Method and system for analyzing polypeptide variants

Abstract

A method of quantitative analysis of charge variants in an analyte can include introducing a sample buffer comprising the analyte into a capillary, separating the charge variants within the sample buffer along an equal gradient, incubating the capillary in a detection antibody, and quantifying a relative abundance of the charge variants based on a signal corresponding to the detection antibody. The method may also include generating an electrophoretogram, wherein the electrophoretogram includes a relational graph of the signal strength produced by the reporter molecule to an isoelectric point along the elevator level of the detected signal.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 269,595, filed on March 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to systems and methods for analyzing polypeptide variants.Some aspects of the present disclosure relate to systems and methods for quantifying charge variants within a sample. Background Art

[0003] Biopharmaceutical products (e.g., antibodies, antibody-drug conjugates, fusion proteins, adeno-associated viruses (AAV), proteins, tissues, cells, peptides, or other biologically derived therapeutic products) are increasingly being used to treat and prevent infectious diseases, genetic diseases, autoimmune diseases, and other diseases. Due to their biological origin, biopharmaceutical products have multiple dimensions of heterogeneity, including charge heterogeneity. For example, various post-translational modifications such as sugar glycosylation, oxidation, or deamidation can lead to charge heterogeneity between products produced biologically under similar or identical production conditions.

[0004] Traditional methods for characterizing charge variants and developing charge variant distribution maps may be limited to understanding intact proteins or multi-protein structures. However, charge variants may form within polypeptides or protein subunits, and characterization of larger structures may lack the detail required to characterize charge variants of a single polypeptide or subunit. In addition, traditional methods for characterizing charge variants may include time and labor intensive chromatography steps that increase the costs associated with characterizing charge variants. Summary of the invention

[0005] Aspects of the present disclosure relate to methods for quantifying charge variants within an analyte. The method may include introducing the analyte into a capillary, separating the charge variants in a sample buffer along an isoelectric gradient, incubating the capillary in a detection antibody, and quantifying the relative abundance of the charge variants based on a signal corresponding to the detection antibody.

[0006] The method may further include incubating the capillary in a reporter molecule. The reporter molecule may include an antibody conjugated to horseradish peroxidase or a streptavidin protein conjugated to horseradish peroxidase. The method may further include introducing a detection agent into the capillary. The detection agent may be luminol-peroxide. The method may further include generating an electropherogram, wherein the electropherogram includes a plot of the intensity of the chemiluminescent signal generated by the reporter molecule versus the isoelectric point along an isoelectric gradient at which the chemiluminescent signal is detected. Quantifying the relative abundance of the charge variant based on the signal corresponding to the detection antibody includes calculating the area under the peak of the electropherogram corresponding to the charge variant. The method may further include immobilizing the charge variant in the capillary after separating the charge variants along the isoelectric gradient and before incubating the capillary in the detection antibody.

[0007] On the other hand, the present disclosure includes a method for quantifying charge variants in an analyte. The method may include reducing and denaturing polypeptides in the analyte to produce reduced and denatured polypeptides, wherein the analyte comprises charge variants of the target polypeptide. The method may further include buffer exchanging the reduced and denatured polypeptide to produce a buffer-exchanged sample, wherein the buffer-exchanged sample comprises reduced and denatured polypeptides. The method may further include preparing a sample buffer comprising the buffer-exchanged sample. In some embodiments, the method includes introducing the sample buffer into a capillary. The method may further include separating the charge variants of the target polypeptide along an isoelectric gradient. In some embodiments, the method may include measuring a signal associated with the abundance of the target polypeptide in a region within the isoelectric gradient in the capillary.

[0008] The sample buffer may include urea, carrier ampholytes, and cellulose. The cellulose may be hydroxypropyl methylcellulose, and the sample buffer may include at least about 1 volume percent of hydroxypropyl methylcellulose. The sample buffer may include a urea concentration of at least about 6M or at least about 8M. The sample buffer may include formamide. The formamide concentration in the sample may be less than or equal to about 30 volume percent.

[0009] On the other hand, the present disclosure includes a method for assessing the level of environmental stress perceived by a sample of a target polypeptide. The method may include generating a charge variant distribution map (curve, distribution curve) of the sample, wherein the charge variant distribution map includes multiple peaks. Each peak in the multiple peaks may be associated with a corresponding charge variant of the target polypeptide; associated with an isoelectric point that is equal to the isoelectric point of the corresponding charge variant of the target polypeptide; and / or include a relative peak area that is associated with the relative abundance of the corresponding charge variant of the target polypeptide. The method may further include comparing the charge variant distribution map of the sample with one or more known charge variant distribution maps. In some embodiments, the method includes determining the level of environmental stress perceived by the sample based on a comparison of the charge variant distribution map of the sample with one or more known charge variant distribution maps.

[0010] The environmental stress level may be associated with the thermal stress level, the stress level caused by the manufacturing process hold time, or the stress level caused by freeze-thaw cycles. The target polypeptide may include an antibody or an adeno-associated virus. Generating a charge variant distribution profile of the sample may include introducing the sample into a capillary and separating the charge variants of the target polypeptide along an isoelectric gradient. Generating a charge variant distribution profile of the sample may further include incubating the capillary in a detection antibody, incubating the capillary in a reporter molecule, and generating an electropherogram, wherein the electropherogram includes a plot of the intensity of the signal generated by the reporter molecule versus the isoelectric point along the isoelectric gradient at which the signal is detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings included in and forming part of this specification illustrate various exemplary embodiments and are used together with the description to explain the principles of the disclosed embodiments. Any features of the embodiments or examples described herein (e.g., compositions, formulations, methods, etc.) may be combined with any other embodiments or examples, and all such combinations are included in the present disclosure. In addition, the described systems and methods are not limited to any single aspect or embodiment thereof, nor to any combination or arrangement of these aspects and embodiments. For the sake of brevity, certain arrangements and combinations are not discussed and / or described separately herein.

[0012] According to various aspects of the present disclosure, Figure 1 An exemplary method for separating and quantifying charge variants of reduced and denatured polypeptides is depicted in flow chart form;

[0013] According to various aspects of the present disclosure, Figure 2 An exemplary method for separating and quantifying charge variants of reduced and denatured polypeptides is depicted in flow chart form;

[0014] According to various aspects of the present disclosure, Figure 3 An exemplary method for quantifying the relative abundance of charge variants is depicted in flowchart form;

[0015] According to various aspects of the present disclosure, Figure 4A and 4B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0016] According to various aspects of the present disclosure, Figure 4C The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0017] According to various aspects of the present disclosure, Figure 4D The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0018] According to various aspects of the present disclosure, Figure 5A and 5B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0019] According to various aspects of the present disclosure, Figure 5C The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0020] According to various aspects of the present disclosure, Figure 5D The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0021] According to various aspects of the present disclosure, Fig. 6A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0022] According to various aspects of the present disclosure, Figure 6B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0023] According to various aspects of the present disclosure, Figure 6C The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0024] According to various aspects of the present disclosure, Fig. 7A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0025] According to various aspects of the present disclosure, Figure 7B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0026] According to various aspects of the present disclosure, Fig. 8A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0027] According to various aspects of the present disclosure, Figure 8BThe charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0028] According to various aspects of the present disclosure, Fig. 9A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0029] According to various aspects of the present disclosure, Fig. 9B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0030] According to various aspects of the present disclosure, Fig. 9C Depicts a comparison based on Fig. 9A and 9B A graph showing the relative peak areas calculated based on the charge variant distribution of the

[0031] According to various aspects of the present disclosure, Fig. 10A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0032] According to various aspects of the present disclosure, Fig. 10B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0033] According to various aspects of the present disclosure, Fig.11A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0034] According to various aspects of the present disclosure, Fig. 11B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0035] According to various aspects of the present disclosure, Fig. 11C Depicts a comparison based on Fig.11A A graph showing the relative peak areas calculated based on the charge variant distribution of the

[0036] According to various aspects of the present disclosure, Fig.11D Depicts a comparison based on Fig. 11B A graph showing the relative peak areas calculated based on the charge variant distribution of the

[0037] According to various aspects of the present disclosure, Fig. 12A The charge variant distribution profiles of native analytes are depicted;

[0038] According to various aspects of the present disclosure, Fig. 12B The charge variant distribution profiles of native analytes are depicted;

[0039] According to various aspects of the present disclosure, Fig.13A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0040] According to various aspects of the present disclosure, Fig. 13BThe charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0041] According to various aspects of the present disclosure, Fig.14A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0042] According to various aspects of the present disclosure, Fig. 14B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0043] According to various aspects of the present disclosure, Fig.15 Western blotting using polyclonal antibodies against viral proteins is depicted;

[0044] According to various aspects of the present disclosure, Fig.16 The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0045] According to various aspects of the present disclosure, Fig.17 The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0046] According to various aspects of the present disclosure, Fig.18 , 19 , 20A, 20B, 21A, 21B, 22A, 22B, 23A to 23C, 24A to 24D, 25A to 25D, and 26A to 26D depict charge variant distribution graphs of reduced and denatured analytes, respectively;

[0047] According to various aspects of the present disclosure, Fig.27A The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0048] According to various aspects of the present disclosure, Fig.27B The charge variant distribution profiles of the reduced and denatured analytes are depicted;

[0049] According to various aspects of the present disclosure, Figures 28A to 28E The charge variant distribution profiles of the reduced and denatured analytes are depicted respectively;

[0050] According to various aspects of the present disclosure, Fig.29 depicts a graph comparing relative peak areas calculated based on the charge variant profiles of FIGS. 28 and 28 ;

[0051] According to various aspects of the present disclosure, Figures 30A to 30D The charge variant distribution profiles of the reduced and denatured analytes are depicted respectively;

[0052] According to various aspects of the present disclosure, Fig.31 Depicts a comparison based on Figures 30A to 30D A graph showing the relative peak areas calculated based on the charge variant distribution of the

[0053] According to various aspects of the present disclosure, Fig.32A , 32B , 33A, 33B, 34A and 34B each depict a charge variant distribution graph of a reduced and denatured analyte; and

[0054] According to various aspects of the present disclosure, Fig.35 , 36 and 37 depict the charge variant distribution profiles of reduced and denatured analytes. DETAILED DESCRIPTION

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any suitable methods and materials (e.g., similar or equivalent to those described herein) can be used in the practice or testing of the present disclosure, specific exemplary methods are described herein. All publications mentioned are incorporated herein by reference.

[0056] As used herein, the terms "include," "comprising," or any other variations thereof are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that includes a list of components includes not only those components but may also include other components not expressly listed or inherent to such process, method, article, or apparatus. The term "exemplary" is used in the sense of "example" rather than "ideal." The terms "for example" and "such as," and their grammatical equivalents, unless expressly stated otherwise, should be understood to follow the term "without limitation."

[0057] As used herein, the term "about" is intended to account for variations due to experimental error. When applied to numerical values, the term "about" may mean a difference of + / - 5% from the disclosed numerical value, unless a different variation has been specified. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In addition, all ranges are understood to include endpoints, for example, 1 centimeter (cm) to 5 cm will include lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0058] It should be noted that all numerical values ​​disclosed or claimed herein (including all disclosed values, limitations, and ranges) may have a variation of + / - 5% from the disclosed numerical values ​​unless a different variation has been specified.

[0059] As used herein, the term "polypeptide" refers to any amino acid polymer having more than about 20 amino acids covalently linked by amide bonds. A protein comprises one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide may be a protein, and a protein may comprise multiple polypeptides to form a single functional biomolecule.

