EA045662B1 - AQUEOUS ELECTROPHORETIC BUFFER (OPTIONS), METHOD FOR DETECTING CONTAMINATIONS OR IMPURITIES IN A PROTEIN DRUG SAMPLE, KIT FOR MICROCHIP CAPILLARY ELECTROPHORESIS - Google Patents

Description

Description of the invention

Aspects of the invention generally relate to the field of capillary electrophoresis, in particular to microarray capillary electrophoresis.

State of the art

Implementing reliable, repeatable, user-friendly technologies is critical to meeting the biologics testing requirements of modern quality control (KK, QC) laboratories. Improvements in methods are necessary to provide increased productivity while maintaining the ability to obtain quality analytical data and attempting to minimize the number of incorrect test results and equipment-related studies. Although electrophoresis has historically been used in KK to determine product purity and fragmentation, the methodology has moved from gel electrophoresis to capillary electrophoresis and later to microarray electrophoresis. Microarray capillary electrophoresis (MCE) can significantly reduce sample analysis time while maintaining the standards of performance and reproducibility required for KK analysis (Ouimet, C, et al., Expert Opin Drug Discov., 12(2): 213-224 (2017 )).

Although MCE has emerged as a promising technique with increasing applications in the pharmaceutical industry for biopharmaceutical characterization, quality control, and drug discovery, it can be subject to analytical interference.

It is therefore an object of the invention to provide improved MCE assay methods and compositions that reduce assay bias.

Another object of the invention is to provide MCE assays and compositions for improving the detection of impurities in a protein pharmaceutical product.

The essence of the invention

MCE assays and reagents are presented for assessing purity and identifying impurities in protein pharmaceutical product samples. Methods for determining analytes in a protein drug sample are presented. Preferred protein drugs include, but are not limited to, antibodies and antigen binding fragments thereof, fusion proteins and recombinant proteins. The analytical methods employ MCE techniques to separate, identify and quantify the protein product and impurities in the protein product. Impurities include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing and non-reducing buffers are also presented.

One embodiment of the invention provides a non-reducing aqueous electrophoretic sample buffer containing an alkylating agent, for example 155 to 175 mM 2-iodoacetamide; from 0.50 to 1.5% lithium dodecyl sulfate; and 65 to 95 mM sodium phosphate, wherein the aqueous electrophoretic 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 contains 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate and 81 mM sodium phosphate.

A reduction buffer is also proposed. In one embodiment of the invention, the reducing buffer is an aqueous electrophoretic sample buffer containing 0.5 to 1.5% lithium dodecyl sulfate, 55 to 85 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoretic sample buffer has a pH greater than 8. In a preferred embodiment of the invention, the pH of the buffer is 9. In one embodiment of the invention, the reduction buffer contains from 135 to 155 mM dithiothreitol. In yet another embodiment, the invention provides a reduction buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate and 142 mM dithiothreitol.

HEPES-based buffers can also be used in the disclosed methods. In one embodiment, the invention provides a non-reducing aqueous HEPES-based electrophoretic sample buffer containing an alkylating agent, for example, 55 to 75 mM 2-iodoacetamide; from 0.1 to 1.0% lithium dodecyl sulfate; 5 to 85 mM HEPES; and from 5 to 115 mM sodium chloride, wherein the aqueous electrophoretic sample buffer has a pH of less than 9. In another embodiment, the pH of the buffer is 8. In yet another embodiment, the aqueous buffer contains 66.4 mM 2-iodoacetamide, 0.32 % lithium dodecyl sulfate, 16.2 mM HEPES and 48.6 mM sodium chloride.

In another embodiment, the invention provides a HEPES-based reducing aqueous electrophoretic sample buffer containing 0.05 to 0.75% lithium dodecyl sulfate, 5 mM to 115 mM sodium chloride, 5 mM to 115 mM HEPES, and a reducing agent, wherein The aqueous electrophoretic sample buffer has a pH greater than 7. In a preferred embodiment, the pH of the buffer is 8. In one embodiment, the reduction buffer contains 35 to 50 mM dithiothreitol. In yet another embodiment, the invention provides a reduction buffer containing 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES and 42.5 mM dithiothreitol.

