Methods and systems for analyzing polypeptide variants - Patents.com

Abstract

A method for quantifying charge variants in an analyte may include introducing a sample buffer containing the analyte into a capillary, separating the charge variants in the 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. The method may further include generating an electropherogram, where 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 was detected.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 269,595, filed March 18, 2022, which is incorporated by reference in its entirety. Field of Disclosure

[0002] SYSTEMS AND METHODS FOR ANALYZING POLYPEPTIDE VARIANTS FIELD OF THE DISCLOSURE Some aspects of the disclosure relate to systems and methods for quantifying charge variants in a sample. [Background technology]

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

[0004] Conventional methods for characterizing charge variants and developing charge variant profiles may be limited to understanding intact proteins or multi-protein structures. However, charge variants may also form within polypeptides or protein subunits, and characterization of larger structures may lack the detail required to characterize charge variants of individual polypeptides or subunits. Furthermore, conventional methods for characterizing charge variants may include time- and labor-intensive chromatography steps, which 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 in an analyte, which 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 streptavidin 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, where the electropherogram includes 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 was detected. Quantifying the relative abundance of the charge variant based on the signal corresponding to the detection antibody may include 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 variant along the isoelectric gradient and prior to incubating the capillary in the detection antibody.

[0007] In another aspect, the disclosure includes a method of quantifying charge variants in an analyte. The method may include reducing and denaturing a polypeptide in the analyte to generate a reduced and denatured polypeptide, where the analyte includes a charge variant of the target polypeptide. The method may further include buffer exchanging the reduced and denatured polypeptide to generate a buffer exchanged sample, where the buffer exchanged sample includes the reduced and denatured polypeptide. The method may further include preparing a sample buffer that includes the buffer exchanged sample. In some embodiments, the method includes introducing a sample buffer into the capillary and introducing the sample buffer into the capillary. The method may further include separating the charge variants of the target polypeptide along an isoelectric gradient. In some embodiments, the method includes measuring a signal that correlates to the abundance of the target polypeptide at a region within the isoelectric gradient in the capillary.

[0008] The sample buffer may include urea, a carrier ampholyte, and cellulose. The cellulose may be hydroxypropylmethylcellulose, and the sample buffer may include at least about 1 volume percent hydroxypropylmethylcellulose. 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 about 30 volume percent or less.

[0009] In another aspect, the disclosure includes a method for evaluating the environmental stress level recognized by a sample of a target polypeptide. The method may include generating a charge variant profile for the sample, where the charge variant profile includes a plurality of peaks. Each peak of the plurality of peaks may include a relative peak area associated with a corresponding charge variant of the target polypeptide, associated with an isoelectric point equivalent to the isoelectric point of the corresponding charge variant of the target polypeptide, and / or associated with the relative abundance of the corresponding charge variant of the target polypeptide. The method may further include comparing the charge variant profile for the sample to one or more known charge variant profiles. In some embodiments, the method includes determining the environmental stress level recognized by the sample based on comparing the charge variant profile for the sample to one or more known charge variant profiles.

[0010] The environmental stress level may be related to a thermal stress level, a level of stress due to manufacturing process hold times, or a level of stress due to freeze-thaw cycles. The target polypeptide may include an antibody or an adeno-associated virus. Generating a charge variant profile for the sample may include introducing the sample into a capillary and separating charge variants of the target polypeptide along an isoelectric gradient. Generating a charge variant profile for the sample may further include incubating the capillary in a detection antibody, incubating the capillary in a reporter molecule, and generating an electropherogram including a plot of the intensity of a 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 accompanying figures, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of an embodiment or example described herein may be combined with any other embodiment or example, and all such combinations are encompassed by this disclosure. Furthermore, the described systems and methods are not limited to any single aspect or embodiment thereof, nor are they limited to any combination or permutation of such aspects and embodiments. For the sake of brevity, certain permutations and combinations are not separately discussed and / or illustrated herein.

[0012] [Figure 1] FIG. 1 illustrates, in flow chart form, an exemplary method for separating and quantifying charge variants of reduced and denatured polypeptides according to an embodiment of the disclosure.

[0013] [Diagram 2] FIG. 2 illustrates, in flow chart form, an exemplary method for separating and quantifying charge variants of reduced and denatured polypeptides according to an embodiment of the disclosure.

[0014] [Diagram 3] FIG. 3 illustrates, in flow chart form, an exemplary method for quantifying the relative abundance of charge variants according to an embodiment of the present disclosure.

[0015] [Figure 4A-4B] 4A and 4B show charge variant profiles of reduced and denatured analytes according to embodiments of the present disclosure.

[0016] [Figure 4C] FIG. 4C shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0017] [Figure 4D]FIG. 4D shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0018] [Figure 5A-5B] 5A and 5B show charge variant profiles of reduced and denatured analytes according to embodiments of the present disclosure.

[0019] [Figure 5C] FIG. 5C shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0020] [Figure 5D] FIG. 5D shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0021] [Figure 6A] FIG. 6A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0022] [Figure 6B] FIG. 6B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0023] [Figure 6C] FIG. 6C shows the charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0024] [Figure 7A] FIG. 7A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0025] [Figure 7B] FIG. 7B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0026] [Figure 8A]FIG. 8A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0027] [Figure 8B] FIG. 8B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0028] [Figure 9A] FIG. 9A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0029] [Figure 9B] FIG. 9B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0030] [Figure 9C] FIG. 9C shows a plot comparing relative peak areas calculated based on the charge variant profiles of FIGS. 9A and 9B, according to an embodiment of the present disclosure.

[0031] [Figure 10A] FIG. 10A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0032] [Figure 10B] FIG. 10B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0033] [Figure 11A] FIG. 11A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0034] [Figure 11B] FIG. 11B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0035] [Figure 11C] FIG. 11C shows a plot comparing the relative peak areas calculated based on the charge variant profile of FIG. 11A.

[0036] [Figure 11D] FIG. 11D shows a plot comparing relative peak areas calculated based on the charge variant profile of FIG. 11B, according to an embodiment of the present disclosure.

[0037] [Figure 12A] FIG. 12A shows a charge variant profile of a native analyte according to an embodiment of the present disclosure.

[0038] [Figure 12B] FIG. 12B shows a charge variant profile of a native analyte according to an embodiment of the present disclosure.

[0039] [Figure 13A] FIG. 13A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0040] [Figure 13B] FIG. 13B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0041] [Figure 14A] FIG. 14A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the present disclosure.

[0042] [Figure 14B] FIG. 14B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0043] [Figure 15] FIG. 15 shows a Western blot of anti-viral protein polyclonal antibodies according to an embodiment of the present disclosure.

[0044] [Figure 16] FIG. 16 shows charge variant profiles of reduced and denatured analytes according to an embodiment of the present disclosure.

[0045] [Figure 17] FIG. 17 shows charge variant profiles of reduced and denatured analytes according to an embodiment of the present disclosure.

[0046] [Figure 18] FIG. 18 shows charge variant profiles of reduced and denatured analytes according to an embodiment of the present disclosure. [Figure 19] FIG. 19 shows charge variant profiles of reduced and denatured analytes according to an embodiment of the present disclosure. [Figure 20A-20B] 20A and 20B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 21A-21B] 21A and 21B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Fig. 22A-22B] 22A and 22B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 23A-23B] 23A-23C show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 23C] 23A-23C show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 24A] 24A-24D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 24B] 24A-24D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 24C] 24A-24D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 24D] 24A-24D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 25A] 25A-25D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 25B] 25A-25D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 25C] 25A-25D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 25D] 25A-25D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 26A] 26A-26D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 26B] 26A-26D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 26C] 26A-26D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 26D] 26A-26D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure.

[0047] [Figure 27A] FIG. 27A shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0048] [Figure 27B] FIG. 27B shows a charge variant profile of a reduced and denatured analyte according to an embodiment of the disclosure.

[0049] [Fig. 28A-28B]28A-28E show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Fig. 28C-28D] 28A-28E show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Figure 28E] 28A-28E show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure.

[0050] [Figure 29] FIG. 29 shows a plot comparing relative peak areas calculated based on the charge variant profiles of FIGS. 28A-28E, according to an embodiment of the present disclosure.

[0051] [Fig. 30A-30B] 30A-30D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Fig. 30C-30D] 30A-30D show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure.

[0052] [Diagram 31] FIG. 31 shows a plot comparing relative peak areas calculated based on the charge variant profiles of FIGS. 30A-30D, according to an embodiment of the present disclosure.

