agent columns containing resins to which thiol-based reducing agents have been immobilized to achieve solid phase reduction of peptide and protein disulfide bonds. Reducing agents suitable for reducing chemical interactions between polypeptides are also contemplated, including oxidants.

[0034] In one embodiment, the reducing buffer contains 135 mM to 155 mM dithiothreitol.

[0035] Another embodiment provides a reducing buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.

[0036] 3.HEPES-based non-reducing buffer

[0037] HEPES-based buffers can 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 55mM to 75mM 2-iodoacetamide; 0.1% to 1.0% lithium dodecyl sulfate; 5mM to 85mM HEPES, and 5mM to 115mM sodium chloride, wherein the aqueous electrophoresis sample buffer has a pH of less than 9. In a preferred embodiment, the pH of the buffer is 8. In another embodiment, the aqueous buffer contains 66.4mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2mM HEPES, and 48.6mM sodium chloride.

[0038] 4.HEPES-based reducing buffer

[0039] Another embodiment provides a reducing HEPES-based aqueous electrophoresis sample buffer containing 0.05% to 0.75% lithium dodecyl sulfate, 5mM to 115mM sodium chloride, 5mM to 115mM 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 contains 35mM to 50mM dithiothreitol. Another embodiment provides a reducing buffer containing 0.28% lithium dodecyl sulfate, 41.5mM sodium chloride, 13.8mM HEPES and 42.5mM dithiothreitol.

[0040] B. Determination

[0041] 1. Non-reducing assay

[0042] One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample, the method comprising adding the protein sample to the above-mentioned non-reducing buffer to form a buffered protein drug sample. The buffered protein drug sample is heated to 65°C to 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. Then a detectable label is added to the denatured buffered protein drug sample and heated at 30°C 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 the added label is heated to 35°C for 30 minutes. Optionally, excess labels are removed from the sample, for example by using a spin filter.

[0043] 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, and the like, as well as those disclosed in U.S. Pat. No. 6,924,372, which is incorporated herein by reference in its entirety.

[0044] The denatured labeled protein drug product is diluted and subjected to MCE on a microchip capillary electrophoresis system to separate the diluted protein drug sample, thereby generating an electropherogram. In one embodiment, the final concentration of the sample starting from 0.5 mg / ml and then injected on the microchip is 9 μg / ml to 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 draws a conclusion by identifying the peaks corresponding to the contaminants or impurities in the electropherogram.

[0045] 2. Reduction assay

[0046] Another embodiment provides a reductive MCE method for identifying contaminants or impurities in a protein drug sample. The method begins by adding the protein drug sample to any of the above-mentioned reducing buffers to form a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered protein drug sample to 65°C to 85°C, preferably to 70°C for 10 minutes to form a denatured protein drug sample. The protein drug sample with the added label is then heated at 30°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample. In a preferred embodiment, the protein drug product sample with the added label is heated to 35°C for 30 minutes. Optionally, for example, excess labels are removed from the sample by using a spin filter. Preferred detectable labels include, but are not limited to, Dyomics DY-631 NHS esters. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, etc., as well as those disclosed in U.S. Patent No. 6,924,372, which is incorporated herein by reference in its entirety.

[0047] In one embodiment, the established assay range for sample concentration is 0.4 mg / ml to 0.6 mg / ml, corresponding to a final analytical 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. The method draws conclusions by identifying peaks in the electropherogram corresponding to contaminants or impurities.

[0048] C. Instruments

[0049] Instruments for performing the disclosed MCE assay are commercially available. In a preferred embodiment, a LabChip GXII or LabChip GXII Touch HT and HT protein expression chip ( The disclosed MCE assay was performed using the HT Protein Express Chip.