In one embodiment of the invention, a non-reducing MCE method is provided for

- 1045662 detecting contaminants or impurities in a protein drug sample, comprising the steps of adding the protein sample to the non-reducing buffer discussed above to form a buffered protein drug sample. The buffered protein drug sample is heated to 65 to 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample. In a preferred embodiment of the invention, the buffered protein drug sample is heated to 70°C for 10 minutes.

The protein drug sample is mixed with a detectable label and heated at 30 to 40° C. for 20 to 40 minutes to form a denatured labeled protein drug sample. Preferred detectable label includes, but is not limited to, NHS Dyomics DY-631 ester. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass spectrometric labels, quantum dots and the like, and those described in US Pat. No. 6,924,372, which is incorporated by reference in its entirety. In a preferred embodiment of the invention, the labeled protein drug sample is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example using a centrifugal filter.

The denatured labeled protein pharmaceutical product is diluted and subjected to MCE to separate the diluted protein drug sample in a microarray capillary electrophoresis system to obtain an electropherogram. In one embodiment of the invention, the final concentration of a sample starting at 0.5 mg/mL that is then injected into the microchip is 9 μg/mL at MCE. The electropherogram contains peaks corresponding to the protein pharmaceutical product and impurities. The method ends with the identification of peaks in the electropherogram corresponding to contaminants or impurities.

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

In one embodiment of the invention, the specified assay concentration range is 0.4 mg/mL to 0.6 mg/mL, corresponding to a final assay concentration of about 7 μg/mL to 11 μg/mL subjected to MCE analysis in a system for microchip capillary electrophoresis to obtain an electropherogram. The method ends with the identification of peaks in the electropherogram corresponding to contaminants or impurities.

Brief description of graphic materials

Fig. 1A illustrates an electropherogram of a typical analysis of an unreduced sample. Fig. 1B illustrates an electropherogram of a typical analysis of a reduced sample. The X axis represents time in minutes and the Y axis represents the fluorescence level in relative units (RFU). An increase in travel time corresponds to an increase in protein size.

Detailed description of the invention

I. Definitions.

The use of terms and similar entities in the singular in the context of the description of the invention claimed herein (especially in the context of the claims) is intended to cover both the singular and plural unless otherwise indicated herein or clearly inconsistent with the context.

The listing of ranges of values in this document is intended only as a guide to separately identify each specific value falling within the range unless otherwise specified herein, and each specific value is included in the description as if it were separately listed herein.

The use of the term about is intended to describe values either above or below a specified value in the range of approx. ±10%; in other embodiments of the invention the values may

- 2 045662 change in value either above or below the declared value in the range of approx. ±5%; in other embodiments of the invention, the values may vary in magnitude or above or below the stated value in the range of approx. ±2%; in other embodiments of the invention, the values may vary in magnitude or above or below the stated value in the range of approx. ±1%. It is assumed that the preceding intervals are made clear by the context, and no further restrictions are implied. All methods presented herein can be performed in any suitable order, unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any and all examples or illustrative phrases (eg, such) presented herein is intended only to better illuminate the invention and does not limit the scope of the invention unless otherwise stated in the claims. Nothing in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

Protein refers to a molecule containing two or more amino acid residues linked to each other by a peptide bond. Protein includes polypeptides and peptides and may also include modifications such as glycosylation, lipid addition, sulfation, gammacarboxylation of glutamic acid residues, alkylation, hydroxylation and ADPribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs, proteins include, among other entities, enzymes, ligands, receptors, antibodies and chimeric or fusion proteins. Proteins are obtained from recombinant cells of various types using well-known cell culture methods, and are generally introduced into the cell using genetic engineering techniques (such as a chimeric protein coding sequence, or a codon-optimized sequence, an intronless sequence, etc. ), where it can be located as an episome or integrated into the cell genome.

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 contains a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CHI, CH2 and CH3. Each light chain contains a light chain variable region and a light chain constant region. The light chain constant region consists of a single domain (CL). The VH and VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term antibody includes both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term antibody includes antibody molecules produced, expressed, generated or isolated by recombinant methods, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes a bispecific antibody, which includes a heterotetrameric immunoglobulin that can bind to more than one different epitopes. Bispecific antibodies are generally described in US Pat. No. 8,586,713, which is incorporated herein by reference.