[0053] [Fig. 32A-32B] 32A and 32B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Fig. 33A-33B] 33A and 33B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure. [Fig. 34A-34B] 34A and 34B show charge variant profiles of reduced and denatured analytes, respectively, according to embodiments of the present disclosure.

[0054] [Diagram 35] FIG. 35 shows charge variant profiles of reduced and denatured analytes according to embodiments of the present disclosure. [Diagram 36] FIG. 36 shows charge variant profiles of reduced and denatured analytes according to embodiments of the present disclosure. [Figure 37] FIG. 37 shows charge variant profiles of reduced and denatured analytes according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary skilled artisans in the technical field to which this disclosure belongs.In carrying out or testing this disclosure, any suitable method and material (for example, similar or equivalent to those described herein) can be used, but specific exemplary methods are described here.All publications mentioned are incorporated herein by reference.

[0056] As used herein, the terms "comprises," "comprising," or any other variations thereof, are intended to include a non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements may not only include those elements, but also other elements 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." With respect to the terms "for example" and "such as," and their grammatical equivalents, the phrase "and without limitation" is understood to follow unless clearly stated otherwise.

[0057] As used herein, the term "about" is meant to account for variations due to experimental error. When applied to a numerical value, the term "about" may indicate a variation of + / - 5% of the disclosed numerical value unless a different variation is specified. As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise. Furthermore, all ranges are understood to include the endpoints, for example, 1 centimeter (cm) to 5 cm would include the length 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, limits, and ranges) may have a variation of + / - 5% of the disclosed numerical value, unless a different variation is specified.

[0059] The term "polypeptide" as used herein refers to any amino acid polymer of greater than about 20 amino acids covalently linked through amide bonds. A protein contains one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide may be a protein, and a protein may contain multiple polypeptides to form a single functional biomolecule.

[0060] Post-translational modifications can modify or alter the structure of a polypeptide. For example, disulfide bridges (e.g., S-S bonds between cysteine ​​residues) can be formed post-translationally in some proteins. Some disulfide bridges are essential for proper structure, function, and interaction of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins can be subjected to 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 glycosyl groups at 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 involving polypeptides.

[0061] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins, as well as 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, and the like. Proteins of interest (POI) can include any polypeptide or protein that is desirable to be isolated, purified, or otherwise prepared. POI can include polypeptides produced by cells, including antibodies.

[0062] The term "antibody" as used herein includes immunoglobulins composed of four polypeptide chains: two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Typically, antibodies have a molecular weight of more than 100 kDa, e.g., between 130 kDa and 200 kDa, e.g., about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a 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 (referred to herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions are further divided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (the heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; the light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

[0063] For example, the immunoglobulin class called immunoglobulin G (IgG) is common in human serum and contains four polypeptide chains, two light chains and two heavy chains. Each light chain is linked to one heavy chain through a cystine disulfide bond, and the two heavy chains are linked to each other through two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. For IgG, there are four subclasses: IgG 1, IgG 2, IgG 3, and IgG 4. Each subclass differs in the constant region and may therefore have different effector functions. In some embodiments described herein, the biopharmaceutical product may contain a target polypeptide that includes IgG. In at least one embodiment, the target polypeptide includes IgG 4.

[0064] The term "antibody", as used herein, also includes antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be obtained from full-length antibodies, for example, using any suitable standard technique, such as proteolytic digestion, or using recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0065] Biopharmaceutical products (e.g., target molecules, polypeptides, antibodies) may be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia spp.), and 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 prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, such as hybridomas or quadromas. In some embodiments, the cells may be human, monkey, ape, hamster, rat or mouse cells. In some embodiments, the cell may be a eukaryotic cell and may 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, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli 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, for example retinal cells that express viral genes (eg, PER.C6™ cells).

[0066] The term "target molecule" may be used herein to refer to a target polypeptide (e.g., an antibody, an antibody fragment, an AAV particle, a viral protein, or other protein or protein fragment) or to refer to other molecules (e.g., an adeno-associated virus (AAV) or other molecules for therapeutic use) that are intended to be produced, isolated, purified, and / or included in a pharmaceutical product. Although the methods according to the present disclosure may refer to a target polypeptide, 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 containing AAV may be subjected to a method according to the present disclosure. Before or after one or more of the following methods of the present disclosure, the mixture containing AAV may be subjected to further methods (e.g., removal of "empty cassettes" or AAV that do not contain target sequences).

[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., an antibody) is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., an anti-Dll4 antibody as described in U.S. Patent No. 9,402,898), an anti-Dll4 antibody, ... No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Patent No. 9,132,192), anti-AngPtl3 antibodies as described in U.S. Patent No. 9,265,827), anti-platelet derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Patent No. 9,132,192), antibodies, or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. US2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibodies (e.g., anti-PCSK9 antibodies as described in U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Patent Nos. 8,871,209 or 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibodies as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., anti-IL4R antibodies as described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibodies (e.g., U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), anti-interleukin 33 (e.g., anti-IL33 antibodies as described in U.S. Patent Application Publication No. US2014 / 0271658A1 or US2014 / 0271642A1), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies as described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti-surface cluster 3 (e.g., anti-IL6R antibodies as described in U.S. Patent Application Publication No. US2014 / 0088295A1 and Nos. 2014 / 0088295A1 and 20150266966A1, and U.S. Patent Application No. 62 / 222,605), anti-cluster of differentiation 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1, and U.S. Patent No. 7,879,984), anti-cluster of differentiation 48 (e.g., anti-CD48 antibodies as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibodies (e.g., as described in U.S. Pat. No. 9,079,948), anti-Middle East respiratory syndrome 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-Zika virus 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 is incorporated herein in its entirety.

[0069] In some embodiments, the targeting molecule (e.g., a bispecific antibody) is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody, an anti-CD3 x anti-mucin 16 bispecific antibody, and an anti-CD3 x anti-prostate specific membrane antigen bispecific antibody. In some embodiments, the targeting molecule is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesbacumab, dupilumab, trevoglumab, evinacumab, and rinukumaab.

[0070] In some embodiments, the target molecule is a recombinant protein (e.g., an Fc fusion protein) containing an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein that contains one or more extracellular domain(s) of a receptor coupled to an Fc portion. In some embodiments, the Fc portion comprises the CH2 and CH3 domains of IgG after the hinge region. In some embodiments, the receptor Fc fusion protein contains two or more separate receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding domain fused to the Il-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety) or VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, which are both incorporated herein by reference in their entirety). In other embodiments, the Fc fusion protein is a ScFv-Fc fusion protein, which contains one or more antigen binding domain(s), such as one or more of the variable heavy and variable light fragments, of an antibody coupled to the Fc portion.

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

[0072] Charge heterogeneity of biopharmaceutical products may be monitored to ensure lot-to-lot consistency throughout the manufacturing process. Some methods of characterizing charge variants and developing charge variant profiles are limited to understanding intact proteins or multi-protein structures. However, charge variants may also form within polypeptides or protein subunits, and characterization of larger structures may lack the detail required to characterize charge variants of individual polypeptides or subunits. For example, a charge variant profile of an antibody's light and / or heavy chains may provide more information than a charge variant profile of an intact antibody. As another example, a charge variant profile of an individual viral protein may provide more information than a charge variant profile of an intact AAV (e.g., intact capsid protein).

[0073] Capillary isoelectric focusing (CIEF) has been used to characterize charge heterogeneity and separate charge variants based on isoelectric point (pI). Depending on the application, CIEF can also be referred to as imaging capillary isoelectric focusing (iCIEF). As described in more detail below, the antibody charge variant profile generated by iCIEF includes a spectrum of peaks arranged by pI, including a predominant main peak, and peaks of acidic and basic species on either side of the main peak.

[0074] Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates polypeptides (e.g., protein subunits) based on isoelectric point in one dimension and size in the second dimension. This two-dimensional separation has been used to understand the resolved charge heterogeneity associated with antibody heavy and light chains. However, 2D-PAGE can only provide a qualitative assessment of charge isoforms and cannot be used to quantify the ratio of individual charge isoforms relative to each other. Furthermore, 2D-PAGE is time- and labor-intensive and can result in streaking, which can reduce the overall resolution of the analysis and the detection of low-abundance isoforms.

[0075] To address the limitations of 2D-PAGE, a method was developed that uses size-exclusion chromatography (SEC) and iCIEF. This method, called ChromiCE, can be used to separately 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 onto a size-exclusion column to separate the heavy and light chain components. The separated heavy and light chains can be individually loaded onto an isoelectric focusing platform and iCIEF can be used to identify and quantify the charge isoforms associated with the heavy and light chains.