[0060] Post-translational modifications can modify or alter the structure of a polypeptide. For example, disulfide bonds (e.g., SS bonds between cysteine ​​residues) can form after translation of certain proteins. Some disulfide bonds are essential for the correct structure, function, and interaction of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins can undergo other post-translational modifications such as lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of sugar groups to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface interactions in which the polypeptide participates.

[0061] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins for research or treatment, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Protein of interest (POI, target protein) may include any polypeptide or protein to be isolated, purified or otherwise prepared. POI may include polypeptides produced by cells, including antibodies.

[0062] As used herein, the term "antibody" includes immunoglobulins composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Typically, antibodies have a molecular weight of more than 100 kDa, such as between 130 kDa and 200 kDa, such as about 140 kDa, 145 kDa, 150 kDa, 155 kDa or 160 kDa. Each heavy chain comprises a heavy chain variable region (heavy chain variable region, abbreviated herein as HCVR or VH) and a heavy chain constant region (heavy chain constant region). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (light chain variable region, abbreviated herein as LCVR or VL) and a light chain constant region (light chain constant region). The light chain constant region comprises one domain CL. The VH and VL regions can be further subdivided into highly variable 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 in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3).

[0063] One class of immunoglobulins, called immunoglobulin G (IgG), for example, is common in human serum and comprises four polypeptide chains—two light chains and two heavy chains. Each light chain is linked to a heavy chain via a cystine disulfide bond, and the two heavy chains are bonded to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. With respect to IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass has a different constant region and therefore may have different effector functions. In some embodiments described herein, a biopharmaceutical product may comprise a target polypeptide, including IgG. In at least one embodiment, the target polypeptide comprises IgG4.

[0064] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtained, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived, for example, from complete antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding variable and optionally constant domains of antibodies. Such DNA is known and / or directly available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced or chemically manipulated by using molecular biological techniques, for example, arranging one or more variable and / or constant domains into a suitable configuration, or introducing codons, generating cysteine ​​residues, modifying, adding or deleting amino acids, and the like.

[0065] Biopharmaceutical products (e.g., target molecules, polypeptides, antibodies) can be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia asp.), or mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotes and eukaryotes (single or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, S. pombe, P. pastoris, P. methylica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, insect cells infected with bacculovirus, Trichoplusiani, etc.), non-human animal cells, human cells, or cell fusions such as fusomas or quadromas. In some embodiments, the cell can be a human, monkey, ape, hamster, rat or mouse cell. In some embodiments, the cell can be a eukaryotic cell and can be selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, mMT 060562, Setley cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells may contain one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6 TM cell).

[0066] The term "target molecule" may be used herein to refer to a target polypeptide (e.g., an antibody, antibody fragment, AAV particle, viral protein, or other protein or protein fragment), or to a target polypeptide that is intended to be manufactured, isolated, purified, and / or included in a pharmaceutical product (e.g., an adeno-associated virus (AAV) or other molecule for therapeutic use). Although the methods according to the present disclosure may be related to target polypeptides, they may be applicable to other target molecules. For example, AAV may be prepared according to a suitable method (e.g., depth filtration, affinity chromatography, etc.), and a mixture including AAV may be subjected to the methods according to the present disclosure. Before or after following one or more methods of the present disclosure, additional procedures may be performed on the mixture including AAV (e.g., removing "empty cassettes" or AAV that does not contain a target sequence).

[0067] In some embodiments, the target molecule is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or a tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0068] In some embodiments, the target molecule (e.g., antibody) is selected from anti-programmed cell death 1 antibody (e.g., anti-PD1 antibody described in U.S. Patent Application Publication No. US2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., anti-PD-L1 antibody described in U.S. Patent Application Publication No. US2015 / 0203580A1), anti-D114 antibody, anti-angiopoetin-2 antibody (e.g., anti-ANG2 antibody described in U.S. Patent No. 9402898), anti-angiopoetin-Like 3 antibody (e.g., anti-AngPtl3 antibody described in U.S. Patent No. 9018356), anti-platelet derived growth factor receptor antibody (e.g., anti-AngPtl3 antibody described in U.S. Patent No. 9018356), anti-D114 antibody, anti-angiopoetin-2 antibody (e.g., anti-ANG2 antibody described in U.S. Patent No. 9402898), anti-angiopoetin-Like 3 antibody (e.g., anti-AngPtl3 antibody described in U.S. Patent No. 9018356), anti-platelet derived growth factor receptor antibody (e.g., anti-D114 antibody), anti-angiopoetin-2 antibody (e.g., anti-ANG2 antibody described in U.S. Patent No. 9402898), anti-angiopoetin-Like 3 antibody (e.g., anti-AngPtl3 antibody described in U.S. Patent No. 9018356), anti-D114 antibody, anti-angiopoetin-2 antibody (e.g., anti-ANG2 antibody described in U.S. Patent No. 9018356), anti-D114 antibody, anti-angiopo Antibodies include, for example, anti-PDGFR antibodies described in U.S. Patent No. 9265827, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies described in U.S. Patent No. 9302015), anti-complement 5 antibodies (e.g., anti-C5 antibodies described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies described in U.S. Patent No. 9132192 or anti-EGFRvIII antibodies described in U.S. Patent Application Publication No. US2015 / 0259423A1), anti-proprotein convertase subtilisin-9 antibodies (e.g., anti-PDGFR antibodies described in U.S. Patent No. 9265827), anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies described in U.S. Patent No. 9302015), anti-complement 5 antibodies (e.g., anti-C5 antibodies described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies described in U.S. Patent No. 9132192 or anti-EGFRvIII antibodies described in U.S. Patent Application Publication No. US2015 / 0259423A1), Kexin-9 antibody) (e.g., anti-PCSK9 antibody described in U.S. Patent No. 8062640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth and differentiation factor-8 antibody (anti-Growth And DifferentiationFactor-8antibody (e.g., anti-GDF8 antibody described in U.S. Patent No. 8871209 or 9260515, also known as anti-myostatin antibody), anti-glucagon receptor (e.g., anti-GCGR antibody described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibody, anti-IL1R antibody, interleukin 4 receptor antibody (interleukin 4 receptor antibody) (e.g., anti-IL4R antibody described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. 8735095 or 8945559), anti-interleukin 6 receptor antibody (anti-interleukin 6 receptor antibody) (e.g., anti-IL6R antibody described in U.S. Patent No. 7582298, 8043617 or 9173880), anti-interleukin 33 (anti-interleukin 33) (e.g., the anti-IL33 antibody described in U.S. Patent Application Publication No. US20140271658A1 or US2014 / 0271642A1), anti-respiratory syncytial virus antibody (e.g., the anti-RSV antibody described in U.S. Patent Application No. US2014 / 0271653A1), anti-differentiation cluster 3 (e.g., the anti-CD3 antibody described in U.S. Patent Application Publication No. US2014 / 0088295A1 and US20150266966A1 and U.S. Application No. 62 / 222605), anti-differentiation cluster 20 (e.g., the anti-CD20 antibody described in U.S. Patent Application Publication No. US2014 / 0088295A1 and US20150266966A1 and U.S. Patent No. 7879984), anti-differentiation cluster 48 ... ofDifferentiation-48) (e.g., anti-CD48 antibody described in U.S. Patent No. 9228014), anti-Fel d1 antibody (such as described in U.S. Patent No. 9079948), anti-Middle East Respiratory Syndrome virus (such as described in U.S. Patent No. 9079948),virus) (e.g., anti-MERS antibodies), anti-Ebola virus antibodies (e.g., Regeneron's REGN-EB3), anti-CD19 antibodies, anti-CD28 antibodies, anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-Erb3 antibodies, anti-Zikavirus antibodies, anti-Lymphocyte Activation Gene 3 (e.g., anti-LAG3 antibodies or anti-CD223 antibodies), and anti-Activin A antibodies. Each of the U.S. patents and U.S. patent publications mentioned in this paragraph are incorporated by reference in their entirety.

[0069] In some embodiments, the target molecule (e.g., bispecific antibody) is selected from anti-CD3 x anti-CD20 bispecific antibody, anti-CD3 x anti-mucin 16 bispecific antibody, and anti-CD3 x anti-prostate specific membrane antigen bispecific antibody. In some embodiments, the target molecule is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab.

[0070] In some embodiments, the target molecule is a recombinant protein (e.g., an Fc fusion protein) comprising an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein, which comprises one or more extracellular domains of a receptor coupled to an Fc portion. In some embodiments, the Fc portion comprises a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains that bind a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as IL-1trap (e.g., rilonacept, which comprises 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, the entire contents of which are incorporated herein by reference), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptor Flk1 fused to the Ig domain 3 of the VEGF receptor Flt1 fused to the Fc of hIgG1; see U.S. Pat. Nos. 7,087,411 and 7,279,159, both of which are incorporated by reference in their entirety). In other embodiments, the Fc fusion protein is a ScFv-Fc-fusion protein, which comprises one or more of one or more antigen binding domains, such as a variable heavy chain fragment and a variable light chain fragment of an antibody coupled to an Fc portion.

[0071] Charge heterogeneity in biopharmaceutical products may affect the safety and efficacy of the resulting drug and may be monitored by biopharmaceutical product manufacturers and regulatory agencies. For example, certain charge variants of an intended biopharmaceutical product may exhibit different pharmacokinetics, biological activity, and / or stability compared to an intended biopharmaceutical product. For example, in the case of antibodies, certain isotypes (e.g., charge variants) may degrade or undergo other changes to form less effective molecules. Improper storage conditions, prolonged storage time, oxidation, or other environmental stresses can affect the effectiveness of biopharmaceutical products and may affect the relative populations of charge variants in a sample.

[0072] The charge heterogeneity of biopharmaceutical products can be monitored to ensure batch-to-batch consistency during the manufacturing process. Some methods for characterizing charge variants and generating charge variant profiles are limited to understanding complete protein or polyprotein structures. However, charge variants can form within polypeptides or protein subunits, and the characterization of larger structures may lack the details necessary for characterizing charge variants of individual polypeptides or subunits. For example, the charge variant profiles of the light and / or heavy chains of an antibody may provide more information than the charge variant profile of a complete antibody. As another example, the charge variant profile of a single viral protein may provide more information than the charge variant profile of a complete AAV (e.g., a complete capsid protein).

[0073] Capillary isoelectric focusing (CIEF) has been used to characterize charge heterogeneity and separate charge variants according to their isoelectric point (pI). Depending on the application, CIEF may also be referred to as imaged capillary isoelectric focusing (iCIEF). As described below, the antibody charge variant distribution profile generated by iCIEF includes a peak spectrum arranged by pI, including a major main peak, as well as peaks of acidic and basic species on both sides of the main peak.

[0074] Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) can separate polypeptides (e.g., protein subunits) based on isoelectric points in one dimension and separate polypeptides based on size in two dimensions. This two-dimensional separation has been used to understand the individual charge heterogeneity associated with the heavy and light chains of antibodies. However, 2D-PAGE can only provide a qualitative assessment of the charge isotypes and cannot be used to quantify the proportions of individual charge isotypes relative to each other. In addition, 2D-PAGE is time-consuming and labor-intensive and can result in streaking, which reduces the overall resolution of analysis and detection of low-abundance isotypes.