Fc fusion proteins contain part or all of two or more proteins, one of which is the Fc part of an immunoglobulin molecule, that are not otherwise found together in nature. The preparation of fusion proteins containing certain heterologous polypeptides fused to various parts of antibody-derived polypeptides (including the Fc domain) has been described, for example: Ashkenazietal., 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). Receptor-Fc fusion proteins comprise one or more extracellular receptor domains fused to an Fc moiety, which in some embodiments comprises a hinge region followed by an immunoglobulin CH2 and CH3 domain. In some embodiments, the Fc fusion protein contains two or more separate receptor chains that are associated with one or more ligands. For example, the Fc fusion protein is a decoy, such as, for example, an IL-1 decoy or a VEGF decoy.

The term MCE or microchip capillary electrophoresis refers to the separation of analytes using capillary electrophoresis (CE) in a microchip format.

II. MCE assays and buffers.

Methods for determining analytes in a protein drug sample are presented. Preferred protein drugs include, but are not limited to, antibodies and antigen binding fragments thereof, fusion proteins and recombinant proteins. The analytical methods employ the MCE method to separate, identify and quantify the protein product and impurities in the protein product. Impurities include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants. Also presented are

- 3 045662 establishing and non-restoring buffers.

A. Buffers.

1. Non-reducing buffers.

In one embodiment, the invention provides a non-reducing aqueous electrophoretic sample buffer containing 155 to 175 mM of an alkylating agent, such as 2-iodoacetamide or NEM; from 0.50 to 1.5% lithium dodecyl sulfate; and 75 to 95 mM sodium phosphate, wherein the aqueous electrophoretic 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 contains 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate and 81 mM sodium phosphate.

2. Restoring buffers.

A reduction buffer is also proposed. In one embodiment of the invention, the reducing buffer is an aqueous electrophoretic sample buffer containing 0.5 to 1.5% lithium dodecyl sulfate, 65 to 95 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoretic sample buffer has a pH greater than 8. In a preferred embodiment of the invention, the pH of the buffer is 9.

Reducing agents are known in the art. Exemplary reducing agents include, but are not limited to, dithiothreitol (DTT, CAS 3483-12-3), betamercaptoethanol (BME, 2BME, 2-ME, b-mer, CAS 60-24-2), 2-aminoethanethiol (2- MEA-HC1, also called cysteamine-HCl, CAS 156-57-0), Tris (2-carboxyethyl)phosphine hydrochloride, (TCEP, CAS 5961-853), cysteine hydrochloride (Cys-HCl, CAS 52-89-1) or 2-mercaptoethanesulfonic acid sodium salt (MESNA). Other methods for reducing bonds in proteins are known in the art, such as an anchored reducing column, which contains a resin onto which a thiol-based reducing agent is anchored to allow solid-phase reduction of disulfide bonds in peptides and proteins. Also included are weakening agents, including oxidizing agents, which are useful for reducing the chemical interaction between polypeptides.

In one embodiment of the invention, the reduction buffer contains from 135 to 155 mM dithiothreitol.

In yet another embodiment, the invention provides a reduction buffer containing 0.69% lithium dodecyl sulfate, 69 mM sodium phosphate and 142 mM dithiothreitol.

3. Non-reducing buffers based on HEPES.

HEPES-based buffers can also be used in the disclosed methods. In one embodiment, the invention provides an aqueous HEPES-based electrophoretic sample buffer containing an alkylating agent, for example, 55 to 75 mM 2-iodoacetamide; from 0.1 to 1.0% lithium dodecyl sulfate; 5 to 85 mM HEPES; and from 5 to 115 mM sodium chloride, wherein the aqueous electrophoretic 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 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES and 48.6 mM sodium chloride.

4. HEPES-based reducing buffers.

In another embodiment, the invention provides a reducing aqueous electrophoretic sample buffer based on HEPES, containing from 0.05 to 0.75% lithium dodecyl sulfate, from 5 mm to 115 mm sodium chloride, from 5 mm to 115 mm HEPES and a reducing agent, which is aqueous the electrophoretic sample buffer has a pH greater than 7. In a preferred embodiment, the pH of the buffer is 8. In one embodiment, the reduction buffer contains 35 to 50 mM dithiothreitol. In yet another embodiment, the invention provides a reduction buffer containing 0.28% lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES and 42.5 mM dithiothreitol.