[0076] Unlike 2D-PAGE, ChromiCE provides quantitative results. However, data collection and analysis from ChromiCE can last as long as a week due to the time required for separation of light and heavy chains. Furthermore, the mobile phase used in the SEC step of ChromiCE utilizes a buffer containing a high concentration of urea (e.g., approximately 8M urea), which can alter the sample and negatively affect the column life and the life of the equipment involved.

[0077] The embodiments of the present disclosure may include methods for identifying and quantifying charge variants of biopharmaceutical products, such as methods that address the aforementioned shortcomings of 2D-PAGE and ChromiCE methods. The 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. Additionally or alternatively, the embodiments of the present disclosure may be used to identify and quantify charge variants of reduced and denatured polypeptides. Reduced and denatured polypeptides that may be analyzed by 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 methods of the present disclosure for identifying and quantifying charge variants of biopharmaceutical products may be referred to as iCIEF-Western methods.

[0078] The method of the present disclosure can include using capillary isoelectric focusing to separate charge variants based on their isoelectric points.The method of the present disclosure (e.g., iCIEF-Western) does not include chromatographic separation (e.g., SEC step).According to an embodiment of the present disclosure, after separating polypeptides based on their isoelectric points, iCIEF-Western utilizes peptide-specific antibodies to quantify and characterize specific portions and charge variants of proteins.

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

[0080] The loading buffer may include one or more components that aid in the 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., hydroxylpropylmethylcellulose), a pI ladder, a base (e.g., urea), formamide, glycerol, ethylene, propylene glycerol, 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 aid in the generation of an electric gradient during charge variant separation. Carrier ampholytes may include those sold under the trademark Pharmalytes, such as Pharmalytes(3-10) and Pharmalytes(5-8). The pI ladder may provide a standardization marking for the results of the CIEF step using molecules of known pI value. The pI ladder and carrier ampholytes may be selected based on the theoretical pI of the analyte. The inclusion of a base, e.g., urea, may prevent species in the loading buffer from converting to other charge variants. Inclusion of a base (eg, urea) in the loading buffer may help maintain fragments of polypeptides (eg, denatured antibody fragments or denatured viral proteins) within the analyte in a denatured state.

[0081] One exemplary sample preparation includes preparing a loading buffer master mix, followed by preparing a sample containing loading buffer and analyte. The preparation of the loading buffer master mix can include dissolving hydroxypropyl methylcellulose in a urea solution (e.g., a dissolution buffer containing at least 6M urea) to produce a 1 weight percent (wt%) hydroxypropyl methylcellulose solution with a urea concentration of at least 6M. For example, a 1 wt% hydroxypropyl methylcellulose solution can include a urea concentration of approximately 8M. The loading buffer master mix can be produced according to the ratios shown in Table 1. Table 1 [Table 1]

[0082] After preparing the loading buffer master mix, a sample for iCIEF-Western analysis may be prepared. The sample may include analyte, water, lysis buffer, loading buffer master mix, or a combination thereof. The sample may have a urea concentration of approximately 8M or more. The sample may have a cellulose (e.g., hydroxylpropylmethylcellulose) concentration of approximately 1% by weight. In an exemplary embodiment, the sample may be prepared according to the ratios shown in Table 2. Table 2 [Table 2]

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

[0084] Analytes may be reduced and denatured prior to analysis as described herein. Reduced and denatured analytes may be buffer exchanged prior to preparation of sample buffer. Buffer exchanged analytes may include urea (e.g., approximately 8M urea), sodium phosphate (e.g., approximately 10 mM sodium phosphate), tris(2-carboxyethyl)phosphine (TCEP) (e.g., approximately 1 mM TCEP), and / or analyte. Another exemplary sample preparation for use with some buffer exchanged analytes is shown in Table 4 below. The sample preparations in Table 4 may be used with reduced and denatured analytes, including, for example, antibodies. Table 4 [Table 4]

[0085] Sample preparation and loading buffers may be adjusted to allow sufficient separation and stability of polypeptides. For example, depending on the characteristics of the analyte (e.g., polypeptide concentration, viscosity, or other physicochemical properties), loading buffers and sample preparation compositions may be adjusted. Additionally or alternatively, parameters of the CIEF separation may be adjusted to accommodate the physicochemical properties of the sample. For example, if the analyte peptides are at higher than average concentration, the resulting sample may have a higher than average viscosity. The loading time and focusing associated with the CIEF separation may be adjusted to account for the increased sample viscosity. As another example, ampholyte selection, ampholyte concentration, sample solution viscosity, sample analyte concentration may need to be adjusted to allow sufficient separation and stability of polypeptide charge variants.

[0086] The disclosed methods may be used to separate and quantify charge variants of reduced and denatured polypeptides. Analytes may be incubated in a solution that reduces and denatures the analyte's component polypeptides prior to iCIEF-Western analysis. In some embodiments, analytes may be incubated in such a solution for approximately 1 hour. For example, analytes may be incubated in a solution containing 6 M guanidine HCl and 10 mM tris(2-carboxyethyl)phosphine (TCEP) to reduce and denature the analyte's polypeptides. In the example of an analyte that includes an antibody, the antibody may be reduced and denatured to yield a solution containing denatured light and heavy chains. Other types of denaturants and / or other types of protein digestion may be used to further characterize the charge variants of the target analyte. In some embodiments, a protease, such as IdeS, papain, or pepsin, may be used to digest the target polypeptide into fragments.

[0087] The reduced and denatured analytes may 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, and about 6 M to about 8 M urea at a pH of about 6.0. The buffer exchanged analytes may 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 analytes may 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 analytes may be combined with other sample buffer components according to the ratios shown in Table 3 or Table 4.

[0088] After preparing the samples, they may be loaded into the capillaries. Samples may be run in parallel, for example in parallel capillaries. In some embodiments, samples are loaded into a well plate and an automated liquid handling system transports the samples and other reagents into the capillaries. When a current is applied to the samples, species within the samples (e.g., ampholytes, pI ladders, charge variants within the samples) may be separated along the capillaries according to their isoelectric points (pI). For example, 21000 μW of power may be applied for 30 minutes. The run time of the isoelectric focusing separation may be adjusted based on the composition of the sample (e.g., conductivity, viscosity, or other properties). Additionally or alternatively, different sample compositions may require different focusing times to achieve optimal separation of charge variants.

[0089] When electrical energy is applied to the capillary, the species comprising the sample may form a gradient according to their isoelectric points, such that the species with the lowest isoelectric point is located at a first end of the capillary and the species with the highest isoelectric point is located at a second end of the capillary. For example, iCIEF separation may be accomplished using an isoelectric focusing platform, such as the PeggySue™ instrument manufactured by Protein Simple.

[0090] After the charge variants in the sample are separated based on their isoelectric points, specific antibodies can be used to detect and quantify the relative abundance of the polypeptides. In some embodiments, the separated charge variants can be immobilized on the capillary walls. The matrix containing the capillaries can be washed to remove excess reagents. Before introducing the detection antibody, the matrix containing the capillaries can be blocked. For example, the matrix can be incubated with bovine serum albumin solution or milk for at least approximately 30 minutes.

[0091] After blocking, the matrix may be washed and a detection antibody may be added. After reducing and denaturing the polypeptide as described previously, charge variants of fragments of the polypeptide may be separately quantified with a specific detection antibody. In an example of an iCIEF-Western analysis of an antibody, a specific anti-human Fc antibody may be used to detect and quantify heavy chain charge variants and an anti-human kappa antibody may be used to detect and quantify light chain charge variants. In an iCIEF-Western analysis of an AAV particle, a specific anti-VP protein may be used to detect and quantify viral protein variants. The incubation in the specific detection antibody may be referred to as a primary incubation. The matrix may be incubated in an antibody specific for one or more polypeptides for at least approximately 60 minutes. In some examples, such as anti-human Fc protein antibody and anti-human kappa protein antibody used in the detection of heavy and light chains of an antibody, a suitable detection antibody concentration may be approximately 1 μg / mL to approximately 10 μg / mL. In some embodiments, a suitable detection antibody concentration may be approximately 2 μg / mL to 400 μg / mL.

