Method for quantifying the relative distribution of glucuronidation, iduronidation, and galacturonidation of polypeptides

Abstract

The present disclosure relates to methods and compositions for quantifying the relative distribution of glucuronidation, iduronidation, and / or galacturonidation of polypeptides, including stereoselective liquid chromatography mass spectrometry (LC-MS) methods that can achieve simultaneous separation and relative quantification of glucuronidation, iduronidation, and / or galacturonidation of polypeptides.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 124,320, filed on December 11, 2020, the entire content of which is incorporated herein by reference.

[0002] Field of the Invention This specification relates to methods and compositions for quantifying the relative distribution of glucuronidation, iduronidation, and galacturonidation of polypeptides. Specifically, this specification relates to a stereoselective liquid chromatography - mass spectrometry (LC - MS) method for achieving simultaneous separation and relative quantification of glucuronidation, iduronidation, and galacturonidation of polypeptides.

Background Art

[0003] Glucuronidation is a post - translational polypeptide modification that results from the covalent attachment of glucuronic acid (also called uronic acid) to a primary amine group such as the side chain of a lysine residue. Naturally occurring glucuronic acids are of five types: glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid, and guluronic acid (Figure 1). In recombinant polypeptides produced by mammalian cells, such as CHO cells, and bacterial cells, such as Escherichia coli, modification of primary amines by glucuronic acid, iduronic acid, and galacturonic acid can occur. In contrast, modification by mannuronic acid and guluronic acid is not expected in recombinant expressed polypeptides because these two glucuronic acids are found in brown algae or in specific types of bacteria that are not generally used for the production of recombinant polypeptides.

[0004] Modification of polypeptides with glucuronic acid, iduronic acid, or galacturonic acid may affect the safety and / or efficacy of such recombinant expression products. For example, modification of lysine residues within the complementarity-determining region of monoclonal antibody drugs with glucuronic acid may inhibit target binding, potentially leading to loss of antibody efficacy. Furthermore, modification of lysine residues within the complementarity-determining region with iduronic acid may cause off-target binding due to the unique structural flexibility of iduronic acid. Additionally, since modification of lysine residues with iduronic acid or galacturonic acid has not been observed in endogenous human polypeptides, iduronic and galacturonic oxidation of recombinant expression polypeptides may form neoepitopes, potentially posing an immunogenicity risk. To ensure the safety and efficacy of recombinant expression polypeptides, it is important to quantify the relative distribution of glucuronic, iduronic, and / or galacturonic oxidation. [Overview of the Initiative]

[0005] In certain embodiments, the disclosure relates to compositions and methods for quantifying the relative distribution of glucuronization, iduronization, and / or galacturonization of polypeptides. In certain embodiments, a method for quantifying the relative distribution of glucuronization, iduronization, and galacturonization of a population of polypeptides includes: a) contacting a population of polypeptides with a protease to produce peptides; b) contacting the peptides with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; and c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of glucuronization, iduronization, and galacturonization of the peptide.

[0006] In certain embodiments, the protease used in connection with a method for quantifying the relative distribution of glucuronization, iduronization, and / or galacturonization of a population of polypeptides described herein is trypsin, lys-C, Glu-C, or Asp-N.

[0007] In certain embodiments, the chromatographic support used in connection with a method for quantifying the relative distribution of glucuronic, iduronic, and / or galacturonic oxidation of a group of polypeptides described herein comprises a support material containing a positively charged surface modifier.

[0008] In certain embodiments, the hydrophobic stationary phase used in connection with a method for quantifying the relative distribution of glucuron, iduron, and / or galacturon oxidation of a group of polypeptides described herein is bonded to the surface modifier. In certain embodiments, the hydrophobic stationary phase comprises alkyl, alkenyl, alkynyl, or aryl functional groups. In certain embodiments, the hydrophobic stationary phase comprises C18 functional groups.

[0009] In certain embodiments, the chromatography support is CSH TM Includes C18 stationary phase.

[0010] In certain embodiments, the separated peptides are analyzed using mass spectrometry.

[0011] In certain embodiments, the methods described herein include the identification of glucuron-oxidized peptides, iduron-oxidized peptides, and / or galacturon-oxidized peptides. In certain embodiments, the peptides are identified by their respective chromatographic retention times and mass spectra.