B. Analyzes 1.

Analyzes under non-reducing conditions.

In one embodiment, the invention provides a non-reducing MCE method for detecting contaminants or impurities in a protein drug sample, the method comprising the steps of adding the protein sample to the non-reducing buffer discussed above to form a buffered protein drug sample. The buffered protein drug sample is heated to 65 to 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample. In a preferred embodiment of the invention, the buffered protein drug sample is heated to 70°C for 10 minutes. A detectable label is then added to the denatured buffered protein drug sample and heated at 30 to 40° C. for 20 to 40 minutes to form a denatured labeled protein drug sample. In a preferred embodiment of the invention, the denatured labeled protein drug sample is heated to 35°C for 30 minutes. Excess label is optionally removed from

- 4 045662 samples, for example using a centrifugal filter.

Preferred detectable label includes, but is not limited to, NHS ester

Dyomics DY-631. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass spectrometric labels, quantum dots and the like, and those described in US Pat. No. 6,924,372, which is incorporated by reference in its entirety.

The denatured labeled protein pharmaceutical product is diluted and subjected to MCE to separate the diluted protein drug sample in a microarray capillary electrophoresis system to obtain an electropherogram. In one embodiment of the invention, the final concentration of a sample starting at 0.5 mg/mL that is then introduced into the microchip is 9 μg/mL at MCE. In another embodiment of the invention, the initial sample concentration is 0.2 mg/ml. The electropherogram contains peaks corresponding to the protein pharmaceutical product and impurities. The method ends with the identification of peaks in the electropherogram corresponding to contaminants or impurities.

2. Analyzes under reducing conditions.

In another embodiment, the invention provides a reductive MCE method for identifying contaminants or impurities in a protein drug sample. The method begins by adding a protein drug sample to any of the reconstitution buffers described above to obtain a buffered protein drug sample. The buffered protein drug sample is denatured by heating the buffered drug sample to 65 to 85°C, preferably 70°C, for 10 minutes to form a denatured protein drug sample. The labeled protein drug sample is then heated at 30 to 40° C. for 20 to 40 minutes to form a denatured labeled protein drug sample. In a preferred embodiment of the invention, a sample of the labeled protein pharmaceutical product is heated to 35°C for 30 minutes. Excess label is optionally removed from the sample, for example using a centrifugal filter. Preferred detectable label includes, but is not limited to, NHS Dyomics DY-631 ester. Other detectable labels that may be used include other dyes, fluorophores, chromophores, mass spectrometric labels, quantum dots and the like, and those described in US Pat. No. 6,924,372, which is incorporated by reference in its entirety.

In one embodiment of the invention, the specified assay concentration range is 0.4 mg/mL to 0.6 mg/mL, which corresponds to a final assay concentration of about 7 μg/mL to 11 μg/mL that is subjected to MCE analysis in the system for microchip capillary electrophoresis to obtain an electropherogram. The method ends with the identification of peaks in the electropherogram corresponding to contaminants or impurities.

C. Equipment.

Equipment to perform the disclosed MCE assays is commercially available. In a preferred embodiment of the invention, the disclosed MCE assays are performed using a LabChip GXII or LabChip GXII Touch HT and a LabChip® HT Rapid Protein Chip Analyzer.

III. Proteins of interest.

The protein of interest, such as a protein pharmaceutical product, that is 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 present designed host cell systems. For example, a protein of interest includes, but is not limited to, an antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a 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 cell surface receptor or fragment thereof. Proteins of interest may be simple polypeptides consisting of a single subunit or complex multisubunit proteins containing two or more subunits. The protein of interest may be a biopharmaceutical product, a food additive or preservative, or any protein product subject to purification and quality standards.

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

- 5 045662 is a chimeric IgG2/IgG4 antibody. In one embodiment of the invention, the antibody is an IgG2/IgG1 chimeric antibody. In one embodiment of the invention, the antibody is an IgG2/IgG1/IgG4 chimeric antibody.