[0075] To address the limitations of 2D-PAGE, a method using size-exclusion chromatography (SEC) and iCIEF was developed. This method, called ChromiCE, can be used to detect and quantify charge variants corresponding to the heavy and light chains of an antibody. ChromiCE involves reducing and denaturing the antibody and loading the reduced and denatured antibody on a size exclusion column to separate the heavy and light chain components. The separated heavy and light chains can be loaded individually on an isoelectric focusing platform, and iCIEF can be used to identify and quantify charge isoforms associated with the heavy and light chains.

[0076] Unlike 2D-PAGE, ChromiCE provides quantitative results. However, due to the time-consuming separation of light and heavy chains, data collection and analysis for ChromiCE may take up to a week. In addition, the mobile phase used in the SEC step of ChromiCE utilizes a buffer with a high urea concentration (e.g., approximately 8 M urea), which may alter the sample and negatively affect the life of the column and the associated instrumentation.

[0077] Embodiments of the present disclosure may include methods for identifying and quantifying charge variants of biopharmaceutical products, such as methods that address the above-mentioned shortcomings of 2D-PAGE and ChromiCE methods. Embodiments of the present disclosure may be used to identify and quantify charge variants of natural polypeptides, such as natural antibodies and / or natural AAV particles. In addition or alternatively, embodiments of the present disclosure may be used to identify and quantify charge variants of reduced and denatured polypeptides. Reduced and denatured polypeptides that can be analyzed using the methods of the present disclosure include antibodies (e.g., heavy chains, light chains, F(ab')2 fragments, Fc fragments, or other polypeptides) and / or reduced and denatured AAV particles (e.g., VP proteins or other polypeptides). The method disclosed herein for identifying and quantifying charge variants of biopharmaceutical products may be referred to as an iCIEF-Western method.

[0078] The methods of the present disclosure may comprise the use of capillary isoelectric focusing to separate charge variants based on their isoelectric points. The methods of the present disclosure (e.g., iCIEF-Western methods) do not comprise chromatographic separation (e.g., SEC steps). According to embodiments of the present disclosure, after separating polypeptides based on their isoelectric points, the iCIEF-Western methods utilize peptide-specific antibodies to quantify and characterize specific portions of a protein and the charge variants of the protein.

[0079] In some embodiments, prior to processing the analyte by the methods of the present disclosure (e.g., iCIEF-Western methods), it may be necessary to prepare the analyte. As used herein, "analyte" may refer to a sample, a solution, a mixture, a pharmaceutical product, or other composition comprising a target polypeptide (e.g., an antibody, an AAV particle, or other polypeptide). Preparing the analyte may include preparing a loading buffer comprising the analyte.

[0080] The loading buffer may include one or more components that facilitate separation of charge variants and / or identification of charge variants. For example, the loading buffer may include one or more carrier ampholytes, one or more celluloses (e.g., hydroxypropylmethylcellulose), a pI ladder, a base (e.g., urea), formamide, glycerol, ethylene, propyleneglycerol, water, or a combination thereof. In some embodiments, the loading buffer may include a lysis buffer (e.g., Bicine / CHAPS lysis buffer). The carrier ampholytes may facilitate generation of electrical gradients during separation of charge variants. The carrier ampholytes may include those sold under the trade name Pharmalytes, such as Pharmalytes (3-10) and Pharmalytes (5-8). The pI ladder may provide standardized labeling for the results of the CIEF step using molecules with known pI values. The pI ladder and carrier ampholytes may be selected based on the theoretical pI of the analyte. Containing a base, such as urea, prevents the conversion of substances in the loading buffer into other charge variants. Including a base (e.g., urea) in the loading buffer may help maintain the denatured state of polypeptide fragments (e.g., denatured antibody fragments or denatured viral proteins) in the analyte.

[0081] An exemplary sample preparation includes preparation of a loading buffer master mix, and then preparation of a sample including the loading buffer and the analyte. Preparation of the loading buffer master mix may include dissolving hydroxypropyl methylcellulose in a urea solution (e.g., a lysis buffer including at least 6 M urea) to produce a 1 weight percent (wt.%) hydroxypropyl methylcellulose solution having a urea solution concentration of at least 6 M. For example, a 1 wt.% hydroxypropyl methylcellulose solution may have a urea concentration of approximately 8 M. The loading buffer master mix may be generated according to the ratios shown in Table 1. Table 1 Reagents Volume percentage (vol.%) 1wt.% Hydroxypropyl methylcellulose solution About 78% Carrier ampholytes About 18% pI ladder About 4%

[0082] After preparing the loading buffer master mix, the sample for iCIEF-Western analysis can be prepared. The sample may include analyte, water, lysis buffer, loading buffer master mix or a combination thereof. The urea concentration of the sample may be greater than or equal to about 8M. The cellulose (e.g., hydroxypropyl methylcellulose) concentration of the sample may be about 1wt.%. In an exemplary embodiment, the sample may be prepared according to the ratio shown in Table 2. Table 2

[0083] Another exemplary sample preparation is shown below in Table 3. The sample preparation in Table 3 can be used with analytes including adeno-associated virus. Table 3

[0084] As described herein, the analyte may be reduced and denatured prior to analysis. Before preparing the sample buffer, the reduced and denatured analyte may be buffer exchanged. The buffer exchanged with the buffer may include urea (e.g., about 8M urea), sodium phosphate (e.g., about 10mM sodium phosphate), Tris (2-carboxyethyl) phosphate (TCEP) (e.g., about 1mM TCEP), and / or the analyte. Another exemplary sample preparation is shown in Table 4 below for use with certain buffer exchanged analytes. For example, the sample preparation in Table 4 may be used, for example, a reduced and denatured analyte comprising an antibody. Table 4 Reagents Volume percentage (vol.%) 1wt.% Hydroxypropyl methylcellulose solution About 46% 12M urea solution About 44% Carrier ampholytes About 2.7% pI ladder About 2.2% Analytes About 2–5%

[0085] Sample preparation and loading buffers can be adjusted to allow for adequate separation and stability of the polypeptides. For example, depending on the characteristics of the analyte (e.g., polypeptide concentration, viscosity, or other physiochemical characteristics), loading buffers and sample preparation components can be adjusted. In addition, the parameters of the CIEF separation can also be adjusted to accommodate the physiochemical characteristics of the sample. For example, if the peptide concentration of the analyte is greater than the average concentration, the viscosity of the resulting sample may be greater than the average. The loading time and focusing associated with the CIEF separation can be adjusted to account for increased sample viscosity. As with other examples, the choice of ampholytes, ampholyte concentrations, sample solution viscosity, and sample analyte concentrations may need to be adjusted to allow for adequate separation and stability of polypeptide charge variants.

[0086] The method disclosed herein can be used to separate and quantify the charge variants of reduced and denatured polypeptides. Before performing iCIEF-Western analysis, the analyte can be incubated in a solution in which the constituent polypeptides of the analyte are reduced and denatured. In some embodiments, the analyte can be cultured (incubated) in this solution for about an hour. For example, the analyte can be incubated in a solution comprising 6M guanidine HCl and 10mM tris(2-carboxyethyl)phosphine (TCEP) to reduce and denature the polypeptides of the analyte. In the example of an analyte including an antibody, the antibody can be reduced and denatured to obtain a solution with denatured light and heavy chains. Other types of denaturants and / or other types of protein digests can be used to further characterize the charge variants of the target analyte. In some embodiments, proteases such as IdeS, papain, or pepsin can be used to digest the target polypeptide into fragments.

[0087] The reduced and denatured analyte can then be buffer exchanged with a buffer comprising about 35 mM to about 50 mM sodium phosphate, about 1 mM to about 10 mM TCEP, about 6 M to about 8 M urea, and a pH of about 6.0. The buffer exchanged analyte can then be combined with a loading buffer and a lysis buffer comprising about 10 M to about 12 M urea. For example, the buffer exchanged analyte can be combined with a loading buffer master mix and a lysis buffer according to the ratios shown in Table 2. Additionally, or alternatively, the buffer exchanged analyte can be combined with other sample buffer components according to the ratios shown in Table 3 or Table 4.

[0088] After preparing the sample, the sample can be loaded into the capillary. The sample can be run in parallel, for example, in parallel capillaries. In some embodiments, the sample is loaded on a well plate, and an automated fluid handling system transports the sample and other reagents to the capillary. Since an electric current is applied to the sample, the types of substances in the sample (e.g., ampholytes, pI ladders, charge variants in the sample) can be separated according to their isoelectric points (PI). For example, 21000 μWatt of electricity can be applied for 30 minutes. The running time of isoelectric focusing separation can be adjusted according to the composition of the sample (e.g., conductivity, viscosity or other characteristics). In addition, or alternatively, different sample compositions may require different focusing times to achieve the best resolution of charge variants.

[0089] As electrical energy is applied to the capillary, species accompanying the sample may form a gradient based on isoelectric points, such that the species with the lowest isoelectric point is located at the first end of the capillary and the species with the highest isoelectric point is located at the second end of the capillary. iCIEF separations can be accomplished using an isoelectric focusing platform, such as the PeggySue TM instrument.

[0090] After separating the charge variants in the sample according to their isoelectric points, specific antibodies can be used to detect the polypeptides and quantify their relative abundance. In some embodiments, the separated charge variants can be immobilized on the walls of the capillaries. The matrix including the capillaries can be washed to remove excess reagents. Prior to introducing the detection antibodies, the matrix including the capillaries may be blocked. For example, the matrix can be incubated in a bovine serum albumin solution for at least 30 minutes.

[0091] After blocking, the matrix can be washed and detection antibodies can be added. As previously described, after the polypeptide is reduced and denatured, the charge variants of the polypeptide fragments can be quantified separately using specific detection antibodies. In the example of iCIEF-Western analysis of antibodies, specific anti-human FC antibodies can be used to detect and quantify heavy chain charge variants, while anti-human Kappa antibodies can be used to detect and quantify light chain charge variants. In iCIEF-Western analysis of AAV particles, specific anti-VP proteins can be used to detect and quantify viral protein variants. Cultivation in specific detection antibodies can be called primary culture. The matrix can be cultured in antibodies specific to one or more polypeptides for at least about 60 minutes. In some examples, such as anti-human FC protein antibodies and anti-human Kappa protein antibodies for detecting the heavy and light chains of antibodies, suitable detection antibody concentrations can be about 1 μg / mL to about 10 μg / mL. In some embodiments, suitable detection antibody concentrations can be about 2 μg / mL to 400 μg / mL.

[0092] Other types of specific detection can be used to further characterize the charge variants of the polypeptide. For example, instead of incubating the substrate in a detection antibody, the substrate can be incubated in an antigen that has specific binding capacity for the target polypeptide.

[0093] After the primary incubation, the matrix can be washed and a secondary incubation can be performed. The secondary incubation step can last for at least about 120 minutes. The secondary incubation can bind one or more reporter molecules to the detection antibody. The reporter molecule may be able to produce a detectable signal (e.g., a chemiluminescent signal) that can be used to quantify the charge variant. The intensity of the detectable signal can be related to the concentration or abundance of the reporter molecule. Due to the specificity of the detection antibody, the concentration or abundance of the reporter molecule (e.g., the intensity of the resulting signal) is related to the concentration or abundance of the target charge variant.

[0094] After the secondary incubation, the matrix can be washed and one or more reporters can be introduced into the capillary. The reporter can interact with the reporter molecule to produce a detectable signal corresponding to each charge variant. Since the substances in the sample have been separated according to their isoelectric points, the position of each signal from the interaction of the reporter with the reporter molecule will indicate the charge variant corresponding to the signal. The intensity of the detectable signal can correspond to the relative abundance of the corresponding charge variant at that position. The intensity of the detectable signal can be plotted according to the isoelectric point at the signal position to generate an electropherogram.