[0012] In certain embodiments, the methods described herein include quantifying the relative distribution of glucuronization, iduronization, and / or galacturonization by integrating the respective peaks of the liquid chromatograms corresponding to the separated peptides, which are monitored by UV absorbance. In certain embodiments, the relative distribution of glucuronization, iduronization, and / or galacturonization is quantified by integrating the respective peaks of the ion chromatograms extracted from mass spectrometry.

[0013] In certain embodiments, the weak acid used in the context of the method described herein is formic acid or acetic acid.

[0014] In certain embodiments, the acidic mobile phase used in the method described herein is at a pH in which more than 50% of the carboxylic acid moiety associated with glucuron, iduron, or galacturon oxidation is in a deprotonated state.

[0015] In certain embodiments, the weakness used in the context of the method described herein Acid transfer The dynamic phase gradient has pH > 3. In certain embodiments, weak Acid transfer The dynamic phase gradient has a pKa value of approximately 4.5.

[0016] In certain embodiments, the mass spectrometry used in the context of the method described herein is electrospray ionization tandem mass spectrometry (ESI-MS / MS).

[0017] In some embodiments, the polypeptide analyzed by the method described herein is an antibody. [Brief explanation of the drawing]

[0018] [Figure 1] This shows the structure of naturally occurring glycuronic acid. [Figure 2] This is an extracted ion chromatogram showing the separation of glucuronized (glua) peptides and iduronized (idoa) peptides. Antibody A was modified by forced glucuronization / iduronization. Subsequently, the sample was digested with trypsin and analyzed by LC-MS / MS using a CSH C18 column. IdoA peptides and GluA peptides eluted at 15.07 minutes and 15.38 minutes, respectively. [Figure 3] The extracted ion chromatogram of the galacturon-oxidized (glaa) peptide is shown. Antibody A was modified by forced galacturon oxidation. Subsequently, the sample was digested with trypsin and analyzed by LC-MS / MS using a CSHTM C18 column. The GlaA peptide eluted in 15.16 minutes. [Figure 4]The BEHTM C18 column indicates that the candidate peptides are not stereoselectively separated.

Best Mode for Carrying Out the Invention

[0019] The following detailed description given by way of example is not intended to limit the presently disclosed subject matter to the specific embodiments described, and can be understood in conjunction with the accompanying drawings.

[0020] This specification relates to methods and compositions for quantifying the relative distribution of glucuronidation, iduronidation, and / or galacturonidation of polypeptides. Specifically, this specification relates to a stereoselective LC-MS method that enables simultaneous separation and relative quantification of glucuronidation, iduronidation, and / or galacturonidation of polypeptides.

[0021] 1. Definitions Unless otherwise specified, all technical terms, notations, and other scientific terms used in this specification are intended to have the meanings commonly understood by those skilled in the technical field to which this disclosure pertains. In some cases, terms having commonly understood meanings are defined in this specification for the purpose of clarification and / or ease of reference, but including such definitions in this specification is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art.

[0022] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)” and variations thereof are intended to be open - ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Also, in the present disclosure, other embodiments that “comprise”, “consist of” and “consist essentially of” the embodiments or elements presented herein are contemplated whether or not explicitly recited.

[0023] As used herein, the term “about” or “approximately” means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within three standard deviations, or more than three standard deviations, depending on the convention used in the art. Alternatively, “about” can mean within up to 20%, preferably up to 10%, more preferably up to 5%, still more preferably up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within five - fold, more preferably within two - fold of a value.

[0024] As used herein, “polypeptide” generally refers to peptides and proteins having more than about 10 amino acids. The polypeptide may be homologous to the host cell or, preferably, may be heterologous, i.e., foreign, to the host cell used, such as a human protein produced by Chinese hamster ovary cells or a yeast polypeptide produced by mammalian cells. In certain embodiments, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, and more preferably those directly secreted into the medium are used.

[0025] The term “protein” refers to a sequence of amino acids having a chain length sufficient to produce a higher-order tertiary and / or quaternary structure. This is to distinguish it from “peptides” or other low molecular weight drugs that do not have such structures. Proteins as used herein typically have a molecular weight of at least about 15–20 kD, preferably at least about 20 kD. Examples of proteins included in the definition herein generally include all mammalian proteins, particularly therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and proteins containing one or more disulfide bonds, including multi-chain polypeptides containing one or more interchain and / or intrachain disulfide bonds.