In some embodiments, the antibody is selected from the group consisting of an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015/0203579A1), an anti-programmed cell death ligand 1 antibody (e.g., an anti-PD1 antibody) PD-L1 as described in US Patent Application Publication No. US2015/0203580A1), anti-D114 antibodies, anti-angiopoietin-2 antibodies (e.g. anti-ANG2 antibody as described in US Patent No. 9402898), anti-angiopoietin-like protein-3 antibodies (e.g. , anti-AngPtl3 antibody as described in US Patent No. 9018356), anti-platelet growth factor receptor antibodies (eg anti-PDGFR antibody as described in US Patent No. 9265827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (eg anti-PRLR antibody , as described in US Patent No. 9302015), anti-complement 5 antibodies (for example, anti-C5 antibody, as described in US Patent Application Publication No. US2015/0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (for example, anti-EGFR antibody, as described in US Patent No. 9132192 or anti-EGFRvIII antibody as described in US Patent Application Publication No. US2015/0259423A1), anti-proprotein convertase subtilisinkexin-9 antibodies (e.g. anti-PCSK9 antibody as described in US Patent No. 8062640 or US Patent No. 9540449 ), anti-growth and differentiation factor-8 antibodies (eg, anti-GDF8, also known as anti-myostatin, as described in US Pat. No. 8,871,209 or 9,260,515), anti-glucagon receptor antibodies (eg, anti-GCGR, as described in US Patent Application Nos. US2015/0337045A1 or US2016/0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, anti-interleukin 4 receptor antibodies (e.g., anti-IL4R antibody as described in US Patent Application Publication No. US2014/0271681A1 or US Patent No. 8735095 or 8,945,559), anti-interleukin 6 receptor antibody (e.g., anti-IL6R antibody as described in US Pat. Nos. 7,582,298, 8,043,617, or 9,173,880), anti-IL1 antibody, anti-IL2 antibody, anti-ILL antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody anti-IL6, anti-IL7, anti-interleukin 33 (e.g., anti-ILRD antibody as described in U.S. Pat. No. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibody (e.g., anti-RSV as described in U.S. Patent Application Publication No. 9,447,173), anti-cluster of differentiation protein 3 antibodies (e.g., anti-CD3 as described in U.S. Pat. Nos. 9,447,173 and 9,447,173 and U.S. Application No. 62/222605), anti-cluster of differentiation 20 proteins (e.g., anti-CD20 as described in in US Patent No. 9657102 and US20150266966A1 and in US Patent No. 7879984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of differentiation protein 48 antibodies (for example, anti-CD48 antibody as described in US Pat. No. 9228014), anti-Fel d1 antibodies ( e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus antibodies (e.g., anti-MERS antibody, as described in U.S. Patent Application Publication No. US2015/0337029A1), anti-Ebola virus antibodies (e.g., as described in Patent Application Publication US No. US2016/0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (eg, anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibodies (eg, anti-NGF antibody, as described in US Patent Application Publication No. US2016/0017029 and US Patent Nos. 8309088 and 9353176) and anti-protein Y antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of bispecific antibodies against CD3 and CD20 (as described in US Patent Application Publications US2014/0088295A1 and US20150266966A1 ), anti-CD3 and mucin 16 bispecific antibody (eg, anti-CD3 and Muc16 bispecific antibody), and anti-CD3 and prostate-specific membrane antigen bispecific antibody (eg, anti-CD3 and PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximabavedontin brodalumab, canakimumab, capromabpendetide, certolizumabapegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtazine, alirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomabatiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxi bakumab, reslizumab, rinukumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, alarmrumab, ustekinumab and vedolizumab.