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

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

[0094] After the second incubation, the matrix may be washed and one or more reporting agents may be introduced into the capillary. The reporting agents may interact with the reporter molecules to generate a detectable signal corresponding to each charge variant. Because the species in the sample have already been separated based on their isoelectric points, the location of each signal resulting from the interaction of the reporting agent with the reporting molecule is indicative of the charge variant to which the signal corresponds. The intensity of the detectable signal may correspond to the relative abundance of the corresponding charge variant at that location. The intensity of the detectable signal may be plotted versus the isoelectric point at the location of the signal to generate an electropherogram.

[0095] The peak area of ​​the electropherogram may be calculated by a Gaussian distribution algorithm. The area of ​​each peak divided by the total area under all peaks in the electropherogram may correspond to the relative abundance of the charge variant corresponding to each peak. The electropherogram may be analyzed with the aid of a processor and / or software, such as, for example, Compass software (e.g., version 4.0.0). The charge variant profile may refer to the electropherogram, or the relative abundance of the charge variant quantified from the electropherogram, and / or the isoelectric point of the charge variant. Further examples of the quantification of charge variants through electropherograms and calculation of peak areas are described below.

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

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

[0098] When developing an iCIEF-Western protocol, some conditions may need to be adjusted based 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. When determining the appropriate detection antibody concentration, application-specific factors such as target polypeptide concentration, range of charge variants, and binding affinity of the detection antibody are taken into account.

[0099] Several iCIEF-Western methods have been described above in the context of antibodies and antibody moieties, but they may be used to characterize the charge variant profile of any proteinaceous biopharmaceutical product. In one embodiment, an iCIEF-Western assay may be used to characterize the charge variant profile of one or more proteins produced by adeno-associated virus (AAV).

[0100] As known to those skilled in the art, adeno-associated virus can also be used to deliver corrective genes into cells or tissues of target organisms.For example, therapeutic genes can be inserted into the AAV genome, and with the assistance of helper viruses, AAV can deliver therapeutic genes to one or more target cells.AAV is non-pathogenic and non-toxic.Depending on the application, different AAV serotypes can be used.Each AAV serotype may have different cell transduction efficiencies for different types of cells, and thus some AAV serotypes are more suitable for certain organ systems, organs, and / or tissues of targets. For example, AAVrhlO 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, and AAV9 and AAVrhlO have the highest systemic cell transduction efficiency.

[0101] There are three main AAV capsid proteins, often referred to as VP1, VP2, and VP3. The sequences of AAV capsid proteins may vary among AAV serotypes. Thus, a detection antibody suitable for use in analyzing the charge variants of a polypeptide produced by one AAV serotype may not be suitable for other serotypes. In some embodiments, the method of the present disclosure includes developing a detection antibody specific to an AAV capsid protein. For example, the method of the present disclosure may include developing a rabbit polyclonal antibody that binds to the capsid protein of a specific AAV serotype.

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

[0103] As described herein, the methods of the present disclosure (e.g., iCIEF-Western) may be used to generate a charge variant profile of a target polypeptide in a sample. The generated charge variant profile may be used to validate the iCIEF-Western, to validate the manufacturing conditions of a lot, and to evaluate the effect of manufacturing or storage conditions on the stability of a biopharmaceutical product.

[0104] For example, charge variants may be quantified for two different lots of the same biopharmaceutical product. Quantification of the relative populations of charge variants, and verification that these match known or theoretical charge variant profiles, can also be used to validate quantification methods developed through iCIEF-Western.

[0105] Additionally, the charge variant profiles of different lots can be compared to determine, confirm or verify the process conditions under which the lots were produced. If the charge profile of the antibody from a first lot under validated manufacturing and environmental conditions is known, it can be compared to the charge variant profile of a newly produced lot to verify that the lot was not exposed to excessive thermal or environmental stress. Additionally, if changes to manufacturing conditions or equipment are made, the charge variant profile of the lot produced under the new conditions can be compared to the charge variant profile of the lot produced under the validated conditions to assess the effectiveness of the changes to the manufacturing conditions and / or equipment.

[0106] Referring to FIG. 1, a flow chart of an exemplary method 100 for separating and quantifying charge variants of a native polypeptide in an analyte is shown. The method 100 may include preparing a sample buffer containing the analyte (step 101). The sample buffer may include one or more carrier ampholytes, one or more celluloses (e.g., hydroxylpropylmethylcellulose), 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 Tables 1 and 2. Additionally or alternatively, the sample buffer may be prepared according to the ratios shown in Tables 3 or 4. The method 100 may include separating the 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 separating the charge variants of a native polypeptide (e.g., an antibody, an AAV particle, or other polypeptide), the method 100 may include incubating the separated charge variants in the presence of a detection antibody (step 103). The detection antibody may be specific to one or more target polypeptides or target amino acid sequences within the analyte. After the detection antibody is conjugated to the charge variants of 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] Referring to FIG. 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. The method 150 may include reducing and denaturing polypeptides in the analyte (step 151). For example, the analyte may be incubated in a solution containing 6 M guanidine HCl and 10 mM tris(2-carboxyethyl)phosphine (TCEP) to reduce and denature the polypeptides of the analyte. The method 150 may include preparing a sample buffer containing the analyte (step 152). The reduced and denatured sample may be buffer exchanged into a buffer containing 8 M urea, 35 mM sodium phosphate, and 1 mM TCEP. A sample buffer may then be prepared with the buffer exchanged sample according to the ratios shown in Tables 1 and 2. Additionally or alternatively, the sample buffer may be prepared according to the ratios shown in Tables 3 or 4.

[0109] Method 150 may include separating the charge variants in a sample buffer using capillary isoelectric focusing (step 153). For example, the sample buffer may be loaded into a capillary and separated using an isoelectric focusing platform. After separating the charge variants of a reduced and denatured polypeptide (e.g., an antibody heavy chain, an antibody light chain, a viral protein, or other polypeptide), 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 conjugated to the charge variants 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 for separating and quantifying charge variants of a polypeptide (e.g., methods 100, 150) can include quantifying the relative abundance of the charge variants based on a signal corresponding to a detection antibody. Referring to Figure 3, a flow chart of an exemplary method 200 for quantifying the relative abundance of charge variants based on a signal corresponding to a detection antibody is shown.

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

[0112] Method 200 may further include incubating the immobilized charge variants in a detection antibody (step 203). Incubating the immobilized charge variants in a detection antibody may refer to a primary incubation. In some embodiments, the capillary (e.g., a matrix including the capillary) may be washed after removal of the blocking buffer and before the primary incubation. As described herein, one or more detection antibodies specific to one or more polypeptides (e.g., a target polypeptide) may be used. For example, a biotinylated goat anti-human IgG Fc-specific polyclonal antibody (pAb) may be used to detect an antibody heavy chain, and a biotinylated goat anti-human kappa pAb may be used to detect an antibody light chain. In other examples, an antibody that specifically binds to a particular viral protein or protein subunit may be used as a detection antibody. During the primary incubation, the detection antibody may be conjugated to the target polypeptide.

[0113] Method 200 may include incubating the fixed charge variant conjugated to a detection antibody in a 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 including the capillary) may be washed between the primary and secondary incubations. The reporter molecule may preferentially bind to the detection antibody. Additionally, 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 produce 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 produces a signal only in the presence of the detection agent. In an example of a reporter molecule that includes HRP conjugated to streptavidin, a signal is only generated when HRP is exposed to luminol-peroxide. Method 200 may further 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 and the reporter molecule (step 206).

[0115] In some embodiments, method 200 may 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 may be divided by the sum of all peak areas to determine the relative abundance of each charge variant. EXAMPLES

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

[0117] In instances where the polypeptides were reduced and denatured, unless otherwise stated, the polypeptides were reduced and denatured in the presence of 6 M GuHCl and 10 mM TCEP for 1 hour at room temperature. The reduced and denatured samples were then buffer exchanged into a buffer containing 8 M urea, 35 mM sodium phosphate, and 1 mM TCEP.