[0050] III. Proteins of Interest

[0051] The protein of interest, e.g., a protein pharmaceutical product, assayed using the disclosed MCE assay and reagents can be any protein of interest suitable for expression in prokaryotic or eukaryotic cells and can be used in the provided engineered host cell systems. For example, the protein of interest includes, but is not limited to, an antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a ScFv or fragment thereof, an Fc fusion protein or fragment thereof, a growth factor or fragment thereof, a cytokine or fragment thereof, or an extracellular domain of a cell surface receptor or fragment thereof. The protein of interest can be a simple polypeptide consisting of a single subunit or a complex multi-subunit protein consisting of two or more subunits. The protein of interest can be a biopharmaceutical product, a food additive or preservative, or any protein product 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 double-chain antibody, a three-chain antibody or a four-chain antibody, 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] In some embodiments, the antibody is selected from anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203579A1), anti-programmed cell death ligand 1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203580A1), anti-D114 antibody, anti-angiopoietin 2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), anti-angiopoietin-like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Patent No. 9,018,356), anti-platelet-derived growth factor receptor antibody (e.g., an anti-PD-L1 antibody as described in U.S. Patent No. 9,265,827), anti-PDGFR antibodies, anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Patent No. 9,132,192 or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. US2015 / 0259423A1), anti-proprotein convertase subtilisin Kexin-9 antibodies (e.g., anti- No. 8,062,640 or U.S. Pat. No. 9,540,449), anti-growth and 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 No. 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibodies as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or No. US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., as described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. No. 8,735,095 or No. 8,945,559), anti-interleukin 6 receptor antibody (e.g., anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 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 antibody as described in U.S. Pat. Nos. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. No. 9,447,173),Anti-differentiation cluster 3 (e.g., anti-CD3 antibodies as described in U.S. Pat. Nos. 9,447,173 and 9,447,173 and in U.S. Application No. 62 / 222,605), anti-differentiation cluster 20 (e.g., anti-CD20 as described in U.S. Pat. Nos. 9,657,102 and US20150266966A1 and in U.S. Pat. No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation cluster 48 (e.g., anti-CD48 antibodies as described in U.S. Pat. No. 9,228,014), anti-Feld1 antibodies (e.g., anti-CD20 antibodies as described in U.S. Pat. No. 9,079,948), ), anti-Middle East respiratory syndrome virus (e.g., anti-MERS antibodies as described in U.S. Patent Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication No. US2016 / 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., anti-NGF antibodies as described in U.S. Patent Application Publication No. US2016 / 0017029 and U.S. Patents Nos. 8,309,088 and 9,353,176), and anti-protein Y antibodies. In some embodiments, the bispecific antibody is selected from an anti-CD3×anti-CD20 bispecific antibody (as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1), an anti-CD3×anti-Mucin 16 bispecific antibody (e.g., anti-CD3×anti-Muc16 bispecific antibody), and an anti-CD3×anti-prostate specific membrane antigen bispecific antibody (e.g., anti-CD3×anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from 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, emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomab tiuxetan), idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratuma b), omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevogrumab,Ustekinumab and vedolizumab.

[0054] In some embodiments, the protein of interest is a recombinant protein (e.g., Fc fusion protein) containing an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor coupled to the Fc portion. In some embodiments, the Fc portion comprises the hinge region of IgG, followed by CH2 and CH3 domains. In some embodiments, the receptor Fc fusion protein contains two or more unique receptor chains that bind to a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as, for example, an IL-1 trap protein (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular region fused to the Fc of hIgG1; see U.S. Pat. No. 6,927,004, which is incorporated herein by reference in its entirety) or a VEGF trap protein (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. Pat. 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 of an antibody coupled to an Fc portion (such as a variable heavy chain fragment and a variable light chain fragment).

[0055] IV. Cell Culture

[0056] The protein drug product assayed with the disclosed MCE assay and reagents is the prepared cell culture. The cell culture is a "fed-batch cell culture" or "fed-batch culture", which refers to a batch culture in which cells and culture medium are initially supplied to a culture vessel, and additional culture nutrients are slowly fed to the culture in discontinuous increments during the culture, with or without regular cell and / or product harvesting before terminating the culture. Fed-batch cultures include "semi-continuous fed-batch cultures", in which the entire culture (possibly including cells and culture medium) is periodically removed and replaced with fresh culture medium. Fed-batch cultures are different from simple "batch cultures", but in batch cultures all components used for cell culture (including animal cells and all culture nutrients) are provided to the culture vessel at the beginning of the culture process. Fed-batch culture may differ from "perfusion culture" in that in a standard fed-batch process, supernatant is not removed from the culture vessel, but in a perfusion culture, cells are confined in culture, for example by filtration, and culture medium is continuously or intermittently introduced into and removed from the culture vessel. However, it is contemplated that samples are removed during a fed-batch cell culture process for testing purposes. The fed-batch process is continued until it is determined that a maximum working volume and / or protein yield has been reached, whereupon the protein is harvested.