[0026] The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence (including polymers of such pairings)), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0027] 2. Method for quantifying the relative distribution of glucuronidation, iduronidation, and galacturonidation of polypeptides In certain embodiments, the methods for the relative distribution and / or identification of glucuronic, iduronic, and / or galacturonic oxidation of polypeptides described herein include: a) contacting the polypeptide with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; and c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; and d) Analyzing the separated peptide to quantify the relative distribution of glucuronization, iduronization, and / or galacturonization of the peptide.

[0028] The methods outlined herein provide direct quantification of the relative distribution of glucuronization, iduronization, and / or galacturonization of polypeptides. These methods are in contrast to other techniques for studying glucuronization, iduronization, and / or galacturonization of polypeptides. For example, techniques that measure the level of glucuronic acid in cell cultures do not necessarily correlate with the level of glucuronization of polypeptides induced by such cultures, much less provide any information regarding the distribution or identity of modified amino acids in such polypeptides. The methods described herein also differ from methods that require the release of glucuronic acid from peptides during the detection process and therefore cannot directly identify the distribution or location of modified amino acids (e.g., Lin et al., Eur. J Biochem, 106:341-351 (1980) and Spiro, JBC, 252(15): 5424-5430 (1977)).

[0029] In certain embodiments, the protease is an aspartate protease, a cysteine ​​protease, a metalloprotease, a serine protease, or a threonine protease. Exemplary proteases useful in connection with this disclosure are known in the art and are described, for example, in Verhamme et al. Proteases: Pivot Points in Functional Proteomics, Methods Mol Biol. 2019;1871:313-392 (the entire text of which is incorporated by reference). In certain embodiments, the protease is trypsin. In certain embodiments, the protease is lys-C. In certain embodiments, the protease is Glu-C. In certain embodiments, the protease is Asp-N.

[0030] In certain embodiments, the chromatography support comprises a support material having a positively charged surface modifier. In certain embodiments, the surface modifier can be attached to the substrate of the chromatography support via derivatization, coating, and / or crosslinking, thereby imparting the chemical properties of the surface modifier to the substrate of the chromatography support. Exemplary modifiers are specified in U.S. Patent No. 7223473 (which is incorporated herein by reference in its entirety). In certain embodiments, the surface modifier may be an organotrihalosilane such as octyltrichlorosilane or octadecyltrichlorosilane. In certain embodiments, the chromatography support comprises a hydrophobic stationary phase. In certain embodiments, the hydrophobic stationary phase is bonded to the surface modifier by covalent or non-covalent bonds. For example, but not limited to, the surface modifier may comprise one or more alkyl, alkenyl, alkynyl, or aryl functional groups as the hydrophobic stationary phase. In certain embodiments, the functional groups include, but are not limited to, C1-C30 alkyl groups such as octyl (C8), octadecyl (C18), and triacontyl (C30); alkaryl groups, such as C1-C4 phenyl groups. In certain embodiments, the chromatography support is a Charged Surface Hybrid (CSH) TM ) is the stationary phase. In certain embodiments, the chromatography support is CSH TM It is a C18 stationary phase.

[0031] In certain embodiments, the acidic mobile phase is at a pH in which more than 50% of the carboxylic acid moieties involved in glucuronic oxidation, iduronic oxidation, and / or galacturonic oxidation are in a deprotonated state. In certain embodiments, the weak acid is formic acid or acetic acid. In certain embodiments, Acid transfer The dynamic phase gradient has a pH greater than 3. In certain embodiments, weak Acid transfer The dynamic phase gradient has a pKa value of approximately 4.5.

[0032] In certain embodiments, the separated peptides are analyzed using mass spectrometry. In certain embodiments, the mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS / MS).

[0033] In certain embodiments, the method of the subject of this disclosure further includes the identification of glucuronized peptides, iduronized peptides, and / or galacturonized peptides. In certain embodiments, the peptides are identified by their respective chromatographic retention times and / or mass spectra. In certain embodiments, the relative distribution of glucuronized, iduronized, and / or galacturonized peptides is quantified by integrating the respective peaks of the liquid chromatograms corresponding to the separated peptides, which are monitored by UV absorbance. In certain embodiments, the relative distribution of glucuronized, iduronized, and / or galacturonized peptides is quantified by integrating the respective peaks of the ion chromatograms extracted from mass spectrometry.

[0034] In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a multispecific antibody, such as a bispecific or triplicate antibody.