In some embodiments, the protein of interest is a recombinant protein that contains an Fc fragment and another domain (eg, an Fc fusion protein). In some embodiments, the Fc fusion protein is a receptor Fc fusion protein that contains one or more extracellular receptor domains linked to an Fc moiety. In some embodiments, the Fc fragment comprises a hinge region followed by an IgG CH2 and CH3 domain. In some embodiments, the receptor Fc fusion protein comprises two or more distinct receptor chains that are bound to either a single ligand or multiple ligands. For example, the Fc fusion protein is a decoy protein, such as, for example, an IL-1 decoy protein (eg, rilonacept, which contains the IL-1RAcP ligand-binding region linked to the Il-1R1 extracellular region bound to the Fc of hIgG1; see US Pat. No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains Flt1 VEGF receptor Ig domain 2 linked to Flk1 VEGF receptor Ig domain 3 , associated with Fc from hlgG1; see US patent No. 7087411 and 7279159). In other embodiments, the Fc fusion protein is a ScFv-Fc fusion protein that contains one or more of one or more antigen binding domains, such as a heavy chain variable fragment and a light chain variable fragment, of an antibody coupled to an Fc fragment.

IV. Cell culture.

The protein pharmaceutical product, which is analyzed using the disclosed MCE assay methods and reagents, is produced using cell cultures. The cell cultures may be a stationary fed-batch cell culture or a stationary fed-batch culture, which refers to a stationary culture in which cells and culture medium are introduced into the culture vessel initially and additional culture nutrients are slowly added, in separate portions, to the culture during cultivation, with or without periodic collection of cells and/or product until completion of culture. Fed-batch stationary cell culture includes a semi-continuous fed-batch cell culture where the entire culture (which may include cells and media) is periodically removed and replaced with fresh media. Fed-batch culture is distinguished from just stationary culture, where all cell culture components (including animal cells and all nutritional components of the culture) are fed into the culture vessel at the beginning of the stationary culture process. Fed-batch culture may differ from flow culture in that the supernatant is not removed from the culture vessel in a standard fed-batch process, whereas in flow culture the cells are retained in the culture, for example by filtration, and the culture medium is maintained continuously or periodically introduced and removed from the culture vessel. However, stationary fed-batch cultivation involves sampling for research purposes. The steady-state fed-batch process continues until it is determined that maximum operating volume and/or protein production has been reached, and the protein is then collected.

Cell culture may be a continuous cell culture, which is a technology used to continuously grow cells, usually at a specific growth phase. For example, if a constant supply of cells is required, or a specific protein of interest is required, the cell culture must be maintained at a specific growth phase. Thus, conditions must be continuously monitored and adjusted accordingly to maintain the cells in that particular phase.

Cells are cultured in cell culture medium. The terms cell culture medium and culture medium refer to a nutrient solution used to grow mammalian cells that typically provides essential nutrients to enhance cell growth, such as carbohydrate energy source, essential (e.g., phenylalanine, valine, threonine, tryptophan, methionine , leucine, isoleucine, lysine and histidine) and non-essential (e.g. alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine) amino acids, trace elements, energy sources, lipids, vitamins, etc. . Cell culture media may contain extracts, such as whey or peptones (hydrolysates), that provide precursors that support cell growth. The medium may contain yeast-derived or soy extracts instead of animal extracts. Chemically defined medium refers to a cell culture medium in which all chemical components are known (i.e., has a known chemical structure). The chemically defined medium contains no animal-derived components at all, such as whey or animal-derived peptones. In one embodiment of the invention, the medium is a chemically defined medium.

The solution may also contain components that enhance growth and/or survival beyond minimal levels, including hormones and growth factors. The solution may be formulated with a pH and salt concentration that is optimal for the survival and proliferation of the particular cell to be cultured.

Cell line refers to a cell or cells that are obtained from a specific line through serial passaging or subculture of cells. The term cell is used

- 7 045662 interchangeable with cell population.

The term cell includes any cell that is 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 quadromas. In some 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) cells (eg, CHO K1, DXB-11 CHO, Veggie-CHO), COS (eg, COS-7), retinal cells, Vero, CV1 , kidney cells (e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocyte, e.g. Jurkat (T-lymphocyte) or Daudi (B lymphocyte), A431 (epidermal), U937, 3T3, L cells, C127 cells, Sp2/0, NS-O, MMT cells, stem cell, tumor cell and a cell line derived from the above cell. In some embodiments, the cell contains one or more viral genes, such as a retinal cell that expresses a viral gene (eg, a PER.C6® cell). In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

V. Sets.

In one embodiment, the invention provides a kit containing one or more disclosed buffers or ingredients for preparing the disclosed buffers. The kit may contain a container for buffers or ingredients. The buffer may be in solution or lyophilized form. The kit optionally also contains a second container containing a diluent or reconstituting solution for the lyophilized formulation; and optionally instructions for use of the solution or reconstituted solution and/or use of lyophilized buffers or powder ingredients.