[0095] The peak area of ​​the electropherogram can be calculated with a Gaussian distribution algorithm. The area of ​​a single peak divided by the total area under all peaks in the electropherogram can correspond to the relative abundance of the charge variants corresponding to the single peak. The electropherogram can be analyzed with the aid of a processor and / or software such as Compass software (e.g., version 4.0.0). The charge variant distribution diagram may refer to an electropherogram, or to the relative abundance of charge variants quantitatively measured from the isoelectric point of an electropherogram and / or a charge variant. More examples of electropherograms and charge variants being quantitatively measured via calculated peak areas are described below.

[0096] In one or more embodiments, an exemplary detection scheme uses horseradish peroxidase (HRP) conjugated to a streptavidin protein as a reporter molecule. For example, after primary incubation in the detection antibody, the matrix can be washed and incubated in a mixture comprising HRP conjugated to a streptavidin protein. The streptavidin protein can preferentially bind to the biotin of the detection antibody, and the HRP can cause a chemiluminescent reaction when luminol-peroxide is introduced. After a secondary incubation and subsequent washing, the luminol-peroxide can be injected into the capillary, thereby generating a chemiluminescent signal from the interaction of the luminol-peroxide with the HRP.

[0097] Because the isotypes of each sample are separated on a pi gradient prior to detection, the chemiluminescence of the measured peak (e.g., at 425 nm for a detection antibody with an HRP conjugate) correlates to the relative abundance of the isotype represented by the peak. The electropherogram generated based on the measured chemiluminescent signal can be used to identify the charge variants of each polypeptide analyzed and quantify the relative abundance of the charge variants.

[0098] When developing an iCIEF-Western protocol, several conditions may need to be adjusted depending on the nature of the sample. For example, the concentration of the detection antibody may need to be adjusted to improve the signal-to-noise ratio. Application-specific factors such as target peptide concentration, range of charge variants, and binding affinity of the detection antibody should be considered when determining the appropriate detection antibody concentration.

[0099] Although some iCIEF-Western methods have been described above for antibodies and portions of antibodies, they can be used to characterize the charge variant profile of any protein biopharmaceutical product. In one embodiment, iCIEF-Western analysis can be used to characterize the charge variant profile of one or more proteins produced by adeno-associated virus (AAV).

[0100] As known to those of ordinary skill in the art, adeno-associated viruses (AAVs) can be used to deliver corrective genes to cells or tissues of a target organism. For example, a therapeutic gene can be inserted into the AAV genome, and with the help of a helper virus, AAV can deliver the therapeutic gene to one or more target cells. AAV is non-pathogenic and non-toxic. Different AAV serotypes can be used depending on the application. Each AAV serotype may have different cell transduction efficiencies for different types of cells, making certain AAV serotypes more suitable for targeting certain organ systems, organs and / or tissues. For example, AAVrh10 and AAV2 have the highest cell transduction efficiency for brain tissue; AAV2 and AAV4 have the highest cell transduction efficiency for eye tissue; AAV6 and AAV1 have the highest cell transduction efficiency for lung tissue; AAV1 and AAV8 have the highest cell transduction efficiency for muscle tissue; AAV8, AAV5 and AAV9 have the highest cell transduction efficiency for liver tissue; AAV9 and AAVrh10 have the highest systemic cell transduction efficiency.

[0101] There are three major AAV capsid proteins, commonly referred to as VP1, VP2, and VP3. The sequence of the AAV capsid protein may vary depending on the AAV serotype. Therefore, a detection antibody suitable for analyzing charge variants of polypeptides produced by one AAV serotype may not be suitable for other serotypes. In some embodiments, the methods of the present disclosure include developing detection antibodies specific for AAV capsid proteins. For example, the methods of the present disclosure may include developing rabbit polyclonal antibodies that bind to the capsid protein of a specific AAV serotype.

[0102] In some cases, the VP2 sequence includes the VP3 sequence, and the VP1 sequence includes the VP2 sequence. In other words, a detection antibody specific for VP3 can also bind to VP1 and VP2, and a detection antibody specific for VP2 can also bind to VP1. For example, one or more recombinant peptides from a portion of the VP1 sequence that is not shared with VP2 and VP3 can be used as rabbit antigens to produce polyclonal antibodies specific to VP1; one or more recombinant peptides from a portion of the VP2 sequence that is not shared with VP3 can be used as rabbit antigens to produce polyclonal antibodies specific to VP2 and VP1; one or more recombinant peptides from VP3 can be used as rabbit antigens to produce polyclonal antibodies specific to VP3, VP2, and VP1. Methods using such detection antibodies can still quantify the charge variants of the AAV capsid protein by comparing the relative electropherograms produced by each detection antibody. Other examples of iCIEF-Western analysis of AAV capsid proteins are described below (e.g., Examples 12 to 25).

[0103] As described herein, the methods disclosed herein (e.g., iCIEF-Western methods) can be used to generate charge variant distribution maps of target polypeptides within a sample. The generated charge variant distribution maps can be used to validate the iCIEF-Western method, validate the manufacturing conditions of a batch, and assess the effects of manufacturing or storage conditions on the stability of a biopharmaceutical product.

[0104] For example, charge variants can be quantified for two different batches of the same biopharmaceutical product. Quantifying the relative amounts of charge variants and verifying that they match known or theoretical charge variant distribution profiles can be used to validate quantitation methods developed via iCIEF-Western.

[0105] In addition, the charge variant profiles of different batches can be compared to determine, confirm or validate the process conditions for manufacturing the batch. If the charge variant profile of the first batch of antibodies under validated production and environmental conditions is known, it can be compared with the charge variant profile of the newly produced batch to confirm that the batch has not been subjected to excessive heat or environmental stress (stress). In addition, if the manufacturing conditions or equipment are changed, the charge variant profile of the batch manufactured under the new conditions will be compared with the charge variant profile of the batch manufactured under the validated conditions to evaluate the effectiveness of the changes in manufacturing conditions and / or equipment.

[0106] Reference Figure 1 , a flow chart of an exemplary method 100 for separating and quantifying charge variants of natural polypeptides within an analyte is shown. Method 100 may include preparing a sample buffer containing an analyte (step 101). The sample buffer may include one or more carrier ampholytes, one or more celluloses (e.g., hydroxypropylmethylcellulose), a pI ladder, a base (e.g., urea), water, or a combination thereof. In some embodiments, the sample buffer is prepared according to the ratios shown in Table 1 and Table 2. Additionally or alternatively, the sample buffer may be prepared according to the ratios shown in Table 3 or Table 4. Method 100 may include separating charge variants in the sample buffer using capillary isoelectric focusing (step 102). For example, the sample buffer may be loaded into a capillary and separated using an isoelectric focusing platform.

[0107] After isolating charge variants of a natural polypeptide (e.g., an antibody, AAV particle, or other polypeptide), method 100 may include incubating the separated charge variants in the presence of a detection antibody (step 103). The detection antibody may be specific for one or more target polypeptides or target amino acid sequences in the analyte. After the charge variant conjugates of the detection antibody and the target polypeptide, the relative abundance of each charge variant may be quantified based on the signal corresponding to the detection antibody (step 104).

[0108] Reference Figure 2, a flow chart of an exemplary method 150 for separating and quantifying charge variants of reduced and denatured polypeptides in an analyte is shown. Method 150 may include reducing and denaturing polypeptides in the analyte (step 151). For example, the analyte may be incubated in a solution comprising 6M guanidine hydrochloride and 10mM Tris (2-carboxyethyl) phosphate (TCEP) to reduce and denature the polypeptides of the analyte. Method 150 may include preparing a sample buffer comprising the analyte (step 152). The reduced and denatured sample may be buffer exchanged into a buffer comprising 8M urea, 35mM sodium phosphate, and 1mM TCEP. The buffer exchanged sample may then be used to prepare a sample buffer according to the proportions shown in Tables 1 and 2. Additionally or alternatively, the sample buffer may be prepared according to the proportions shown in Tables 3 or 4.

[0109] Method 150 may include using capillary isoelectric focusing to separate charge variants in a sample buffer (step 153). For example, a sample buffer may be loaded into a capillary and separated using an isoelectric focusing platform. After the charge variants of reduced and denatured polypeptides (e.g., antibody heavy chains, antibody light chains, viral proteins, or other polypeptides) are separated, method 150 may include incubating the separated charge variants in the presence of a detection antibody (step 154). The detection antibody may be specific to one or more target polypeptides or target amino acid sequences in the analyte. After the detection antibody is engaged with the charge variant of the target polypeptide, the relative abundance of each charge variant may be quantified based on the signal corresponding to the detection antibody (step 155).

[0110] As described herein, methods of separating and quantifying polypeptide charge variants (eg, methods 100, 150) can include quantifying the relative abundance of the charge variants based on a signal corresponding to a detection antibody. Figure 3 , a flow chart showing an exemplary method 200 for quantifying the relative abundance of charge variants based on a signal corresponding to a detection antibody.

[0111] Method 200 may include immobilizing the separated charge variants within a capillary (step 201). For example, after separating the charge variants along an isoelectric gradient within the capillary, the charge variants can be irreversibly immobilized on the wall of the capillary. The method may include incubating the immobilized charge variants in a blocking buffer (step 202). In some embodiments, the capillary (e.g., a matrix comprising the capillary) may be washed prior to incubating the immobilized charge variants in a blocking buffer.

[0112] Method 200 may further include incubating the fixed charge variant in a detection antibody (step 203). Incubating the fixed charge variant in the detection antibody may be referred to as a primary incubation. In some embodiments, the capillary (e.g., a matrix comprising the capillary) may be washed after removing the blocking buffer and before the primary incubation. As described herein, one or more detection antibodies may be used that are specific for one or more polypeptides (e.g., a target polypeptide). For example, a biotinylated goat anti-human IgG Fc-specific polyclonal antibody (pAb) may be used to detect antibody heavy chains, while a biotinylated goat anti-human kappa pAb may be used to detect antibody light chains. In other examples, antibodies that specifically bind to certain viral proteins or protein subunits may be used as detection antibodies. During the initial incubation, the detection antibody may be coupled to the target polypeptide.

[0113] Method 200 may include incubating the immobilized charge variant bound to the detection antibody in the reporter molecule (step 204). The incubation in the reporter molecule may be referred to as a secondary incubation. In some embodiments, the capillary (e.g., a matrix comprising the capillary) may be washed between the primary incubation and the secondary incubation. The reporter molecule may preferably bind to the detection antibody. In addition, the reporter molecule may have a functional group or subunit capable of generating a signal (e.g., a bioluminescent or chemiluminescent signal). In some embodiments, the functional group or subunit capable of generating a signal may generate a signal in response to an external stimulus (e.g., a chemical or physical stimulus).

[0114] Method 200 may further include introducing a detection agent into the capillary (step 205). In some examples, the reporter molecule generates a signal only in the presence of the detection agent. In the example of a reporter molecule comprising HRP conjugated to a streptavidin protein, a signal is generated only when the HRP is exposed to luminol-peroxide. Method 200 may also include calculating an isoelectric point corresponding to each charge variant and a peak area corresponding to each charge variant based on the signal generated by the interaction of the detection agent with the reporter molecule (step 206).