[0118] For each example described herein, unless otherwise indicated, the iCIEF-Western analytes were included in a sample buffer prepared with lysis buffer, carrier ampholytes, pI ladder, and hydroxylpropylmethylcellulose according to the ratios shown in Tables 1 and 2. For examples involving reduced and denatured polypeptides, the buffer-exchanged samples were included in a sample buffer prepared with lysis buffer, carrier ampholytes, pI ladder, and hydroxylpropylmethylcellulose according to the ratios 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 capillary of the iCIEF instrument. In the examples described herein, the iCIEF separation and analysis were performed using a PeggySue™ instrument manufactured by Protein Simple. The samples were electrophoresed by applying 21000 μWatts for 30 minutes. Example 1

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

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

[0121] Charge variant profiles were generated for reduced and denatured antibodies from the same production lot of antibody 1 using iCIEF-Western and are shown in Figure 4C. A biotinylated goat anti-human IgG Fc-specific polyclonal antibody (pAb) was used as the detection antibody for the heavy chain of the reduced and denatured antibodies. A biotinylated goat anti-human kappa pAb was used as the detection antibody for the light chain of the reduced and denatured antibodies. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent. In Figure 4C, the signals detected from the Fc pAb (e.g., signals corresponding to the heavy chain charge variants) are shown as solid lines and the signals detected from the kappa pAb (e.g., signals corresponding to the light chain charge variants) are shown as dotted lines.

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

[0123] Referring to FIG. 4D, iCIEF-Western was used to generate charge variant profiles for reduced and denatured antibodies from the same manufacturing lot of Antibody 1. Anti-human H+L antibody was used as a detection antibody with no specificity for a particular portion of the analyte antibody. As can be seen in FIG. 4D, the peak for the heavy chain charge variants (peak 301) overlaps with the peak for the light chain charge variants (peak 312). Using multiple detection antibodies in the iCIEF-Western method can improve the detectability of the peaks. Additionally, the use of multiple detection antibodies can improve the resolution of adjacent peaks. Example 2

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

[0125] Charge variant profiles were generated for reduced and denatured antibodies from the same production lot of antibody 2 using iCIEF-Western and are shown in Figure 5C. Biotinylated goat anti-human IgG Fc specific pAb was used as detection antibody for the heavy chain of reduced and denatured antibodies. Biotinylated goat anti-human kappa pAb was used as detection antibody for the light chain of reduced and denatured antibodies. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent. In Figure 5C, the signals detected from the Fc pAb (e.g., signals corresponding to the heavy chain charge variants) are shown as solid lines and the signals detected from the kappa pAb (e.g., signals corresponding to the light chain charge variants) are shown as dotted lines.

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

[0127] Referring to FIG. 5D, iCIEF-Western was used to generate charge variant profiles for reduced and denatured antibodies from the same manufacturing lot of antibody 2. Anti-human H+L antibody was used as a detection antibody with no specificity for a particular portion of the analyte antibody. As can be seen in FIG. 5D, some known peaks of the analyte (e.g., peaks 333 and 321) were not detected. In the iCIEF-Western method, the use of multiple detection antibodies can improve the detectability of peaks. Furthermore, the use of multiple detection antibodies can improve the resolution of adjacent peaks. Example 3

[0128] Charge variant profiles were generated using ChromiCE for reduced and denatured antibodies from a single manufacturing lot of Antibody 3 and are shown in Figures 6A and 6B. ChromiCE analyses were performed in triplicate and electropherograms from each run were overlaid on top of each other. The electropherogram of the heavy chain antibody is shown in Figure 6A and the electropherogram of the light chain antibody is shown in Figure 6B. The electropherograms generated using ChromiCE also showed 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 were generated for reduced and denatured antibodies from the same production lot of antibody 3 using iCIEF-Western and are shown in Figure 6C. Biotinylated goat anti-human IgG Fc specific pAb was used as detection antibody for the heavy chain of reduced and denatured antibodies. Biotinylated goat anti-human kappa pAb was used as detection antibody for the light chain of reduced and denatured antibodies. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent. In Figure 6C, the signals detected from the Fc pAb (e.g., signals corresponding to the heavy chain charge variants) are shown as solid lines and the signals detected from the kappa pAb (e.g., signals corresponding to the light chain charge variants) are shown as dotted lines.

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

[0131] In contrast to the reduction and denaturation methods described above, the polypeptides (e.g., antibodies) in the analyte may be digested by a protease (e.g., IdeS). IdeS cleaves the antibody into F(ab')2 and Fc fragments. An analyte containing antibody 3 from the same production lot used in Example 3 was digested using IdeS. The charge variant profiles of the F(ab')2 and Fc fragments were generated using ChromiCE and are shown in Figure 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. The peaks corresponding to the Fc fragments do not overlap with the peaks corresponding to the F(ab')2 fragments because the Fc fragment generally has a lower isoelectric point than the F(ab')2 fragment. The electropherogram generated using ChromiCE also showed peaks at isoelectric points of 5.85 and 9.50, which correspond to the isoelectric markers included in the sample buffer.

[0132] Charge variant profiles of F(ab')2 and Fc fragments were also generated using iCIEF-Western and are shown in FIG. 7B. Anti-human H+L antibody was used as a detection antibody with no specificity for a particular portion of the analyte antibody. In FIG. 7B, 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 (FIG. 7B) contains the same peaks (e.g., peaks 361, 362, 363, 364, 365, 366, 371, 372, 373 and 374) at the same positions as the electropherogram generated using ChromiCE (FIG. 7A). Example 5

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

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

[0135] The electropherogram generated using iCIEF-Western (Figure 8B) contains the same peaks (e.g., peaks 381, 382, ​​383, 384, 385, and 386) at the same positions as the electropherogram generated using ChromiCE (Figure 8A). In addition, the electropherogram generated using iCIEF-Western has better resolution than the electropherogram generated using ChromiCE. Furthermore, iCIEF-Western was used to characterize the heavy chain charge variants without the use of a chromatography step. Without being limited by theory, the poorer heavy chain resolution associated with ChromiCE may be due to the degradation of the analytes due to the high concentration of urea in the size exclusion chromatography (SEC) buffer and the long process time of the SEC step. Example 6

[0136] Using ChromiCE, a charge variant profile was generated for the reduced and denatured antibody heavy chains from a single production lot of Antibody 5 and is shown in Figure 9 A. The electropherogram generated using ChromiCE also showed 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] Charge variant profiles were generated using iCIEF-Western for reduced and denatured antibody heavy chains from the same production lot of antibody 5 and are shown in Figure 9B. Biotinylated goat anti-human IgG Fc specific pAb was used as the detection antibody for reduced and denatured antibody heavy chains. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent.

[0138] The electropherogram generated using iCIEF-Western (FIG. 9B) contains the same peaks (e.g., peaks 391, 392, 393, 394, and 395) in the same positions as the electropherogram generated using ChromiCE (FIG. 9A). Peak 393 corresponds to the predominant species of the heavy chain of antibody 5; peaks 391 and 392 correspond to acidic variants of the heavy chain of antibody 5; and peaks 394 and 395 correspond to basic variants of the heavy chain of antibody 5.

[0139] The peak area of ​​each peak was also calculated for the electropherograms generated using ChromiCE (Figure 9A) and iCIEF-Western (Figure 9B). The peak areas were plotted as a percentage of the total area under all peaks and are shown in Figure 9C. The peak areas calculated based on the ChromiCE electropherograms are indicated by squares and dotted lines, and the peak areas calculated based on the iCIEF-Western electropherograms are indicated by circles and solid lines. As shown in Figure 9C, the peak areas calculated based on the ChromiCE electropherograms (Figure 9A) are similar to the peak areas calculated based on the iCIEF-Western electropherograms (Figure 9B). Example 7

[0140] The charge variant profile generated via iCIEF-Western for a first lot of antibody may be compared to a known charge variant profile or to a charge variant profile for a second lot of antibody. Comparison of the charge variant profile of the first lot of antibody may validate the process conditions under which the first lot of antibody was produced.

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

[0142] An electropherogram generated based on an Fc pAb containing heavy chain charge variants is shown in FIG. 10A, and an electropherogram generated based on a kappa pAb containing light chain charge variants is shown in FIG. 10B. The charge variant profile for lot A is shown as a grey line, and the charge variant profile for lot B is shown as a black line. Still referring to FIGS. 10A and 10B, the charge variant profile for lot A is the same as the charge variant profile for lot B. For example, the electropherogram for lot A contains the same peaks as the electropherogram for lot B (e.g., peaks 401, 402, 403, 404, 405, 406, 407, 408, 411, 412, and 413). Additionally, the peak areas for lot A may be compared to lot B to ensure that the proportions of each charge variant between lots A and B are the same. Example 8

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

[0144] An electropherogram generated based on the Fc pAb containing heavy chain charge variants is shown in Figure 11A, and an electropherogram generated based on the kappa pAb containing light chain charge variants is shown in Figure 11B. The charge variant profile for lot A is shown as a grey line, and the charge variant profile for lot B is shown as a black line. Still referring to Figures 10A and 10B, the charge variant profile for lot A is the same as the charge variant profile for lot B. For example, the electropherogram for lot A contains the same peaks as the electropherogram for lot B (e.g., peaks 421, 422, 423, 424, 425, 431, and 432).