The kit may further contain additional reagents necessary to perform the disclosed MCE assay methods, including one or more buffering agents, diluents, and filters. The buffer and reagents may be contained in a bottle, vial, or tube.

Example

Example 1: MCE analysis to determine the purity and impurities of therapeutic proteins.

Methods and materials:

Materials:

LabChip GXII or LabChip GXII Touch HT and LabChip® HT Rapid Protein Chip Analyzer were used for capillary electrophoretic separation and data acquisition (PerkinElmer). For MCE analysis, the non-reducing and reducing denaturing buffers described above were used.

Techniques:

Table 1 shows the sequence of steps to obtain a sample for MCE analysis. Briefly, protein samples were diluted to 0.5 mg/mL. 1 μL of non-reducing (NR) or reducing (R) denaturing buffer and 4 μL of diluted sample were added to a 96-well plate. The sample was mixed, centrifuged and heated for 10 minutes at the temperature specified for the product, usually 75°C. Samples were then labeled with 5 μM of a commercially available dye (e.g., NHS Dyomics ester DY-631). The samples were mixed, centrifuged, and then heated at 35°C for 30 min. The labeled sample was then diluted with 105 μL of diluted stop solution. Samples were separated using LabChip GXII or LabChip GXII Touch HT.

Buffers.

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

Non-reducing buffer was prepared by adding 34 µl 1 M iodoacetamide (IAM) (prepared fresh in Milli-Q® water) + 166 µl 100 mM sodium phosphate, 1% DSL, pH 6 + 5 µl Milli-Q ® water. The final concentrations were 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.

Reducing buffer was prepared by adding 68 μl of 10x reducing agent (500 mM dithiothreitol (DTT) + 166 μl 100 mM sodium phosphate, 1% DSL, pH 9 + 6 μl Milli-Q® water). Final concentrations were 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate and 142 mM dithiothreitol.

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Claims (17)