[0115] In some embodiments, method 200 can further include determining the relative abundance of each charge variant based on the calculated peak areas (step 207). For example, the peak area corresponding to each charge variant can be divided by the sum of all peak areas to determine the relative abundance of each charge variant.

[0116] In the examples described herein, unless otherwise specified, the monoclonal antibodies may comprise IgG1 and IgG4, and / or be derived from CHO cells.

[0117] In the case where the polypeptide was reduced and denatured, the polypeptide was reduced and denatured in the presence of 6M guanidine hydrochloride and 10mM TCEP at room temperature for one hour unless otherwise stated. The reduced and denatured sample buffer was then exchanged into a buffer containing 8M urea, 35mM sodium phosphate and 1mM TCEP.

[0118] For each embodiment described herein, unless otherwise specified, the iCIEF-Western analytes were contained in a sample buffer prepared with lysis buffer, carrier ampholytes, pI ladder and hydroxypropyl methylcellulose in the proportions shown in Tables 1 and 2. For the examples containing reduced and denatured polypeptides, the buffer-exchanged samples were contained in a sample buffer prepared with lysis buffer, carrier ampholytes, pI ladder and hydroxypropyl methylcellulose in the proportions shown in Tables 1 and 2. The sample buffer, detection antibody, reporter molecule and luminol-peroxide mixture were loaded into a 384-well plate and then automatically injected into the capillaries of the iCIEF instrument. In the examples described herein, the iCIEF separation and analysis were performed using a PeggySue™ instrument manufactured by ProteinSimple. The samples were electrophoresed by applying 21000 μWatts for 30 minutes. Example 1

[0119] As previously described, the iCIEF-Western method can be used to generate a charge variant profile of an analyte. The charge variant profile of an analyte (e.g., a manufacturing batch of a biopharmaceutical product) can be compared to a known charge variant profile of the analyte to validate the iCIEF-Western method.

[0120] Charge variant profiles for reduced and denatured antibodies were generated using ChromiCE from a single manufacturing batch of Antibody 1 and are shown in Figure 4A and 4B As mentioned above, ChromiCE requires a chromatography step to separate the heavy and light chains of antibodies. The electrophoresis of the heavy chain antibody is shown in Figure 4A , and the electrophoresis diagram of the light chain antibody is shown in Figure 4B The electropherogram generated using ChromiCE also showed peaks at isoelectric points 5.85 and 8.40, which correspond to the isoelectric point markers included in the sample buffer.

[0121] Charge variant profiles of reduced and denatured antibodies were generated from the same manufacturing batch of Antibody 1 using the iCIEF-Western method and are shown in Figure 4CBiotinylated goat anti-human IgG Fc specific polyclonal antibody (pAb) was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent. Figure 4C In the graph, the signal detected from the Fc pAb (eg, the signal corresponding to the heavy chain charge variant) is shown as a solid line, while the signal detected from the kappa pAb (eg, the signal corresponding to the light chain charge variant) is shown as a dashed line.

[0122] Electropherograms generated using iCIEF-Western Figure 4C ) in the electropherogram generated using ChromiCE ( Figure 4A and 4B ) include the same peaks at the same positions (e.g., peaks 301, 302, 303, 304, 305, 311 and 312).

[0123] Reference Figure 4D , charge variant profiles of reduced and denatured antibodies generated from the same manufacturing batch of Antibody 1 using iCIEF-Western. Anti-human H+L antibody was used as the detection antibody, which is not specific for a particular portion of the analyte antibody. Figure 4D As can be seen, the peak of the heavy chain charge variant (peak 301) overlaps with the peak of the light chain charge variant (peak 312). Using multiple detection antibodies in the iCIEF-Western method can improve the detectability of the peak. In addition, using multiple detection antibodies can improve the resolution of adjacent peaks. Example 2

[0124] Charge variant profiles for reduced and denatured antibodies were generated using ChromiCE from a single manufacturing batch of Antibody 2 and are shown in Figure 5A and 5B The electrophoresis diagram of the heavy chain antibody is shown in Figure 5A , and the electrophoresis diagram of the light chain antibody is shown in Figure 5B The electropherogram generated using ChromiCE also showed peaks at isoelectric points 4.65 and 8.40, which correspond to the isoelectric markers included in the sample buffer.

[0125] Charge variant profiles for reduced and denatured antibodies were generated from the same manufacturing batch of Antibody 2 using the iCIEF-Western method and are shown in Figure 5CBiotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain, and biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporter agent. Figure 5C In the graph, the signal detected from the Fc pAb (eg, the signal corresponding to the heavy chain charge variant) is shown as a solid line, while the signal detected from the kappa pAb (eg, the signal corresponding to the light chain charge variant) is shown as a dashed line.

[0126] Electropherograms generated using iCIEF-Western Figure 5C ) in the electropherogram generated using ChromiCE ( Figure 5A and 5B ) include the same peaks at the same positions (e.g., peaks 321, 322, 323, 324, 325, 331, 332 and 333).

[0127] Reference Figure 5D , charge variant profiles of reduced and denatured antibodies generated using iCIEF-Western from the same manufacturing batch of antibody 2. Anti-human H+L antibody was used as the detection antibody, which is not specific for a particular portion of the analyte antibody. Figure 4D As can be seen, some known peaks of the analyte (e.g., peaks 333 and 321) were not detected. Using multiple detection antibodies in the iCIEF-Western method can improve the detectability of the peaks. In addition, using multiple detection antibodies can improve the resolution of adjacent peaks. Example 3

[0128] Charge variant profiles for reduced and denatured antibodies were generated using ChromiCE from a single manufacturing batch of Antibody 3 and are shown in Fig. 6A and 6B ChromiCE analysis was performed in triplicate and the electropherograms from each run were superimposed. Fig. 6A As shown, the electrophoresis diagram of the light chain antibody is as follows Figure 6B The electropherogram generated using ChromiCE also shows peaks at isoelectric points of approximately 5.85 and approximately 9.25, which correspond to the isoelectric point markers included in the sample buffer.

[0129] Charge variant profiles for reduced and denatured antibodies were generated from the same manufacturing batch of Antibody 3 using the iCIEF-Western method and are shown in Figure 6CBiotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain, and biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporter agent. Figure 6C In the graph, the signal detected from the Fc pAb (eg, the signal corresponding to the heavy chain charge variant) is shown as a solid line, while the signal detected from the kappa pAb (eg, the signal corresponding to the light chain charge variant) is shown as a dashed line.

[0130] Electropherograms generated using iCIEF-Western Figure 6C ) in the electropherogram generated using ChromiCE ( Fig. 6A and 6B ) include the same peaks at the same positions (e.g., peaks 341, 342, 343, 344, 345, 346, 351, 352, 353 and 354). Example 4

[0131] In contrast to the reduction and denaturation methods described above, polypeptides (e.g., antibodies) in the analyte can be digested by a protease (e.g., IdeS). IdeS cleaves the antibody into F(ab')2 and Fc fragments. IdeS was used to digest the analyte including Antibody 3 from the same manufacturing batch as used in Example 3. A charge variant distribution profile of the F(ab')2 and Fc fragments was generated using ChromiCE and is shown in Fig. 7A Peaks 361, 362, 363, 364, 365 and 366 correspond to charge variants of the Fc fragment. Peaks 371, 372, 373 and 374 correspond to charge variants of the F(ab')2 fragment. Since the isoelectric point of the Fc fragment is generally lower than that of the F(ab')2 fragment, the peaks corresponding to the Fc fragment do not overlap with the peaks corresponding to the F(ab')2 fragment. The electropherogram generated using ChromiCE also shows peaks at isoelectric points of 5.85 and 9.50, which correspond to the isoelectric point markers included in the sample buffer.

[0132] Charge variant profiles of F(ab')2 and Fc fragments were also generated using the iCIEF-Western method, such as Figure 7B As shown. Anti-human H+L antibody was used as detection antibody, which is not specific for a particular part of the analyte antibody. Figure 7B In the figure, the signal detected from the Fc fragment is shown as a solid line, and the signal detected from the F(ab')2 fragment is shown as a dotted line. The electropherogram generated using iCIEF-Western ( Figure 7B ) in the electropherogram generated using ChromiCE ( Fig. 7A) include the same peaks at the same positions (e.g., peaks 361, 362, 363, 364, 365, 366, 371, 372, 373 and 374). Example 5

[0133] Charge variant profiles for reduced and denatured antibody were generated using ChromiCE from two manufacturing batches (Batch A and B) of Antibody 4 and are shown in Fig. 8A Only the electropherogram of the heavy chain antibody is shown. The electropherogram generated using ChromiCE also shows peaks at isoelectric points 6.14 and 9.22, which correspond to the isoelectric marker included in the sample buffer.

[0134] Charge variant profiles for reduced and denatured antibodies from manufacturing batches A and B were generated using the iCIEF-Western method and are shown in Figure 8B Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent. Figure 8B In the figure, the electropherogram corresponding to batch A is shown as a dotted line, while the electropherogram corresponding to batch B is shown as a solid line.

[0135] Electropherograms generated using iCIEF-Western Figure 8B ) in the electropherogram generated using ChromiCE ( Fig. 8A ) include the same peaks (e.g., peaks 381, 382, ​​383, 384, 385, and 386) at the same positions. In addition, the electropherograms generated using iCIEF-Western have better resolution than those generated using ChromiCE. In addition, the charge variants of the heavy chain can be characterized using iCIEF-Western without the need for a chromatography step. Without being limited by theory, the poor heavy chain resolution associated with ChromiCE may be due to analyte degradation by the high concentration of urea in the size-selective chromatography (SEC) buffer and the long processing time of the SEC step. Example 6

[0136] Charge variant profiles of the heavy chain of reduced and denatured antibodies were generated using ChromiCE from a single manufacturing batch of antibody 5, as shown in Fig. 9A The electropherogram generated using ChromiCE also shows peaks at isoelectric points of approximately 5.85 and approximately 8.4, which correspond to the isoelectric point markers included in the sample buffer.

[0137] The charge variant distribution profiles of the heavy chain of the reduced and denatured antibody were generated using iCIEF-Western from the same manufacturing batch of antibody 5 and are shown in Fig. 9BBiotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0138] Electropherograms generated using iCIEF-Western Fig. 9B ) in the electropherogram generated using ChromiCE ( Fig. 9A ) include the same peaks (e.g., peaks 391, 392, 393, 394, and 395) at the same positions. Peak 393 corresponds to the main species of the heavy chain of Antibody 5; Peaks 391 and 392 correspond to the acidic variants of the heavy chain of Antibody 5; Peaks 394 and 395 correspond to the basic variants of the heavy chain of Antibody 5.

[0139] Electropherograms generated using ChromiCE were also calculated ( Fig. 9A ) and the electropherogram generated using iCIEF-Western ( Fig. 9B The peak areas are plotted as a percentage of the total area under all peaks and are shown in Fig. 9C The peak area calculated based on the ChromiCE electropherogram is represented by a square and a dotted line, and the peak area calculated based on the iCIEF-Western electropherogram is represented by a circle and a solid line. Fig. 9C As shown, based on the ChromiCE electropherogram ( Fig. 9A ) calculated based on the peak area and iCIEF-Western electrophoresis ( Fig. 9B )The peak areas calculated are similar. Example 7

[0140] The charge variant profile of the first batch of antibodies produced by iCIEF-Western can be compared to a known charge variant profile or to a charge variant profile of a second batch of antibodies. Comparison of the charge variant profiles of the first batch of antibodies can verify the process conditions for making the first batch of antibodies.