[0057] The cell culture may be a "continuous cell culture," which is a technique for culturing cells continuously, typically in a specific growth phase. For example, if a continuous supply of cells is required, or if a specific protein of interest needs to be produced, the cell culture may need to be maintained in a specific growth phase. Therefore, conditions must be continuously monitored and adjusted accordingly to maintain the cells in that specific growth phase.

[0058] Cells are cultured in cell culture medium. The terms "cell culture medium" and "culture medium" refer to a nutrient solution for culturing mammalian cells, which generally provides the necessary nutrients to enhance the growth of cells, such as carbohydrate energy, essential amino acids (such as phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine) and non-essential amino acids (such as alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine), trace elements, energy, lipids, vitamins, etc. The cell culture medium may contain extracts, such as serum or peptone (hydrolysate), which may provide raw materials to support cell growth. The culture medium may contain extracts or soy extracts derived from yeast, rather than extracts derived from animals. A chemically defined medium refers to a cell culture medium in which all chemical components are known (i.e., have a known chemical structure). A chemically defined medium is completely free of animal-derived components (such as serum-derived or animal-derived peptones). In one embodiment, the culture medium is a chemically defined medium.

[0059] The solution may also contain components that enhance growth and / or survival above a minimum rate, including hormones and growth factors.The solution may be formulated to have an optimal pH and salt concentration for the survival and proliferation of the particular cells being cultured.

[0060] "Cell line" refers to one or more cells derived from a particular lineage by serial passage or subculture of cells. The term "cell" can be used interchangeably with "cell population."

[0061] 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, such as, for example, hybridomas or quadruple hybridomas. 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-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, 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 cells), U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes, such as retinal cells expressing viral genes (e.g., In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHOK1 cell.

[0062] V. Kit

[0063] One embodiment provides a kit comprising one or more of the disclosed buffers or ingredients for preparing the disclosed buffers. The kit may include a container for the buffer or ingredient. The buffer may be a solution or a lyophilized form. The kit optionally further comprises a second container containing a diluent or a reconstitution solution for the lyophilized formulation; and optionally containing instructions for use of the solution or the reconstitution and / or the use of the lyophilized buffer or powdered ingredient.

[0064] The kit may also include other reagents required to perform the disclosed MCE assay, including one or more of a buffer, a diluent, and a filter. The buffer and reagents may be in bottles, vials, or test tubes.

[0065] This application also includes the following implementation methods:

[0066] Embodiment 1. An aqueous electrophoresis sample buffer comprising:

[0067] 155mM to 175mM 2-iodoacetamide;

[0068] 0.50% to 1.5% lithium lauryl sulfate; and

[0069] 75mM to 95mM sodium phosphate,

[0070] Wherein the aqueous electrophoresis sample buffer has a pH of less than 7.

[0071] Embodiment 2. An aqueous buffer according to embodiment 1, wherein the pH is 6.

[0072] Embodiment 3. The aqueous buffer according to embodiment 1 or 2, comprising 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate and 81 mM sodium phosphate.

[0073] Embodiment 4. An aqueous electrophoresis sample buffer, consisting of:

[0074] 166mM 2-iodoacetamide,

[0075] 0.81% lithium dodecyl sulfate, and

[0076] 81mM sodium phosphate,

[0077] Wherein the aqueous electrophoresis sample buffer has a pH of 6.0.