[0035] 3. Exemplary Embodiments A-1. In a particular embodiment, the present disclosure is a method for quantifying the relative distribution of glucuronization of a population of polypeptides, the method comprising: a) contacting a population of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of glucuronization of the peptide.

[0036] A-2. In a particular embodiment, the present disclosure relates to a method for quantifying the relative distribution of iduronic oxidation of a group of polypeptides, the method comprising: a) contacting a group of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of iduronic oxidation of the peptide.

[0037] A-3. In certain embodiments, the disclosure relates to a method for quantifying the relative distribution of galacturon oxidation of a group of polypeptides, the method comprising: a) contacting a group of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of galacturon oxidation of the peptide.

[0038] A-4. In certain embodiments, the disclosure relates to a method for quantifying the relative distribution of glucuronization and iduronization of a population of polypeptides, the method comprising: a) contacting a population of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of glucuronization and iduronization of the peptide.

[0039] A-5. In certain embodiments, the disclosure relates to a method for quantifying the relative distribution of glucuronization and galacturonization of a population of polypeptides, the method comprising: a) contacting a population of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of glucuronization and galacturonization of the peptide.

[0040] A-6. In certain embodiments, the disclosure relates to a method for quantifying the relative distribution of iduronic and galacturonic oxidation of a group of polypeptides, the method comprising: a) contacting a group of polypeptides with a protease to produce a peptide; b) contacting the peptide with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support. Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of iduronic and galacturonic oxidation of the peptide.

[0041] A-7. In certain embodiments, the disclosure relates to a method for quantifying the relative distribution of glucuron, iduron, and galacturon oxidation of a population of polypeptides, the method comprising: a) contacting a population of polypeptides with a protease to produce peptides; b) contacting the peptides with a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) weakening the chromatographic support Acid transfer d) Separating the peptide by contacting it with a dynamic phase gradient; d) Analyzing the separated peptide to quantify the relative distribution of glucuronization, iduronization, and galacturonization of the peptide.

[0042] B-1. In specific embodiments A-1 to A-7, the protease is selected from the group consisting of trypsin, lys-C, Glu-C, and Asp-N. In specific embodiments A-1 to A-7, the protease is trypsin. In specific embodiments A-1 to A-7, the protease is lys-C. In specific embodiments A-1 to A-7, the protease is Glu-C. In specific embodiments A-1 to A-7, the protease is Asp-N.

[0043] C-1. In specific embodiments A-1 to A-7, the chromatography support comprises a support material having a positively charged surface modifier.

[0044] C-2. In specific embodiments A-1 to A-7, B-1, and C-1, the hydrophobic stationary phase is bonded to a surface modifier.

[0045] C-3. In a specific embodiment C-2, the hydrophobic stationary phase comprises an alkyl, alkenyl, alkynyl, or aryl functional group.

[0046] C-4. In a specific embodiment C-3, the hydrophobic stationary phase contains a C18 functional group.

[0047] C-5. In a specific embodiment C-3, the chromatography support is CSH TM Includes C18 stationary phase.

[0048] D-1. In specific embodiments A-1 to A-7, B-1, and C-1 to C-5, the separated peptides are analyzed by mass spectrometry. In specific embodiment D-1, the mass spectrometry is electrospray ionized tandem mass spectrometry (ESI-MS / MS). In specific embodiment D-1, the relative distribution of glucuronization is quantified by integrating each peak of the ion chromatogram extracted from the mass spectrometry. In specific embodiment D-1, the relative distribution of iduronization is quantified by integrating each peak of the ion chromatogram extracted from the mass spectrometry. In specific embodiment D-1, the relative distribution of galacturonization is quantified by integrating each peak of the ion chromatogram extracted from the mass spectrometry. In specific embodiment D-1, the relative distributions of glucuronization and iduronization are quantified by integrating each peak of the ion chromatogram extracted from the mass spectrometry. In a specific embodiment D-1, the relative distribution of glucuronization and galacturonization is quantified by integrating the respective peaks of the ion chromatogram extracted from mass spectrometry. In a specific embodiment D-1, the relative distribution of iduronization and galacturonization is quantified by integrating the respective peaks of the ion chromatogram extracted from mass spectrometry. In a specific embodiment D-1, the relative distribution of glucuronization, iduronization, and galacturonization is quantified by integrating the respective peaks of the ion chromatogram extracted from mass spectrometry.