[0145] Additionally, the peak areas for lot A may be compared to lot B to ensure that the ratios of each charge variant are the same between lots A and B. The relative peak areas for the heavy chain charge variants of lots A and B are shown in Figure 11C. The relative peak areas for the light chain charge variants of lots A and B are shown in Figure 11D. Example 9

[0146] The charge variant profile of the native antibody from a first lot of antibody 8 (Lot A) was compared to the charge variant profile of the native antibody from a second lot of antibody 8 (Lot B). Lot A was subjected to heat stress. Specifically, Lot A was stored at 40° C. for 3 months. Biotinylated goat anti-human IgG Fc-specific pAb was used as detection antibody for the heavy chain of reduced and denatured antibodies. Biotinylated goat anti-human kappa pAb was used as detection antibody for the light chain of reduced and denatured antibodies. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent.

[0147] Electropherograms based on Fc pAb (heavy chain detection) are shown in Figure 12A and electropherograms based on kappa pAb detection are shown in Figure 12B. For both Figures 12A and 12B, the electropherograms of Lot A (heat stressed lot) are shown by grey lines and the electropherograms of Lot B (non-heat stressed lot) are shown by black lines. From the charge variant profiles of the native antibodies it is clear that the relative proportions of charge variants differ between heat stressed and non-heat stressed lots. However, there is no baseline separation of the peaks and it is not possible to reliably quantitate the individual isotypes.

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

[0149] The electropherogram based on the Fc pAb (heavy chain detection) is shown in Figure 13 A and the electropherogram based on the kappa pAb (light chain detection) is shown in Figure 13 B. For both Figures 13A and 13B, the electropherogram of Lot A (heat stressed lot) is shown by the grey line and the electropherogram of Lot B (non-heat stressed lot) is shown by the black line.

[0150] The isoelectric points of the heavy and light chains upon dissociation are different from that of the intact antibody. As a result, the iCIEF-Western method was able to generate charge variant profiles with baseline resolution and quantify the individual charge isotypes of the light and heavy chains.

[0151] With reference to Figure 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. With reference to Figure 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. Based on the electropherograms for Lot A (heat stressed) and Lot B (not heat stressed), the peak areas were calculated 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 [Table 5]

[0152] Heat stressed lots have a decreased relative abundance of the major isotypes and an increased abundance of acidic isotypes for both light and heavy chains compared to non-heat stressed lots. A charge variant profile may be generated for an unknown lot of antibody and compared to the charge variant profiles of heat stressed and non-heat stressed lots. Comparison of the charge variant profiles may provide information as to whether the unknown lot has been subjected to heat stress or other undesirable 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 detection antibody. Each combination of target polypeptide and detection antibody may require a different concentration of detection antibody during the first incubation step. iCIEF-Western analysis may be performed at different concentrations of detection antibody to determine the appropriate concentration of detection antibody.

[0154] Charge variant profiles were generated for reduced and denatured analytes containing antibody 9 using iCIEF-Western at different concentrations of detection antibody and are shown in Figures 14A and 14B. The method used to generate the charge variant profile shown in Figure 14A included a primary incubation in detection antibody at an antibody concentration of approximately 100 μg / mL. The method used to generate the charge variant profile shown in Figure 14B included a primary incubation in detection antibody at an antibody concentration of approximately 2.5 μg / mL. A biotinylated goat anti-human IgG Fc-specific pAb was used as the detection antibody for the heavy chain of the reduced and denatured antibodies. A biotinylated goat anti-human kappa pAb was used as the detection antibody for the light chain of the reduced and denatured antibodies. Streptavidin conjugated to HRP was used as the reporter molecule and luminol-peroxide was used as the reporting agent. In Figures 14A and 14B, signals detected from Fc pAbs (e.g., signals corresponding to heavy chain charge variants) are shown as solid lines, and signals detected from kappa pAbs (e.g., signals corresponding to light chain charge variants) are shown as dotted lines.

[0155] With reference to Figure 14A, iCIEF-Western analysis performed with excessive concentrations of detection antibody may result in excessive baseline noise, false peak detection, poor resolution, and / or distorted or asymmetric peaks. With reference to Figure 14B, iCIEF-Western analysis performed with appropriate concentrations of detection antibody may include a consistent baseline with limited background noise, and isolated peaks resolved to the baseline. Adjustment of the detection antibody concentration may reduce baseline noise and thus increase the measured signal-to-noise ratio, facilitating improved detection of low-abundance charge variants over a dynamic range. Example 11

[0156] To properly characterize VP proteins in iCIEF-Western, VP protein-specific antibodies may be developed. Antibodies with specificity for VP proteins were developed for AAV8 serotype AAV. A recombinant portion from the portion of the VP1 sequence not shared by VP2 and VP3 was used as a rabbit antigen to generate a VP1-specific polyclonal antibody. Two recombinant peptides from the portion of the VP2 sequence not shared by VP3 were used as rabbit antigens to generate a polyclonal antibody with specificity for VP2 and VP1. A recombinant peptide from VP3 was used as a rabbit antigen to generate a polyclonal antibody with specificity for VP3, VP2, and VP1. Briefly, a polyclonal antibody that binds with specificity for VP3, VP2, and VP1 may be referred to as anti-VP3 / VP2 / VP1, a polyclonal antibody that binds with specificity for VP2 and VP1 may be referred to as anti-VP2 / VP1 antibody, and a polyclonal antibody that binds with specificity for VP1 may be referred to as anti-VP1 antibody.

[0157] Western blots were used to confirm that the developed anti-VP3 / VP2 / VP1, anti-VP2 / VP1, and anti-VP1 polyclonal antibodies bind to their intended targets. The results of the western blots are shown in Figure 15. The leftmost blot (A) used anti-VP1 antibody as the primary antibody, the middle blot (B) used anti-VP1 / VP2 antibody as the primary antibody, and the rightmost blot (C) used anti-VP1 / VP2 / VP3 antibody as the primary antibody. The molecular weights of the bands in the reference ladder are shown in kilodaltons (kDa) on the left side of Figure 15. Bands corresponding to VP1, VP2, and VP3 proteins are marked on the right side of Figure 15. Anti-VP1 pAb binds only to VP1, anti-VP1 / VP2 pAb binds to VP1 and VP2, and anti-VP1 / VP2 / VP3 antibodies bind to VP1, VP2, and VP3. Example 12

[0158] iCIEF-Western may 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, the anti-VP3 / VP2 / VP1 antibody binds to VP1, VP2 and VP3, and the chemiluminescence profile generated by the anti-VP3 / VP2 / VP1 antibody may be compared to the profiles generated by other antibodies to determine which peaks correspond to the VP1 charge variant, the VP2 charge variant and the VP3 charge variant.

[0159] Referring to FIG. 16, the iCIEF-Western method was used to generate charge variant profiles for reduced and denatured analytes containing AAV8 serotype AAV. The rabbit polyclonal antibody described in Example 11 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 reporting agent. Line 461 shows an electropherogram generated using anti-VP1 as the detection antibody, line 462 shows an electropherogram generated using anti-VP1 / VP2 as the detection antibody, and line 463 shows an electropherogram generated using anti-VP1 / VP2 / VP3 antibody as the detection antibody. The profile generated from the anti-VP3 / VP2 / VP1 antibody (line 463) may be compared to the profiles generated from the 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. As shown in FIG. 16, 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 help associate VP protein charge variants with chemiluminescence peaks. For example, for VP proteins from AAV8 serotype AAV: the VP1 major charge variant has an isoelectric point of 6.01, the VP2 major charge variant has an isoelectric point of 6.87, and the VP3 major charge variant has an isoelectric point of 6.31. Example 13

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

[0162] Referring to Figure 17, the iCIEF-Western method was used to generate charge variant profiles for reduced and denatured analytes containing AAV8 serotype AAV. The anti-VP1 pAb, anti-VP1 / VP2 pAb, and anti-VP1 / VP2 / VP3 pAb described above in connection with Examples 11 and 12 were used as detection antibodies to generate charge variant profiles labeled 463. Anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. The commercially available anti-VP antibody designed against AAV2 serotype AAV was used to generate charge variant profiles labeled 464.