[0078] Embodiment 5. An aqueous electrophoresis sample buffer comprising:

[0079] 0.50% to 1.5% lithium lauryl sulfate;

[0080] 45mM to 75mM sodium phosphate, and

[0081] reducing agent,

[0082] Wherein the aqueous electrophoresis sample buffer has a pH greater than 8.

[0083] Embodiment 6. An aqueous buffer according to embodiment 5, wherein the pH is 9.

[0084] Embodiment 7. The aqueous buffer according to embodiment 5 or 6, comprising 135 mM to 155 mM dithiothreitol.

[0085] Embodiment 8. An aqueous buffer according to any one of embodiments 5 to 7, comprising 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate and 142 mM dithiothreitol.

[0086] Embodiment 9. An aqueous electrophoresis sample buffer, consisting of:

[0087] 0.69% lithium dodecyl sulfate;

[0088] 69 mM sodium phosphate, and

[0089] 142mM dithiothreitol,

[0090] Wherein the aqueous electrophoresis sample buffer has a pH of 9.0.

[0091] Embodiment 10. A method for identifying contaminants or impurities in a protein drug sample, the method comprising the following steps:

[0092] adding the protein drug sample to the buffer according to any one of embodiments 1-4 to form a buffered protein drug sample;

[0093] heating the buffer protein drug sample to 65° C. to 85° C. for 5 to 15 minutes to form a denatured buffer protein drug sample;

[0094] adding a detectable label to the denatured buffered protein drug sample and heating it at 30°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample;

[0095] diluting the denatured labeled protein drug sample and subjecting it to MCE on a microchip capillary electrophoresis system to separate the diluted protein drug sample, thereby generating an electropherogram; and

[0096] Peaks in the electropherogram corresponding to contaminants or impurities are identified.

[0097] Embodiment 11. The method according to embodiment 10, wherein the buffer protein drug sample is heated at 70°C for 10 minutes.

[0098] Embodiment 12. The method according to embodiment 10 or 11, wherein the labeled protein drug sample is heated at 35°C for 30 minutes.

[0099] Embodiment 13. The method according to any one of embodiments 10 to 12, wherein the diluted protein drug sample is 9 μg / ml.

[0100] Embodiment 14. A method for identifying contaminants or impurities in a protein drug sample, the method comprising the following steps:

[0101] adding a protein sample to a buffer according to any one of embodiments 5-9 to form a buffered protein drug sample;

[0102] heating the buffered protein drug sample to 65° C. to 85° C. for 5 to 15 minutes to form a denatured protein drug sample;

[0103] adding a detectable label to the denatured protein drug sample and heating it at 30°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample;

[0104] diluting the denatured labeled protein drug sample and subjecting it to MCE analysis on a microchip capillary electrophoresis system to generate an electropherogram; and

[0105] Peaks in the electropherogram corresponding to contaminants or impurities are identified.

[0106] Embodiment 15. The method according to embodiment 14, wherein the buffer protein drug sample is heated at 70°C for 10 minutes.

[0107] Embodiment 16. The method of embodiment 14 or 15, wherein the sample is heated at 35°C for 30 minutes.

[0108] Embodiment 17. The method of any one of embodiments 14 to 16, wherein the diluted protein drug sample is 9 μg / ml.

[0109] Embodiment 18. The method of any one of embodiments 10 to 17, wherein the detectable label is DY-631 N-hydroxysuccinimide ester.

[0110] Embodiment 19. A kit comprising the buffer according to any one of embodiments 1 to 9, and

[0111] Written instructions for preparing samples for electrophoresis in the described buffer.

[0112] Embodiment 20. An aqueous electrophoresis sample buffer comprising:

[0113] 55 mM to 75 mM 2-iodoacetamide;

[0114] 0.1% to 1.0% lithium lauryl sulfate;

[0115] 5mM to 115mM sodium chloride, and

[0116] 5mM to 85mM HEPES,

[0117] The aqueous electrophoresis sample buffer has a pH of less than 9.

[0118] Embodiment 21. An aqueous electrophoresis sample buffer comprising:

[0119] 66.4 mM 2-iodoacetamide;

[0120] 0.32% lithium dodecyl sulfate;

[0121] 48.6 mM NaCl, and

[0122] 16.2 mM HEPES,

[0123] The aqueous electrophoresis sample buffer has a pH of less than 9.