[0049] E-1. In specific embodiments A-1 to A-7, B-1, and C-1 to C-5, the separated peptides are analyzed by their respective chromatographic retention times. In specific embodiment E-1, the separated peptides are analyzed by integrating each peak of the liquid chromatogram corresponding to the separated peptide, which is monitored by UV absorbance. In specific embodiment E-1, the relative distribution of glucuron oxidation is quantified by integrating each peak of the liquid chromatogram corresponding to the separated peptide, which is monitored by UV absorbance. In specific embodiment E-1, the relative distribution of iduron oxidation is quantified by integrating each peak of the liquid chromatogram corresponding to the separated peptide, which is monitored by UV absorbance. In specific embodiment E-1, the relative distribution of galacturon oxidation is quantified by integrating each peak of the liquid chromatogram corresponding to the separated peptide, which is monitored by UV absorbance. In a specific embodiment E-1, the relative distribution of glucuronization and iduronization is quantified by integrating the respective peaks of the liquid chromatogram corresponding to the separated peptide, which are monitored by UV absorbance. In a specific embodiment E-1, the relative distribution of iduronization and galacturonization is quantified by integrating the respective peaks of the liquid chromatogram corresponding to the separated peptide, which are monitored by UV absorbance. In a specific embodiment E-1, the relative distribution of glucuronization and galacturonization is quantified by integrating the respective peaks of the liquid chromatogram corresponding to the separated peptide, which are monitored by UV absorbance. In a specific embodiment E-1, the relative distribution of glucuronization, iduronization, and galacturonization is quantified by integrating the respective peaks of the liquid chromatogram corresponding to the separated peptide, which are monitored by UV absorbance.

[0050] F-1. In specific embodiments A-1 to A-7, B-1, C-1 to C-5, D-1, and E-1, the method includes the identification of glucuronized peptides, iduronized peptides, or galacturonized peptides. In specific embodiment F-1, the method includes the identification of glucuronized peptides. In specific embodiment F-1, the method includes the identification of iduronized peptides. In specific embodiment F-1, the method includes the identification of galacturonized peptides. In specific embodiment F-1, the method includes the identification of glucuronized peptides and iduronized peptides. In specific embodiment F-1, the method includes the identification of iduronized peptides and galacturonized peptides. In specific embodiment F-1, the method includes the identification of glucuronized peptides and galacturonized peptides. In specific embodiment F-1, the method includes the identification of glucuronized peptides, iduronized peptides, and galacturonized peptides.

[0051] F-2. In a specific embodiment F-1, the peptides are identified by their respective chromatographic retention times. In a specific embodiment F-1, the peptides are identified by their respective mass spectra. In a specific embodiment F-1, the peptides are identified by their respective chromatographic retention times and mass spectra.

[0052] G-1. In specific embodiments A-1 to A-7, B-1, C-1 to C-5, D-1, E-1, and F-1 to F-2, the weak acid is formic acid or acetic acid.

[0053] G-2. In specific embodiments A-1 to A-7, B-1, C-1 to C-5, D-1, E-1, and F-1 to F-2, the acidic mobile phase is at a pH in which more than 50% of the carboxylic acid moiety related to glucuronization, iduronization, or galacturonization is in a deprotonated state. In certain embodiments, the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with glucuron oxidation is in a deprotonated state; the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with iduron oxidation is in a deprotonated state; the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with galacturon oxidation is in a deprotonated state; the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with glucuron oxidation and iduron oxidation is in a deprotonated state; the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with glucuron oxidation and galacturon oxidation is in a deprotonated state; the acidic mobile phase is at a pH where more than 50% of the carboxylic acid moiety associated with glucuron oxidation, iduron oxidation, and galacturon oxidation is in a deprotonated state. In certain embodiment G-2, Acid transfer The dynamic phase gradient has a pH greater than 3. In a specific embodiment G-2, weak Acid transfer The dynamic phase gradient has a pKa value of approximately 4.5.