Table 1 Method of sample preparation for MCE analysis ____________________________Sample preparation____________________________ ____________NR__________________________R_____________ 4 µl 0.5 mg/ml sample 4 µl 0.5 mg/ml sample ________1 µl NR buffer________________________1 µl R buffer____________ Stir, centrifuge, heat at indicated temperature for 10 __________________________ minutes___________________________ __________________________Specimen labeling___________________________ ______________________5 µl denatured sample_________________________ _______________________5 µl 5 µM PICO dye____________________ _____Stir , centrifuge, heat at 35°C for 30 minutes________ ________________________Final dilution___________________________ 5 µl labeled sample ________________________ 105 µl diluted stop solution ___________________________ ___________________________Separation procedure ___________________________ ______________________ Rapid Protein Analysis HT PICO 200 ______________________ Results: Microarray capillary electrophoresis (MCE) can significantly reduce sample analysis time while maintaining the standards of throughput and reproducibility required for KK analysis. The MCE assay method was developed using the non-reducing and reducing denaturing buffers disclosed herein. Fig. 1A-1B illustrate a typical electropherogram demonstrating protein analysis in unreduced samples and reduced samples. Although the foregoing description of the present invention has been presented in connection with specific embodiments thereof, and many details have been provided for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of additional embodiments and that some of the details described herein may vary significantly without deviating from the basic principles of the invention. All sources cited in this document are incorporated by reference in their entirety. The present invention may embrace other specific forms without departing from its spirit or essential features, and accordingly the appended claims, rather than the foregoing description, should be relied upon as defining the scope of the invention. CLAIM 1. An aqueous electrophoretic buffer for a protein drug sample containing: 155 to 175 mM 2-iodoacetamide; from 0.50 to 1.5% lithium dodecyl sulfate; and 75 to 95 mM sodium phosphate, wherein the aqueous electrophoretic sample buffer has a pH of less than 7. 2. Aqueous electrophoretic sample buffer according to claim 1, characterized in that the pH is 6. 3. An aqueous electrophoretic sample buffer according to claim 1 or 2, containing 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate and 81 mM sodium phosphate. 4. An aqueous electrophoretic buffer for a protein drug sample, consisting of: 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate and 81 mM sodium phosphate, the aqueous electrophoretic sample buffer having a pH of 6.0. 5. A method for detecting contaminants or impurities in a sample of a protein drug, the method comprising the steps of: adding a protein drug sample to an aqueous electrophoretic buffer containing: 155 to 175 mM 2-iodoacetamide; from 0.50 to 1.5% lithium dodecyl sulfate; and 75 to 95 mM sodium phosphate, wherein the aqueous electrophoretic sample buffer has a pH of less than 7, to obtain a buffered protein drug sample; heating the buffered protein drug sample to 65 to 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample; - 9 045662 adding a detectable label to a denatured buffered protein drug sample and heating it at 30 to 40° C. for 20 to 40 minutes to form a denatured labeled protein drug sample; diluting the denatured labeled protein drug sample and subjecting it to microchip capillary electrophoresis (MCE) to separate the diluted denatured labeled protein drug sample in the microchip capillary electrophoresis system to obtain an electropherogram; and identifying peaks in the electropherogram corresponding to contaminants or impurities. 6. The method according to claim 5, characterized in that the buffered sample of the protein drug is heated at 70°C for 10 minutes. 7. The method according to claim 5 or 6, characterized in that the labeled protein drug sample is heated at 35°C for 30 minutes. 8. The method according to any one of claims 5-7, characterized in that the diluted sample of the protein drug has a concentration of 9 μg/ml. 9. A method for detecting contaminants or impurities in a sample of a protein drug, the method comprising the steps of: adding a protein sample to an aqueous electrophoretic buffer containing: 155 to 175 mM 2-iodoacetamide; from 0.50 to 1.5% lithium dodecyl sulfate; and 75 to 95 mM sodium phosphate, wherein the aqueous electrophoretic sample buffer has a pH of less than 7, to provide a buffered protein drug sample; heating the buffered protein drug sample to 65 to 85°C for 5 to 15 minutes to form a denatured buffered protein drug sample; adding a detectable label to the denatured protein drug sample and heating it at 30 to 40°C for 20 to 40 minutes to form a denatured labeled protein drug sample; diluting the denatured labeled protein drug sample and subjecting it to microchip capillary electrophoresis (MCE) to separate the diluted denatured labeled protein drug sample in the microchip capillary electrophoresis system to obtain an electropherogram; and identifying peaks in the electropherogram corresponding to contaminants or impurities. 10. The method according to claim 9, characterized in that the buffered sample of the protein drug is heated at 70°C for 10 minutes. 11. Method according to claim 9 or 10, characterized in that the denatured sample of the protein drug is heated at 35°C for 30 minutes. 12. The method according to any one of claims 9-11, characterized in that the diluted sample of the protein drug has a concentration of 9 μg/ml. 13. Method according to any one of claims 5 to 12, characterized in that the detectable label is N-hydroxysuccinimidyl ester DY-631. 14. A microarray capillary electrophoresis (MCE) kit containing an aqueous protein drug sample electrophoretic buffer containing 155 to 175 mM 2-iodoacetamide; from 0.50 to 1.5% lithium dodecyl sulfate; and 75 to 95 mM sodium phosphate, wherein the aqueous electrophoretic sample buffer has a pH less than 7, and written instructions for preparing the sample for microarray capillary electrophoresis in the aqueous electrophoretic buffer to detect contaminants or impurities. 15. An aqueous electrophoretic buffer for a protein drug sample containing: 55 to 75 mM 2-iodoacetamide; from 0.1 to 1.0% lithium dodecyl sulfate; 5 to 115 mM sodium chloride and 5 to 85 mM HEPES, wherein the aqueous electrophoretic sample buffer has a pH less than 9. 16. An aqueous electrophoretic buffer for a protein drug sample containing: 66.4 mM 2-iodoacetamide; 0.32% lithium dodecyl sulfate; 48.6 mM NaCl and 16.2 MM HEPES, with the aqueous electrophoretic sample buffer having a pH less than 9. 17. Aqueous buffer according to claim 15 or 16, characterized in that the pH is 8. -