[0124] Embodiment 22. An aqueous buffer according to embodiment 20 or 21, wherein the pH is 8.

[0125] Embodiment 23. An aqueous electrophoresis sample buffer comprising:

[0126] 0.05% to 0.75% lithium lauryl sulfate;

[0127] 5mM to 115mM NaCl;

[0128] 5mM to 85mM HEPES, and

[0129] 35mM to 50mM dithiothreitol,

[0130] Wherein the aqueous electrophoresis sample buffer has a pH greater than 7.

[0131] Embodiment 24. An aqueous electrophoresis sample buffer comprising:

[0132] 0.28% lithium dodecyl sulfate;

[0133] 41.5mM NaCl

[0134] 13.8 mM HEPES, and

[0135] 42.5 mM dithiothreitol,

[0136] Wherein the aqueous electrophoresis sample buffer has a pH greater than 7.

[0137] Embodiment 25. An aqueous buffer according to embodiment 23 or 24, wherein the pH is 8.

[0138] Example

[0139] Example 1: MCE assay for purity and impurity analysis of therapeutic proteins.

[0140] Methods and Materials :

[0141] Material:

[0142] Connect the LabChip GXII or LabChip GXII Touch HT and HT protein expression chips were used for capillary electrophoresis separation and data collection (Perkin Elmer). Non-reducing and reducing denaturing buffers disclosed above were used for the MCE assay.

[0143] method:

[0144] Table 1 shows the workflow for preparing samples for MCE determination. In short, the protein sample is diluted to 0.5 mg / ml. 1 μl of non-reducing denaturing buffer (NR) or reducing denaturing buffer (R) and 4 μl of diluted sample are added to a 96-well plate. The sample is mixed, centrifuged and heated at the temperature specified by the product (usually 75°C) for 10 minutes. The sample is then labeled with 5 μM of a commercially available dye (e.g., Dyomics DY-631 NHS ester). The sample is mixed, centrifuged and then heated at 35°C for 30 minutes. The labeled sample is then diluted with 105 μl of dilution stop solution. The sample is separated using a LabChip GXII or LabChip GXIITouch HT.

[0145] Buffer

[0146] Prepare a stock solution of 200 mM sodium dihydrogen phosphate monohydrate, 200 mM sodium dihydrogen phosphate heptahydrate, and 10% lithium dodecyl sulfate (LDS). Use this stock solution and Water, prepare solutions of 100 mM sodium phosphate 1% LDS pH 6 and 100 mM sodium phosphate 1% LDS pH 9.

[0147] The mixture was then quenched by adding 34 μL of 1 M iodoacetamide (IAM) (freshly prepared in Water) + 166 μL 100 mM sodium phosphate 1% LDS pH 6 + 5 μL Prepare a non-reducing buffer with water. Final concentrations are 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

[0148] The solution was prepared by adding 68 μL 10× reducing agent (500 mM dithiothreitol (DTT) + 166 μL 100 mM sodium phosphate 1% LDS pH 9 + 6 μL Prepare reducing buffer with water. Final concentrations are 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate and 142 mM dithiothreitol.

[0149] Table 1. Sample preparation method for MCE assay.

[0150]

[0151] result :

[0152] Microchip capillary electrophoresis (MCE) can significantly reduce sample analysis time while maintaining the performance and reproducibility standards required for QC analysis. An MCE assay was developed using the non-reducing and reducing denaturing buffers disclosed herein. Figure 1A-1B Representative electropherograms showing analysis of proteins in non-reduced and reduced samples are shown.

[0153] Although the present invention has been described in the foregoing specification with respect to certain embodiments of the invention and many details have been presented for the purpose of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to other embodiments and that certain details described herein may be modified considerably without departing from the basic principles of the invention.

[0154] All references cited herein are incorporated herein by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes of the invention, and therefore, reference should be made to the appended claims, rather than to the foregoing specification, to indicate the scope of the invention.