[0163] As shown in Figure 17, the pAb developed for use with AAV8 serotype AAV had greater sensitivity, detecting 15 peaks (e.g., peaks 473a-473o) compared to the 10 peaks detected by the commercially available antibody (e.g., peaks 474a-474j). Example 14

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

[0165] Using iCIEF-Western, charge variant profiles for reduced and denatured analytes containing AAV1 serotype AAV were generated and are shown in FIG. 18. A polyclonal antibody with specificity for AAV1 serotype VP2 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 reporting agent. As shown in FIG. 18, the polyclonal antibody with specificity for AAV1 serotype VP2 detected the polypeptide in the AAV1 serotype analyte. Example 15

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

[0167] Using iCIEF-Western, charge variant profiles for reduced and denatured analytes containing AAV5 serotype AAV were generated and are shown in FIG. 19. A polyclonal antibody with specificity for AAV5 serotype VP2 was used as the detection antibody. Anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. As shown in FIG. 19, the polyclonal antibody with specificity for AAV5 serotype VP2 detected the polypeptide in the AAV5 serotype analyte. Example 16

[0168] Using the iCIEF-Western method, charge variant profiles were generated for reduced and denatured analytes containing AAV8 serotype AAV, and are shown in Figures 20A and 20B. The charge variant profile shown in Figure 20A was generated using a polyclonal antibody with specificity for AAV1 serotype VP2 as the detection antibody. The charge variant profile shown in Figure 20B was generated using a polyclonal antibody with specificity for AAV5 serotype VP2 as the detection antibody. For both Figures 20A and 20B, an anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent.

[0169] Referring to Figure 20A, the polyclonal antibody specific to AAV1 serotype VP2 detected a charge variant peak, indicating that it has some sensitivity to AAV8 serotype viral proteins. Referring to Figure 20B, no charge variant peak was detected, indicating that the polyclonal antibody specific to AAV5 serotype VP2 cannot detect AAV8 serotype viral proteins. Example 17

[0170] Charge variant profiles were generated for the reduced and denatured analytes containing AAV1 serotype AAV, and are shown in Figures 21A and 21B. The charge variant profile shown in Figure 21A was generated using a polyclonal antibody with specificity for AAV8 serotype VP2 (e.g., the anti-VP1 / VP2 antibody described in Examples 11 and 12) as the detection antibody. The charge variant profile shown in Figure 21B was generated using a polyclonal antibody with specificity for AAV1 serotype VP2 as the detection antibody. For both Figures 21A and 21B, an anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. As can be seen by comparing the charge variants in Figures 21A and 21B, the polyclonal antibody with specificity for AAV8 serotype VP2 was unable to detect AAV1 serotype VP2 protein. Example 18

[0171] Charge variant profiles were generated for the reduced and denatured analytes containing AAV1 serotype AAV, and are shown in Figures 22A and 22B. The charge variant profile shown in Figure 22A was generated using a polyclonal antibody with specificity for AAV8 serotype VP2 (e.g., the anti-VP1 / VP2 antibody described in Examples 11 and 12) as the detection antibody. The charge variant profile shown in Figure 22B was generated using a polyclonal antibody with specificity for AAV5 serotype VP2 as the detection antibody. For both Figures 22A and 22B, an anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. As can be seen by comparing the charge variant profiles in Figures 22A and 22B, the polyclonal antibody with specificity for AAV8 serotype VP2 was unable to detect AAV5 serotype VP2 protein. Example 19

[0172] As described above, charge variant profiles of biopharmaceutical product lots can also be generated to characterize the environmental stress experienced by the lot. For example, charge variant profiles may be generated for heat-stressed and non-heat-stressed lots. The charge variant profile of an unknown lot may then be compared to known charge variant profiles to determine whether the unknown lot was under an unacceptable level of stress.

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

[0174] The charge variant profile for lot A is shown in Figure 23A, the charge variant profile for lot B is shown in Figure 23B, and the charge variant profile for lot C is shown in Figure 23C. As can be seen from Figures 23A-23C, there is an acidic shift in the charge variant profile as the AAV is subjected to more heat stress. Example 20

[0175] Charge variant profiles were generated for four lots of biopharmaceutical products containing AAV8 serotype AAV using the iCIEF-Western method. 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 the reporter molecule, and luminol-peroxide was used as the reporting agent. The charge variant profiles generated using anti-VP1 pAb as detection antibody are shown in Figures 24A-24D, the charge variant profiles generated using anti-VP1 / VP2 pAb as detection antibody are shown in Figures 25A-25D, and the charge variant profiles generated using anti-VP1 / VP2 / VP3 pAb as detection antibody are shown in Figures 26A-26D.

[0176] The first lot (Lot A) was not heat stressed, the second lot (Lot B) was stored at 40° C. for 15 days, the third lot (Lot C) was stored at 40° C. for 1 month, and the fourth lot (Lot D) was stored at 40° C. for 2 months. The charge variant profile of Lot A is shown in Figures 24A, 25A, and 26A, the charge variant profile of Lot B is shown in Figures 24B, 25B, and 26B, the charge variant profile of Lot C is shown in Figures 24C, 25C, and 26C, and the charge variant profile of Lot D is shown in Figures 24D, 25D, and 26D. As can be seen in Figures 24A-26D, there is an acidic shift in the charge variant profile as the AAV is subjected to more heat stress. Additionally, 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 may allow for the detection of 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 the environmental stress of a biopharmaceutical product lot compared to monitoring the charge variant profile of all viral proteins.

[0178] Referring to Figure 27A, charge variant profiles were generated for four lots of assays containing reduced and denatured AAV. The first lot (Lot A) was not heat stressed, the second lot (Lot B) was stored at 40°C for 15 days, the third lot (Lot C) was stored at 40°C for 1 month, and the fourth lot (Lot C) was stored at 40°C for 2 months. Anti-VP1 / VP2 / VP3 pAb described in Examples 11 and 12 was used as the detection antibody, anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. The charge variant profile of Lot A is shown by line 465, the charge variant profile of Lot B is shown by line 466, the charge variant profile of Lot C is shown by line 467, and the charge variant profile of Lot D is shown by line 468.

[0179] Further referring to Figure 27A, as AAV is subjected to more heat stress, there is an acidic shift in the charge variant profile. For example, the height (e.g., and 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 examples, based on the data shown in Figure 27A, the relative peak areas of peaks 488 and 487 can be used to determine the level of environmental stress of an unknown lot.

[0180] Referring to Figure 27B, charge variant profiles were generated for lots A, B, C, and D of assays containing reduced and denatured AAV. Anti-VP1 pAb described in Examples 11 and 12 was used as the detection antibody, anti-rabbit antibody conjugated to HRP was used as the reporter molecule, and luminol-peroxide was used as the reporting agent. The charge variant profile for lot A is shown by line 465, the charge variant profile for lot B is shown by line 466, the charge variant profile for lot C is shown by line 467, and the charge variant profile for lot D is shown by line 468. The charge variant profiles shown in Figure 27B show the same acidic shift as those shown in Figure 27A. Example 22

[0181] In addition to heat stress, charge variant profiles generated using the iCIEF method may be used to assess the impact of other manufacturing or storage conditions on the efficacy of a biopharmaceutical product that contains a polypeptide.

[0182] Using the iCIEF-Western method, charge variant profiles were generated for five different lots of reduced and denatured analytes containing biopharmaceutical products, including AAV. The first lot (Lot A) was not subjected to a hold time during manufacture, the second lot (Lot B) was subjected to an 8 hour process hold time during manufacture, the third lot (Lot C) was subjected to a 24 hour process hold time during manufacture, the fourth lot (Lot D) was subjected to a 48 hour hold time during manufacture, and the fifth lot (Lot E) was subjected to a 72 hour hold time during manufacture. The charge variant profile for Lot A is shown in Figure 28A, the charge variant profile for Lot B is shown in Figure 28B, the charge variant profile for Lot C is shown in Figure 28C, the charge variant profile for Lot D is shown in Figure 28D, and the charge variant profile for Lot E is shown in Figure 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 in Figures 28A-28E.

[0183] The relative peak areas were calculated for each peak in each charge variant profile and are summarized in FIG. Example 23

[0184] Using the iCIEF-Western method, charge variant profiles were generated for four different lots of reduced and denatured analytes containing biopharmaceutical products, including AAV. The first lot (Lot A) was subjected to one freeze-thaw cycle (e.g., freezing the biopharmaceutical product and then thawing), the second lot (Lot B) was subjected to one freeze-thaw cycle and kept at 4° C. for 7 days, the third lot (Lot C) was subjected to three freeze-thaw cycles, and the fourth lot (Lot D) was subjected to five freeze-thaw cycles. The charge variant profile for Lot A is shown in FIG. 30A, the charge variant profile for Lot B is shown in FIG. 30B, the charge variant profile for Lot C is shown in FIG. 30C, and the charge variant profile for Lot 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 in Figures 30A-30D.