[0054] H-1. In specific embodiments A-1 to A-7, B-1, C-1 to C-5, D-1, E-1, F-1 to F-2, and G-1 to G-2, the polypeptide is an antibody. [Examples]

[0055] Example 1: Assay for quantifying the relative distribution of glucuronization, iduronization, and galacturonization of polypeptides. The antibody A polypeptide was digested using proteases (trypsin, lys-C, Glu-C, Asp-N, etc.). The resulting peptide was processed using CSH. TMSeparation was performed using a C18 column. Stereoselective separation was based on the intercharge interaction between the negative charge of glucuronic acid and the positive surface charge of the stationary phase, and the hydrophobic interaction between the glucuronide-oxidized peptide and the hydrophobic stationary phase. A mobile phase gradient using a weak acid (e.g., formic acid or acetic acid, but not trifluoroacetic acid) was employed to achieve separation of glucuronide-oxidized peptide, iduronide-oxidized peptide, and galacturonide-oxidized peptide. At pH > 3, most of the carboxyl groups (pKa = approximately 4.5) of the glucuronic acid moiety are negatively charged, which promotes stereoselective intercharge interaction between the glucuronic acid moiety and the positively charged stationary phase; therefore, a weak acid was selected.

[0056] The antibody A polypeptide was digested using proteases (trypsin, lys-C, Glu-C, Asp-N, etc.). The resulting peptide was obtained using BEH, where the stationary phase is not positively charged. TM Analysis was performed using a C18 column. A mobile phase gradient (pH < 3) using trifluoroacetic acid was employed as the mobile phase. Trifluoroacetic acid is one of the commonly used ion-pairing reagents to improve peak shape for chromatographic separation. As shown in Figure 4, BEH TM Stereoselective separation cannot be achieved with a C18 column.

[0057] Subsequently, the eluted peptides were analyzed by ESI-MS / MS. The identity of glucuronized peptides, iduronized peptides, and galacturonized peptides was confirmed by the retention times of each peptide in chromatographic separation (Figures 2 and 3) and the respective peptide mass spectra collected by ESI-MS / MS analysis. The relative distribution of glucuronized, iduronic acid, and galacturonized peptides was quantified by integrating the respective peaks in the liquid chromatogram (monitored by UV absorbance) or the extracted ion chromatogram (based on molecular weight, Figures 2 and 3).

Claims

1. A method for quantifying the relative distribution of glucuronization, iduronization, and galacturonization of a polypeptide population, a) The process of producing peptides by contacting a group of polypeptides with a protease; b) Contacting the peptide with a chromatography support containing a hydrophobic stationary phase and a positive surface charge; c) Separating the peptide by bringing the chromatography support into contact with a weak acid mobile phase gradient; d) Analyze the separated peptides to quantify the relative distribution of glucuronization, iduronization, and / or galacturonization of the peptides. Methods that include...

2. The method according to claim 1, wherein the protease is trypsin, lys-C, Glu-C, or Asp-N.

3. The method according to claim 1, wherein the chromatography support comprises a support material having a positively charged surface modifier.

4. The method according to claim 3, wherein the hydrophobic stationary phase is bonded to the surface modifier.

5. The method according to claim 4, wherein the hydrophobic stationary phase comprises an alkyl, alkenyl, alkynyl, or aryl functional group.

6. The method according to claim 5, wherein the hydrophobic stationary phase contains a C18 functional group.

7. The method according to claim 1, wherein the separated peptide is analyzed by mass spectrometry.

8. The method according to claim 1, comprising the identification of glucuron-oxidized peptides, iduron-oxidized peptides, and galacturon-oxidized peptides.

9. The method according to claim 7, wherein the peptides are identified by their respective chromatographic retention times and mass spectra.

10. The method according to claim 1, wherein the relative distribution of glucuronization, iduronization, and galacturonization is quantified by integrating the respective peaks of the liquid chromatograms corresponding to the separated peptides, which are monitored by UV absorbance.

11. The method according to claim 1, wherein the relative distributions of glucuronization, iduronization, and galacturonization are quantified by integrating the respective peaks of an ion chromatogram extracted from mass spectrometry.

12. The method according to claim 1, wherein the weak acid is formic acid or acetic acid.

13. The method according to claim 1, wherein the weak acid mobile phase is at a pH in which more than 50% of the carboxylic acid moiety related to glucuronization, iduronization, or galacturonization is in a deprotonated state.

14. The method according to claim 1, wherein the weak acid mobile phase gradient has pH > 3.

15. The method according to claim 1, wherein the weak acid mobile phase gradient has a pKa value of approximately 4.

5.

16. The method according to claim 1, wherein the mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS / MS).

17. The method according to claim 1, wherein the polypeptide is an antibody.

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