Claims

1. An aqueous electrophoresis sample buffer comprising: 0.50% to 1.5% lithium lauryl sulfate; 65mM to 95mM sodium phosphate, and a reducing agent selected from the group consisting of dithiothreitol, β-mercaptoethanol, 2-aminoethanethiol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine ​​hydrochloride and 2-mercaptoethanesulfonic acid sodium salt, Wherein the aqueous electrophoresis sample buffer has a pH greater than 8.

2. The aqueous electrophoresis sample buffer according to claim 1, wherein the pH is 9.

3. The aqueous electrophoresis sample buffer of claim 1, wherein the buffer comprises 135 mM to 155 mM dithiothreitol.

4. An aqueous electrophoresis sample buffer, consisting of: 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate, and 142 mM dithiothreitol.

5. A method for identifying contaminants or impurities in a protein drug sample, the method comprising the following steps: Adding the protein drug sample to an aqueous electrophoresis sample buffer to form a buffered protein drug sample, wherein the aqueous electrophoresis sample buffer comprises 0.50% to 1.5% lithium dodecyl sulfate; 65mM to 95mM sodium phosphate; and a reducing agent selected from the group consisting of dithiothreitol, β-mercaptoethanol, 2-aminoethanethiol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine ​​hydrochloride, and 2-mercaptoethanesulfonic acid sodium salt; heating the buffer protein drug sample to 65° C. to 85° C. for 10 minutes to form a denatured buffer protein drug sample; adding a detectable label to the denatured buffered protein drug sample and heating it at 30°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample; subjecting the denatured labeled protein drug sample to microchip capillary electrophoresis to generate an electropherogram; and Peaks in the electropherogram corresponding to contaminants or impurities are identified.

6. The method according to claim 5, wherein the buffer protein drug sample is heated at 70°C for 10 minutes.

7. The method of claim 5, wherein the denatured buffered protein drug sample is heated at 35°C for 30 minutes.

8. The method of claim 5, wherein the detectable label is DY-631 N-hydroxysuccinimide ester.

9. An aqueous electrophoresis sample buffer comprising: 0.05% to 0.75% lithium lauryl sulfate; 5mM to 115mM sodium chloride; 5mM to 115mM HEPES, and a reducing agent selected from the group consisting of dithiothreitol, β-mercaptoethanol, 2-aminoethanethiol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine ​​hydrochloride and 2-mercaptoethanesulfonic acid sodium salt, Wherein the aqueous electrophoresis sample buffer has a pH greater than 7.

10. The aqueous electrophoresis sample buffer according to claim 9, wherein the pH is 8.

11. The aqueous electrophoresis sample buffer of claim 9, wherein the buffer comprises 35 mM to 50 mM dithiothreitol.

12. An aqueous electrophoresis sample buffer comprising: 0.28% lithium dodecyl sulfate; 41.5 mM NaCl; 13.8 mM HEPES, and 42.5 mM dithiothreitol.

13. A method for identifying contaminants or impurities in a protein drug sample, the method comprising the following steps: adding the protein drug sample to an aqueous electrophoresis sample buffer to form a buffered protein drug sample, wherein the aqueous electrophoresis sample buffer comprises 0.05% to 0.75% lithium dodecyl sulfate; 5mM to 115mM sodium chloride; 5mM to 115mM HEPES and a reducing agent selected from the group consisting of dithiothreitol, β-mercaptoethanol, 2-aminoethanethiol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine ​​hydrochloride and 2-mercaptoethanesulfonic acid sodium salt; heating the buffered protein drug sample to 65° C. to 85° C. for 10 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°C to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample; subjecting the denatured labeled protein drug sample to microchip capillary electrophoresis to generate an electropherogram; as well as Peaks in the electropherogram corresponding to contaminants or impurities are identified.

14. The method of claim 13, wherein the buffer protein drug sample is heated at 70°C for 10 minutes.

15. The method of claim 13, wherein the denatured buffer protein drug sample is heated at 35°C for 30 minutes.

16. The method of claim 13, wherein the detectable label is DY-631 N-hydroxysuccinimide ester.

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