[0185] The relative peak areas were calculated for each peak in each charge variant profile and are summarized in FIG. Example 24

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

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

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

[0189] Comparing the charge variant profiles in Figures 32A, 33A, and 34A with those in Figures 32B, 33B, and 34B, less baseline noise is observed and therefore a better signal-to-noise ratio is achieved with sample buffers containing higher urea concentrations. Example 25

[0190] Charge variant profiles were prepared for analytes containing empty capsid AAV according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer containing approximately 4.3 M urea and approximately 30% formamide by volume, and the charge variant profile generated from this method is shown by line 541 in FIG. 35. The second iCIEF-Western method included a sample buffer containing approximately 5.6 M urea and approximately 15% formamide by volume, and the charge variant profile generated from this method is shown by line 542 in FIG. 35. The third iCIEF-Western method included a sample buffer containing approximately 7.3 M urea and approximately 0% formamide by volume, and the charge variant profile generated from this method is shown by line 543 in FIG. 35.

[0191] Charge variant profiles were prepared for analytes containing partially intact capsid AAV according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer containing approximately 4.3 M urea and approximately 30% formamide by volume, and the charge variant profile generated from this method is shown by line 544 in Figure 36. The second iCIEF-Western method included a sample buffer containing approximately 5.6 M urea and approximately 15% formamide by volume, and the charge variant profile generated from this method is shown by line 545 in Figure 36. The third iCIEF-Western method included a sample buffer containing approximately 7.3 M urea and approximately 0% formamide by volume, and the charge variant profile generated from this method is shown by line 546 in Figure 36.

[0192] Charge variant profiles were prepared for analytes containing intact capsid AAV according to three different iCIEF-Western methods. The first iCIEF-Western method included a sample buffer containing approximately 4.3 M urea and approximately 30% formamide by volume, and the charge variant profile generated from this method is shown by line 547 in Figure 37. The second iCIEF-Western method included a sample buffer containing approximately 5.6 M urea and approximately 15% formamide by volume, and the charge variant profile generated from this method is shown by line 548 in Figure 37. The third iCIEF-Western method included a sample buffer containing approximately 7.3 M urea and approximately 0% formamide by volume, and the charge variant profile generated from this method is shown by line 549 in Figure 37.

[0193] Comparing the charge variant profiles in Figures 35-37, less baseline noise is observed in sample buffers containing formamide in the presence of urea, and therefore a better signal-to-noise ratio is achieved.

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

[0195] Item 1. A method for quantifying charge variants in an analyte, comprising: Introducing an analyte into the capillary; Separating the charge variants in the sample buffer along an isoelectric gradient; incubating the capillary in a detection antibody; Quantifying the relative abundance of the charge variants based on the signal corresponding to the detection antibody; The above method.

[0196] Item 2. The method of item 1, further comprising 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 streptavidin conjugated to horseradish peroxidase.

[0198] Item 4. The method of item 2, further comprising introducing a detection agent into the capillary.

[0199] Item 5. The method of item 4, wherein the detection agent is luminol peroxide.

[0200] Item 6. The method of item 2, further comprising generating an electropherogram, the electropherogram comprising 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 quantification of the relative abundance of the charge variants based on the signal corresponding to the detection antibody comprises calculating the area under the electropherogram peak corresponding to the charge variant.

[0202] Item 8. The method of item 1, further comprising immobilizing the charge variants in the capillary after separating the charge variants along the isoelectric gradient and prior to incubating the capillary in a detection antibody.

[0203] Item 9. A method for quantifying charge variants in an analyte, comprising: reducing and denaturing a polypeptide within the analyte to produce a reduced and denatured polypeptide, wherein the analyte comprises a charge variant of the target polypeptide; buffer exchanging the reduced and denatured polypeptide to generate a buffer exchanged sample, wherein the buffer exchanged sample comprises the reduced and denatured polypeptide; preparing a sample buffer containing a buffer exchanged sample; introducing a sample buffer into the capillary; separating charge variants of the target polypeptide along an isoelectric gradient; measuring a signal in a region within the isoelectric gradient in the capillary that correlates with the abundance of the target polypeptide; The above method.

[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 hydroxypropyl methylcellulose and the sample buffer comprises at least about 1 volume percent hydroxypropyl methylcellulose.

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

[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 approximately 30 volume percent or less.

[0210] Item 16. A method for assessing the level of environmental stress recognized by a sample of a target polypeptide, comprising: generating a charge variant profile for the sample, the charge variant profile comprising a plurality of peaks, each peak comprising: associated with a corresponding charge variant of the target polypeptide; associated with an isoelectric point equivalent to that of the corresponding charge variant of the target polypeptide; The relative peak areas, which are related to the relative abundance of the corresponding charge variants of the target polypeptide, And, comparing the charge variant profile for the sample to one or more known charge variant profiles; determining a level of environmental stress perceived by the sample based on a comparison of the charge variant profile for the sample to one or more known charge variant profiles; The above method.

[0211] Item 17. The method of item 16, wherein the level of environmental stress is related to a level of thermal stress, a level of stress due to manufacturing process hold times, or a level of stress due to 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. Generation of a charge variant profile for a sample: Introducing a sample into the capillary; Separating charge variants of the target polypeptide along an isoelectric gradient; The method of item 16, including

[0214] Item 20. Generation of a charge variant profile for a sample: incubating the capillary in a detection antibody; incubating the capillary in a reporter molecule; generating an electropherogram, the electropherogram including 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; 20. The method of item 19, further comprising:

[0215] Those skilled in the art will appreciate that the conception on which this disclosure is based may be readily used as a basis for the designing of other methods and systems for carrying out some of the purposes of the present disclosure, and therefore the claims are not to be deemed limited by the foregoing description.

Claims

1. 1. A method for quantifying charge variants in an analyte, comprising: introducing the analyte into a capillary; separating charge variants in said sample buffer along an isoelectric gradient; incubating the capillary in a detection antibody; quantitating the relative abundance of charge variants based on the signal corresponding to said detection antibody; The method comprising:

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

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

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

5. The method of claim 4, wherein the detection agent is luminol peroxide.

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

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

8. After separating charge variants along the isoelectric gradient and prior to incubating the capillary in the detection antibody. Immobilizing charge variants in the capillary 2. The method of claim 1, further comprising:

9. 1. A method for quantifying charge variants in an analyte, comprising: reducing and denaturing a polypeptide within the analyte to produce a reduced and denatured polypeptide, wherein the analyte comprises a charge variant of a target polypeptide; buffer exchanging 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 containing the buffer exchanged sample; introducing the sample buffer into a capillary; separating charge variants of said target polypeptide along an isoelectric gradient; measuring a signal in a region within the isoelectric gradient in the capillary that correlates to the abundance of the target polypeptide; The above method.

10. 10. The method of claim 9, wherein the sample buffer comprises urea, a carrier ampholyte, and cellulose.

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

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

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

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

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

16. 1. A method for assessing the level of environmental stress perceived by a sample of a target polypeptide, comprising: generating a charge variant profile for the sample, the charge variant profile comprising a plurality of peaks, each peak comprising: associated with a corresponding charge variant of said target polypeptide; relative to the isoelectric point equivalent to the isoelectric point of the corresponding charge variant of the target polypeptide; and a relative peak area associated with the relative abundance of the corresponding charge variant of the target polypeptide. And, comparing the charge variant profile for the sample to one or more known charge variant profiles; determining a level of environmental stress perceived by the sample based on a comparison of the charge variant profile for the sample to the one or more known charge variant profiles; The method comprising:

17. 17. The method of claim 16, wherein the level of environmental stress is related to a level of thermal stress, a level of stress due to manufacturing process hold times, or a level of stress due to freeze-thaw cycles.

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

19. 2. The method of claim 1, further comprising: introducing said sample into a capillary; separating charge variants of said target polypeptide along an isoelectric gradient; 17. The method of claim 16, comprising:

20. 2. The method of claim 1, further comprising: incubating the capillary in a detection antibody; incubating the capillary in a reporter molecule; generating an electropherogram, said electropherogram comprising a plot of the intensity of the signal generated by said reporter molecule versus the isoelectric point along said isoelectric gradient at which said signal is detected; 20. The method of claim 19, further comprising:

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