[0141] The charge variant profile of the reduced and denatured antibody of Antibody 6 from the first batch (Batch A) was compared to the charge variant profile of the reduced and denatured antibody of Antibody 6 from the second batch (Batch B). Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0142] The electropherogram of Fc-based pAb generation containing heavy chain charge variants is shown in Fig. 10A, and the electropherogram generated based on kappa pAb containing light chain charge variants is shown in Fig. 10B The charge variant distribution of batch A is shown as a gray line, and the charge variant distribution of batch B is shown as a black line. Fig. 10A and 10B , the charge variant profile of batch A is the same as the charge variant profile of batch B. For example, the electropherogram of batch A includes the same peaks (e.g., peaks 401, 402, 403, 404, 405, 406, 407, 408, 411, 412, and 413) as the electropherogram of batch B. In addition, the peak areas of batch A can be compared to batch B to ensure that the ratios of each charge variant are the same between batch A and batch B. Example 8

[0143] The charge variant profile of the reduced and denatured antibody of Antibody 7 from the first batch (Batch A) was compared to the charge variant profile of the reduced and denatured antibody of Antibody 7 from the second batch (Batch B). Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0144] The electropherogram of Fc-based pAb generation containing heavy chain charge variants is shown in Fig.11A , and the electropherogram generated based on kappa pAb containing light chain charge variants is shown in Fig. 11B The charge variant distribution of batch A is shown as a gray line, and the charge variant distribution of batch B is shown as a black line. Fig.11A and 11B , the charge variant distribution profile of batch A is the same as the charge variant distribution profile of batch B. For example, the electropherogram of batch A includes the same peaks as the electropherogram of batch B (e.g., peaks 421, 422, 423, 424, 425, 431, and 432).

[0145] In addition, the peak areas of batch A can be compared with batch B to ensure that the ratio of each charge variant is the same between batch A and batch B. The relative peak areas of the heavy chain charge variants of batches A and B are shown in Fig. 11C The relative peak areas of light chain charge variants of batches A and B are shown in Fig.11D . Example 9

[0146] The charge variant profile of the reduced and denatured antibody of Antibody 8 from the first batch (Batch A) was compared with the charge variant profile of the reduced and denatured antibody of Antibody 8 from the second batch (Batch B). Batch A was subjected to thermal stress. Specifically, Batch A was stored at 40°C for three months. Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0147] The electrophoresis diagram based on Fc pAb (heavy chain detection) is shown in Fig. 12A , and the electrophoresis diagram based on kappa pAB detection is shown in Fig. 12B .for Fig. 12A and 12B In both, the electropherogram of batch A (the batch subjected to thermal stress) is represented by the gray line, while the electropherogram of batch B (the batch not subjected to thermal stress) is represented by the black line. It is obvious from the charge variant distribution diagram of the native antibody that the relative proportions of the charge variants are different between the batch subjected to thermal stress and the batch not subjected to thermal stress. However, without baseline separation of the peaks, it is not possible to reliably quantify the individual isotypes.

[0148] The charge variant profile of the reduced and denatured antibody of Antibody 8 from Batch A was compared to the charge variant profile of the reduced and denatured antibody of Antibody 8 from Batch B. Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0149] The electrophoresis diagram based on Fc pAb (heavy chain detection) is shown in Fig.13A , while the electrophoresis diagram based on kappa pAB (light chain detection) is shown in Fig. 13B .for Fig.13A and 13B In both cases, the electropherogram of batch A (batch subjected to thermal stress) is represented by the grey line, and the electropherogram of batch B (batch not subjected to thermal stress) is represented by the black line.

[0150] The isoelectric points of the dissociated heavy and light chains are different from those of the intact antibody. Therefore, the iCIEF-Western method is able to generate charge variant profiles with baseline resolution and quantify the individual charge isotypes of the light and heavy chains.

[0151] Reference Fig.13A, peak 442 corresponds to the major charge variant of the heavy chain of antibody 8, peak 441 corresponds to the acidic charge variant of the heavy chain, and peak 443 corresponds to the basic charge variant of the heavy chain. Fig. 13B , peak 452 corresponds to the major charge variant of the light chain of antibody 8, and peak 451 corresponds to the acidic charge variant of the light chain of antibody 8. The peak areas were calculated based on the electropherograms of batch A (heat stressed) and batch B (not heat stressed) and are summarized in Table 5. The area of ​​each peak was integrated and divided by the total area of ​​all peaks to determine the relative peak area corresponding to each peak (e.g., corresponding to each detected charge variant). Table 5

[0152] For both light and heavy chains, the batches subjected to heat stress had lower relative abundance of the major isotype and higher abundance of the acidic isotype compared to the batches not subjected to heat stress. A charge variant profile can be generated for an unknown batch of antibodies and compared to the charge variant profiles of the batch subjected to heat stress and the batch not subjected to heat stress. Comparison of the charge variation profiles can provide information on whether the unknown batch was subjected to heat stress or other adverse storage conditions. Example 10

[0153] When developing an iCIEF-Western method for characterizing charge variants of a polypeptide, it may be useful to adjust the concentration of the detection antibody. Each combination of a target polypeptide and a detection antibody may require a different concentration of the detection antibody in the primary incubation step. iCIEF-Western analysis may be performed using different concentrations of the detection antibody to determine the appropriate detection antibody concentration.

[0154] Using the iCIEF-Western method with different concentrations of detection antibody, charge variant distribution profiles were generated for reduced and denatured analytes including antibody 9 and are shown in Fig.14A and 14B . Used to generate Fig.14A The method for the charge variant profile shown involves a primary incubation in the detection antibody at a concentration of approximately 100 μg / mL. Fig. 14B The method for the charge variant profile shown included a primary incubation in the detection antibody at a concentration of approximately 2.5 μg / mL. Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for the reduced and denatured antibody heavy chain. Biotinylated goat anti-human kappa pAb was used as the detection antibody for the reduced and denatured antibody light chain. Streptavidin protein conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent. Fig.14A and 14BIn the graph, the signal detected from the Fc pAb (eg, the signal corresponding to the heavy chain charge variant) is shown as a solid line, while the signal detected from the kappa pAb (eg, the signal corresponding to the light chain charge variant) is shown as a dashed line.

[0155] Reference Fig.14A iCIEF-Western analysis with excessive concentrations of detection antibody may result in excessive baseline noise, detection of false peaks, poor resolution, and / or skewed or asymmetric peaks. Fig. 14B , iCIEF-Western analysis performed with appropriate concentrations of detection antibody may include a consistent baseline with limited background noise, and separated peaks resolved to the baseline. Adjusting the detection antibody concentration can reduce baseline noise, thereby increasing the signal-to-noise ratio of the measurement, facilitating improved detection of low-abundance charge variants within the dynamic range. Example 11

[0156] In order to correctly characterize the VP protein using iCIEF-Western, antibodies specific to the VP protein can be developed. Antibodies specific to the VP protein were developed for the AAV8 serotype AAV. A recombinant portion from a portion of the VP1 sequence that is not shared with VP2 and VP3 was used as a rabbit antigen to produce a polyclonal antibody specific to VP1. Two recombinant peptides from a portion of the VP2 sequence that is not shared with VP3 were used as rabbit antigens to produce polyclonal antibodies specific to VP2 and VP1. Recombinant peptides from VP3 were used as rabbit antigens to produce polyclonal antibodies specific to VP3, VP2 and VP1. In short, a polyclonal antibody that specifically binds to VP3, VP2 and VP1 can be referred to as an anti-VP3 / VP2 / VP1 antibody, a polyclonal antibody that specifically binds to VP2 and VP1 can be referred to as an anti-VP2 / VP1 antibody, and a polyclonal antibody that specifically binds to VP1 can be referred to as an anti-VP1 antibody.

[0157] Western blot analysis was used to confirm that the developed anti-VP3 / VP2 / VP1 antibodies, anti-VP2 / VP1 antibodies, and anti-VP1 polyclonal antibodies bind to their intended targets. The results of Western blot analysis are shown in Fig.15 The leftmost dot (A) uses anti-VP1 antibody as primary antibody, the middle dot (B) uses anti-VP1 / VP2 antibody as primary antibody, and the rightmost dot (c) uses anti-VP1 / VP2 / VP3 antibody as primary antibody. The molecular weights of the bands in the reference ladder are shown in Fig.15 The bands corresponding to VP1, VP2, and VP3 proteins are marked on the left side of Fig.15The anti-VP1 pAb binds only to VP1, the anti-VP1 / VP2 pAb binds to VP1 and VP2, and the anti-VP1 / VP2 / VP3 antibody binds to VP1, VP2, and VP3. Example 12

[0158] iCIEF-Western can be used to identify and quantify the charge variants of VP1, VP2, and VP3 by using all three VP protein-specific rabbit antibodies described in Example 11. For example, even if the anti-VP3 / VP2 / VP1 antibody binds to VP1, VP2, and VP3, the chemiluminescence profile produced by the anti-VP3 / VP2 / VP1 antibody can be compared with the profiles produced by the other antibodies to determine which peak corresponds to the VP1 charge variant, the VP2 charge variant, and the VP3 charge variant.

[0159] Reference Fig.16 , using the iCIEF-Western method to generate a charge variant distribution map of a reduced and denatured analyte containing AAV8 serotype AAV. The rabbit polyclonal antibody described in Example 11 was used as a detection antibody. An anti-rabbit antibody conjugated to HRP was used as a reporter molecule, and luminol-peroxide was used as a reporter. Line 461 represents the electropherogram generated using anti-VP1 as the detection antibody, line 462 represents the electropherogram generated using anti-VP1 / VP2 as the detection antibody, and line 463 represents the electropherogram generated using anti-VP1 / VP2 / VP3 as the detection antibody. The distribution map generated by anti-VP3 / VP2 / VP1 antibody (line 463) can be compared with the distribution maps generated by anti-VP3 / VP2 antibody (line 462) and anti-VP1 antibody (line 461) to determine which peaks are associated with VP1, which peaks are associated with VP2, and which peaks are associated with VP3. Fig.16 As shown, there are five charge variant peaks associated with VP1, three charge variant peaks associated with VP3, and three charge variant peaks associated with VP3.

[0160] The theoretical pI of each analyte polypeptide can also aid in the association of chemiluminescent peaks with charge variants of the VP proteins. For example, for VP proteins from AAV8 serotype AAV: the major charge variant of VP1 is 6.01, the major charge variant of VP2 is 6.87, and the major charge variant of VP3 is 6.31. Example 13

[0161] As previously described, viral proteins of different AAV serotypes have different a sequences. Therefore, antibodies designed for one AAV serotype may not be effective against different AAV serotypes. The anti-VP1 pAb, anti-VP1 / VP2 pAb, and anti-VP1 / VP2 / VP3 pAb described above in Examples 11 and 12 were developed for use with AAV8 serotype AAV. Compared to the anti-VP antibodies described in Examples 11 and 12, commercially available anti-VP antibodies, such as anti-VP antibodies that can be used for AAV2 serotype AAV, may be less effective in characterizing viral proteins from AAV8 serotype AAV.

[0162] Reference Fig.17 , using the iCIEF-Western method to generate charge variant profiles of reduced and denatured analytes comprising AAV8 serotype AAV. The charge variant profile labeled 463 was generated using the anti-VP1 pAb, anti-VP1 / VP2 pAb, and anti-VP1 / VP2 / VP3 pAb described above with respect to Examples 11 and 12 as detection antibodies. An anti-rabbit antibody conjugated to HRP was used as a reporter molecule and luminol-peroxide was used as a reporter. The charge variant profile labeled 464 was generated using a commercially available anti-VP antibody designed for AAV2 serotype AAV.

[0163] like Fig.17 As shown, the pAb developed for the AAV8 serotype AAV has higher sensitivity and detects 15 peaks (eg, peaks 473a to 473o) compared to 10 peaks detected by the commercially available antibody (eg, peaks 474a to 474j). Example 14

[0164] Antibodies specific for the VP protein of AAV1 serotype AAV were developed. Two recombinant peptides from portions of the VP2 sequence were used as rabbit antigens to generate polyclonal antibodies specific for AAV1 serotype VP2.

[0165] Charge variant profiles of reduced and denatured analytes comprising AAV1 serotype AAV were generated using the iCIEF-Western method and are shown in Fig.18 A polyclonal antibody specific for VP2 of AAV1 serotype was used as the detection antibody. An anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporter agent. Fig.18 As shown, a polyclonal antibody specific for AAV1 serotype VP2 detected polypeptides in the AAV1 serotype analyte. Example 15

[0166] Antibodies specific for the VP protein of AAV5 serotype AAV were developed. Two recombinant peptides from portions of the VP2 sequence were used as rabbit antigens to generate polyclonal antibodies specific for AAV5 serotype VP2.

[0167] Charge variant profiles of reduced and denatured analytes comprising AAV5 serotype AAV were generated using the iCIEF-Western method and are shown in Fig.19 A polyclonal antibody specific for VP2 of AAV5 serotype was used as the detection antibody. An anti-rabbit antibody conjugated with HRP was used as the reporter molecule, and luminol-peroxide was used as the reporter agent. Fig.19 As shown, a polyclonal antibody specific for AAV5 serotype VP2 detected polypeptides in the AAV5 serotype analyte. Example 16

[0168] Charge variant profiles of reduced and denatured analytes comprising AAV8 serotype AAV were generated using the iCIEF-Western method and are shown in Fig. 20A and 20B . Fig. 20A The charge variant profiles shown were generated using a polyclonal antibody specific for AAV1 serotype VP2 as the detection antibody. Fig. 20B The charge variant distribution diagram shown in was generated using a polyclonal antibody specific for AAV5 serotype VP2 as the detection antibody. Fig. 20A and 20B In both, anti-rabbit antibody conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent.

[0169] Reference Fig. 20A , the charge variant peak was detected, indicating that the polyclonal antibody specific to AAV1 serotype VP2 has a certain sensitivity to AAV8 serotype viral protein. Fig. 20B , no charge variant peak was detected, indicating that the polyclonal antibody specific for AAV5 serotype VP2 could not detect AAV8 serotype viral protein. Example 17

[0170] Charge variant distribution plots were generated for reduced and denatured analytes comprising AAV1 serotype AAV and are shown in Fig.21A and 21B . Fig.21A The charge variant profiles shown were generated using a polyclonal antibody specific for AAV8 serotype VP2 (eg, the anti-VP1 / VP2 antibodies described in Examples 11 and 12) as the detection antibody. Fig. 21BThe charge variant distribution profiles shown were generated using a polyclonal antibody specific for AAV1 serotype VP2 as the detection antibody. Fig.21A and 21B In both cases, anti-rabbit antibody conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent. Fig.21A and 21B As can be seen from the charge variant distribution diagram in, the polyclonal antibody specific for AAV8 serotype VP2 is unable to detect AAV1 serotype VP2 protein. Example 18

[0171] Charge variant distribution plots were generated for reduced and denatured analytes comprising AAV1 serotype AAV and are shown in Fig.22A and 22B . Fig.22A The charge variant profiles shown were generated using a polyclonal antibody specific for AAV8 serotype VP2 (eg, the anti-VP1 / VP2 antibodies described in Examples 11 and 12) as the detection antibody. Fig. 22B The charge variant distribution profiles shown were generated using a polyclonal antibody specific for AAV5 serotype VP2 as the detection antibody. Fig.22A and 22B In both cases, anti-rabbit antibody conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporter agent. Fig.22A and 22B As can be seen from the charge variant distribution diagram in, the polyclonal antibody specific for AAV8 serotype VP2 is unable to detect AAV5 serotype VP2 protein. Example 19

[0172] As previously described, a charge variant profile of a batch of a biopharmaceutical product can be generated to characterize the environmental stresses experienced by the batch. For example, a charge variation profile of a batch that has been subjected to thermal stress and a batch that has not been subjected to thermal stress can be generated. The charge variant profile of an unknown batch can then be compared to the known charge variant profile to determine whether the unknown batch is under an unacceptable stress level.

[0173] Using the iCIEF-Western method, charge variant profiles were generated for three batches of a biopharmaceutical product containing AAV8 serotype AAV. The anti-VP1 pAb described in Examples 11 and 12 was used as a detection antibody, an anti-rabbit antibody conjugated to HRP was used as a reporter molecule, and luminol-peroxide was used as a reporter agent. The first batch (Batch A) was not subjected to heat stress, the second batch (Batch B) was stored at 40°C for one week, and the third batch (Batch C) was stored at 40°C for two weeks.

[0174] The charge variant distribution diagram for batch A is shown in Fig.23A The charge variant distribution diagram of batch B is shown in Fig. 23B The charge variant distribution diagram of batch C is shown in Fig.23C .like Figures 23A to 23C As shown, as AAV is subjected to more thermal stress, the charge variant profile undergoes an acidic shift. Example 20

[0175] Charge variant profiles were generated using the iCIEF-Western method for four batches of a biopharmaceutical product containing AAV8 serotype AAV. The anti-VP1 pAb, anti-VP1 / VP2 pAb, and anti-VP1 / VP2 / VP3 pAb described in Examples 11 and 12 were used as detection antibodies, anti-rabbit antibody conjugated to HRP was used as a reporter molecule, and luminol-peroxide was used as a reporter. The charge variant profiles generated using anti-VP1 pAb as the detection antibody are shown in Figures 24A to 24D The charge variant distribution diagram generated using anti-VP1 / VP2pAb as the detection antibody is shown in Figures 25A to 25D , while the charge variant distribution generated using anti-VP1 / VP2 / VP3 pAb as detection antibody is shown in Figures 26A to 26D .

[0176] The first batch (Batch A) was not subjected to thermal stress, the second batch (Batch B) was stored at 40°C for fifteen days, the third batch (Batch C) was stored at 40°C for one month, and the fourth batch (Batch D) was stored at 40°C for two months. The charge variant distribution diagram of Batch A is shown in Fig.24A , 25A and 26A; the charge variant distribution diagram of batch B is shown in Fig. 24B , 25B and 26B; the charge variant distribution diagram of batch C is shown in Fig.24C , 25C and 26C; the charge variant distribution diagram of batch D is shown in Fig.24D , 25D and 26D. Figures 24A to 26D As can be seen, the charge variant profile undergoes an acidic shift as AAV is subjected to more heat stress. In addition, some peaks corresponding to VP1 charge variants do not appear to decrease under heat stress. Example 21

[0177] As previously described, the use of a combination of anti-VP pAbs can detect charge variants of a single viral protein. In some embodiments, monitoring the charge variant profile of a single viral protein may be more useful in determining environmental stress of a large number of biopharmaceutical products than monitoring the charge variant profile of all viral proteins.

[0178] Reference Fig.27A, charge variant distribution profiles were generated for four batches of analytes comprising reduced and denatured AAV. The first batch (Batch A) was not subjected to heat stress, the second batch (Batch B) was stored at 40°C for 15 days, the third batch (Batch C) was stored at 40°C for one month, and the fourth batch (Batch D) was stored at 40°C for two months. The anti-VP1 / VP2 / VP3 pAb described in Examples 11 and 12 was used as a detection antibody, an anti-rabbit antibody conjugated to HRP was used as a reporter molecule, and luminol-peroxide was used as a reporter. The charge variant distribution profile for batch A is shown by line 465, the charge variant distribution profile for batch B is shown by line 466, the charge variant distribution profile for batch C is shown by line 467, and the charge variant distribution profile for batch D is shown by line 468.

[0179] Still refer to Fig.27A As the AAV is subjected to more heat stress, the charge variant distribution profile shifts toward an acidic state. For example, the height (e.g., relative area) of peak 488 decreases as the level of heat stress increases, and the height of peak 487 increases as the level of heat stress increases. In some cases, based on Fig.27A Based on the data shown in , the relative peak areas of peaks 488 and 487 can be used to determine the environmental stress level of an unknown batch.

[0180] Reference Fig.27B , charge variant distribution profiles were generated for analytes of batches A, B, C, and D comprising reduced and denatured AAV. The anti-VP1 pAb described in Examples 11 and 12 was used as the detection antibody, the anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporter. The charge variant distribution profile for batch A is shown by line 465, the charge variant distribution profile for batch B is shown by line 466, the charge variant distribution profile for batch C is shown by line 467, and the charge variant distribution profile for batch D is shown by line 468. Fig.27B The charge variant distribution diagram shown is similar to Fig.27A The same acid conversion is shown. Example 22

[0181] In addition to thermal stress, charge variant profiles generated using the iCIEF method can be used to assess the impact of other manufacturing or storage conditions on the effectiveness of biopharmaceutical products, including polypeptides.

[0182] The iCIEF-Western method was used to generate charge variant distribution graphs of reduced and denatured analytes of five different batches of biopharmaceutical products including AAV. The first batch (Batch A) did not undergo a holding time during the manufacturing process, the second batch (Batch B) experienced an 8-hour process holding time during the manufacturing process, the third batch (Batch C) experienced a 24-hour process holding time during the manufacturing process, the fourth batch (Batch D) experienced a 48-hour holding time during the manufacturing process, and the fifth batch (Batch E) experienced a 72-hour holding time during the manufacturing process. The charge variant distribution graph of Batch A is shown in Fig.28A The charge variant distribution diagram of batch B is shown in Fig.28B The charge variant distribution diagram of batch C is shown in Fig.28C The charge variant distribution diagram of batch D is shown in Fig.28D The charge variant distribution diagram of batch E is shown in Fig.28E Peaks 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511 and 512 each correspond to a different charge variant and are labeled Figures 28A to 28E .

[0183] The relative peak area is calculated for each peak in each charge variant profile. The calculated relative peak areas are summarized in Fig.29 . Example 23

[0184] The iCIEF-Western method was used to generate charge variant distribution graphs of reduced and denatured analytes of four different batches of biopharmaceutical products including AAV. The first batch (Batch A) was subjected to one freeze-thaw cycle (e.g., the biopharmaceutical product was frozen and then thawed), the second batch (Batch B) was subjected to one freeze-thaw cycle and kept at 4°C for 7 days, the third batch (Batch C) was subjected to three freeze-thaw cycles, and the fourth batch (Batch D) was subjected to five freeze-thaw cycles. The charge variant distribution graph of Batch A is shown in Fig. 30A The charge variant distribution diagram of batch B is shown in Fig. 30B The charge variant distribution diagram of batch C is shown in Fig. 30C The charge variant distribution diagram of batch D is shown in Fig.30D Peaks 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, and 532 each correspond to a different charge variant and are labeled Figures 30A to 30D .

[0185] The relative peak area is calculated for each peak in each charge variant profile. The calculated relative peak areas are summarized in Fig.31 . Example 24

[0186] Charge variant profiles of analytes containing empty capsid AAV were prepared according to two different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer that included a relatively small amount of urea, and the charge variant profiles generated from this method are shown in Fig.32A The second iCIEF-Western method included a sample buffer comprising at least 6 M urea, and the charge variant distribution profiles generated from this method are shown in Fig.32B .

[0187] Charge variant profiles of analytes containing partially intact capsid AAV were prepared according to two different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer that included a relatively small amount of urea, and the charge variant profiles generated from this method are shown in Fig.33A The second iCIEF-Western method included a sample buffer comprising at least 6 M urea, and the charge variant distribution profiles generated from this method are shown in Fig.33B .

[0188] Charge variant profiles of analytes containing intact capsid AAV were prepared according to two different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer that included a relatively small amount of urea, and the charge variant profiles generated from this method are shown in Fig.34A The second iCIEF-Western method included a sample buffer comprising at least 6 M urea, and the charge variant distribution profiles generated from this method are shown in Fig.34B .

[0189] Will Fig.32A , 33A The charge variant distribution of 34A and Fig.32B , 33B Compared with the charge variant distribution profiles of 34B and 34B, less baseline noise was observed, thus, a better signal-to-noise ratio could be achieved by using a sample buffer containing a higher urea concentration. Example 25

[0190] Charge variant profiles of analytes containing empty capsid AAV were prepared according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer comprising approximately 4.3 M urea and approximately 30 vol.% formamide, and the charge variant profiles generated from this method consisted of Fig.35 The second iCIEF-Western method comprises a sample buffer comprising about 5.6 M urea and about 15 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.35The third iCIEF-Western method comprises a sample buffer comprising about 7.3 M urea and about 0 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.35 Line 543 in FIG.

[0191] Charge variant profiles of analytes containing partially intact capsid AAV were prepared according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer comprising approximately 4.3 M urea and approximately 30 vol.% formamide, and the charge variant profiles generated from this method consisted of Fig.36 The second iCIEF-Western method comprises a sample buffer comprising about 5.6 M urea and about 15 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.36 The third iCIEF-Western method comprises a sample buffer comprising about 7.3 M urea and about 0 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.36 Line 546 in FIG.

[0192] Charge variant profiles of analytes containing intact capsid AAV were prepared according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer comprising approximately 4.3 M urea and approximately 30 vol.% formamide, and the charge variant profiles generated from this method consisted of Fig.37 The second iCIEF-Western method comprises a sample buffer comprising about 5.6 M urea and about 15 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.37 The third iCIEF-Western method comprises a sample buffer comprising about 7.3 M urea and about 0 vol.% formamide, and the charge variant distribution profile generated from this method is represented by Fig.37 Line 549 in FIG.

[0193] Will Figures 35 to 37 Compared with the charge variant profiles of 100 μM MgCl2, less baseline noise was observed, thus, a better signal-to-noise ratio was achieved using a sample buffer containing formamide in the presence of urea.

[0194] The present disclosure is further described by the following non-limiting items.

[0195] Item 1: A method for quantifying charge variants in an analyte, comprising: introducing the analyte into a capillary; Separation of charge variants within the sample buffer along an isoelectric gradient; incubating the capillary in a detection antibody; and Based on the signal corresponding to the detection antibody, the relative abundance of the charge variants is quantified.

[0196] Item 2: The method of Item 1 further comprises incubating the capillary in a reporter molecule.

[0197] Item 3: The method of Item 2, wherein the reporter molecule comprises an antibody conjugated to horseradish peroxidase or a streptavidin protein conjugated to horseradish peroxidase.

[0198] Item 4: The method of Item 2 further comprises introducing a detection agent into the capillary.

[0199] Item 5: A method as in Item 4, wherein the detection agent is luminol-peroxide.

[0200] Item 6: The method of Item 2 further comprises generating an electropherogram, wherein the electropherogram comprises a plot of the intensity of the chemiluminescent signal generated by the reporter molecule versus the isoelectric point along the isoelectric gradient at which the chemiluminescent signal is detected.

[0201] Item 7: The method of Item 6, wherein quantifying the relative abundance of the charge variant based on the signal corresponding to the detection antibody comprises calculating the area under the peak of the electropherogram corresponding to the charge variant.

[0202] Item 8: The method of Item 1, after separating the charge variants along the isoelectric gradient and before incubating the capillary in the detection antibody, further comprises: fixing the charge variants in the capillary.

[0203] Item 9: A method for quantifying charge variants in an analyte, comprising: reducing and denaturing polypeptides in the analyte to produce reduced and denatured polypeptides, wherein the analyte comprises a charge variant of a target polypeptide; performing a buffer exchange on the reduced and denatured polypeptide to produce a buffer-exchanged sample, wherein the buffer-exchanged sample comprises the reduced and denatured polypeptide; preparing a sample buffer comprising the buffer-exchanged sample; introducing the sample buffer into the capillary; separating the charge variants of the target polypeptide along an isoelectric gradient; and A signal is measured, the signal correlating to the abundance of the target polypeptide in a region within the isoelectric gradient in the capillary.

[0204] Item 10: The method of Item 9, wherein the sample buffer comprises urea, a carrier ampholyte, and cellulose.

[0205] Item 11: The method of Item 10, wherein the cellulose is hydroxypropylmethylcellulose, and the sample buffer comprises at least about 1 volume percent of hydroxypropylmethylcellulose.

[0206] Item 12: The method of Item 10, wherein the sample buffer comprises a urea concentration of at least about 6 M.

[0207] Item 13: The method of Item 12, wherein the urea concentration of the sample buffer is at least about 8M.

[0208] Item 14: The method of Item 9, wherein the sample buffer comprises formamide.

[0209] Item 15: The method of Item 14, wherein the formamide concentration in the sample is less than or equal to approximately 30 volume percent.

[0210] Item 16: A method for evaluating the level of environmental stress perceived by a sample of a target polypeptide, comprising: generating a charge variant distribution profile for the sample, wherein the charge variant distribution profile comprises a plurality of peaks, and wherein each peak: associating with a corresponding charge variant of said target polypeptide; is associated with an isoelectric point that is equal to the isoelectric point of the correspondingly charged variant of the subject polypeptide; and comprising relative peak areas, the relative peak areas being correlated to the relative abundance of the corresponding charge variants of the target polypeptide; comparing the charge variant profile of the sample to one or more known charge variant profiles; Based on a comparison of the charge variant profile of the sample to the one or more known charge variant profiles, a level of environmental stress sensed by the sample is determined.

[0211] Item 17: The method of Item 16, wherein the environmental stress level is associated with a thermal stress level, a stress level caused by a manufacturing process hold time, or a stress level caused by freeze-thaw cycles.

[0212] Item 18: The method of Item 16, wherein the target polypeptide comprises an antibody or an adeno-associated virus.

[0213] Item 19: The method of Item 16, wherein generating a charge variant distribution profile of the sample comprises: introducing the sample into a capillary tube; and The charge variants of the target polypeptide are separated along an isoelectric gradient.

[0214] Item 20: The method of Item 19, wherein generating a charge variant distribution profile of the sample further comprises: incubating the capillary in a detection antibody; incubating the capillary in a reporter molecule; and An electropherogram is generated, wherein the electropherogram comprises a plot of the intensity of the signal generated by the reporter molecule versus the isoelectric point along the isoelectric gradient at which the signal is detected.

[0215] Those skilled in the art will appreciate that the concepts on which the present disclosure is based can be readily used as a basis for designing other methods and systems for carrying out the several purposes of the present disclosure. Therefore, the scope of the patent application should not be considered to be limited to the foregoing description.

Claims

1. A method for quantifying charge variants in an analyte, characterized in that include: introducing the analyte into a capillary; Separation of charge variants within sample buffer along an isoelectric gradient; incubating the capillary in a detection antibody; and Based on the signal corresponding to the detection antibody, the relative abundance of the charge variants is quantified.

2. The method of claim 1, further comprising incubating the capillary in a reporter molecule.

3. The method according to claim 2, wherein the reporter molecule comprises an antibody conjugated to horseradish peroxidase or a streptavidin protein conjugated to horseradish peroxidase.

4. The method of claim 2, further comprising introducing a detection agent into the capillary.

5. The method according to claim 4, wherein the detection agent is luminol-peroxide.

6. The method of claim 2, further comprising generating an electropherogram, wherein the electropherogram comprises a plot of the intensity of the chemiluminescent signal generated by the reporter molecule versus the isoelectric point along the isoelectric gradient at which the chemiluminescent signal is detected.

7. The method of claim 6, wherein quantifying the relative abundance of the charge variant based on the signal corresponding to the detection antibody comprises calculating the area under the electropherogram peak corresponding to the charge variant.

8. The method of claim 1, further comprising, after separating the charge variants along the isoelectric gradient and before incubating the capillary in the detection antibody: The charge variant is immobilized in the capillary.

9. A method for quantifying charge variants in an analyte, characterized in that include: reducing and denaturing polypeptides in the analyte to produce reduced and denatured polypeptides, wherein the analyte comprises a charge variant of a target polypeptide; performing a buffer exchange on the reduced and denatured polypeptide to produce a buffer-exchanged sample, wherein the buffer-exchanged sample comprises the reduced and denatured polypeptide; preparing a sample buffer comprising the buffer-exchanged sample; introducing the sample buffer into the capillary; separating the charge variants of the target polypeptide along an isoelectric gradient; and A signal is measured, the signal being correlated to the abundance of the target polypeptide in a region within the isoelectric gradient in the capillary.

10. The method according to claim 9, wherein the sample buffer comprises urea, carrier ampholytes and cellulose.

11. The method of claim 10, wherein the cellulose is hydroxypropylmethylcellulose and the sample buffer comprises at least about 1 volume percent of hydroxypropylmethylcellulose.

12. The method of claim 10, wherein the sample buffer comprises a urea concentration of at least about 6 M.

13. The method of claim 12, wherein the urea concentration of the sample buffer is at least about 8 M.

14. The method of claim 9, wherein the sample buffer comprises formamide.

15. The method of claim 14, wherein the concentration of formamide in the sample is less than or equal to about 30 volume percent.

16. A method for evaluating the level of environmental stress perceived by a target polypeptide sample, characterized in that include: generating a charge variant distribution profile for the sample, wherein the charge variant distribution profile comprises a plurality of peaks, and wherein each peak: associating with a corresponding charge variant of said target polypeptide; is associated with an isoelectric point equivalent to that of the corresponding charged variant of the subject polypeptide; and comprising a relative peak area, the relative peak area being correlated to the relative abundance of the corresponding charge variant of the target polypeptide; comparing the charge variant profile of the sample to one or more known charge variant profiles; Based on a comparison of the charge variant profile of the sample to the one or more known charge variant profiles, a level of environmental stress sensed by the sample is determined.

17. The method of claim 16, wherein the environmental stress level is associated with a thermal stress level, a stress level caused by a manufacturing process hold time, or a stress level caused by freeze-thaw cycles.

18. The method of claim 16, wherein the target polypeptide comprises an antibody or an adeno-associated virus.

19. The method of claim 16, wherein generating the charge variant profile of the sample comprises: introducing the sample into a capillary tube; and The charge variants of the target polypeptide are separated along an isoelectric gradient.

20. The method of claim 19, wherein generating the charge variant distribution profile of the sample further comprises: incubating the capillary in a detection antibody; incubating the capillary in a reporter molecule; and An electropherogram is generated, wherein the electropherogram comprises a plot of the intensity of the signal generated by the reporter molecule versus the isoelectric point along the isoelectric gradient at which the signal is detected.

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