METHODS FOR DETERMINING THE RELATIVE DISTRIBUTION OF GLUCURONIDATION, IDURONIDATION, AND GALACTURONIDATION OF POLYPEPTIDES

Abstract

The present disclosure relates to methods and compositions for determining the relative distribution of glucuronidation, iduronidation, and/or galacturonidation of polypeptides, including stereoselective liquid chromatography-mass spectrometry (LC-MS) methods capable of achieving the simultaneous separation and relative quantitation of glucuronidation, iduronidation, and/or galacturonidation of polypeptides.

Claims

1. A method for determining the relative distribution of glucuronidation, iduronidation, and galacturonidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of glucuronidation, iduronidation, and/or galacturonidation of the peptides.

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

3. The method of claim 1, wherein the chromatographic support comprises a support material with a positively charged surface modifier.

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

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

6. The method of claim 5, wherein the hydrophobic stationary phase comprises a C18 functional group.

7. The method of claim 1, wherein the chromatographic support a CSH™ C18 stationary phase.

8. The method of claim 1, wherein the separated peptides are analyzed by using mass spectrometry.

9. The method of claim 1, comprising the identification of glucuronidated, iduronidated, and galacturonidated peptides.

10. The method of claim 8, wherein the peptides are identified by their respective chromatographic retention time and mass spectra.

11. The method of claim 1, wherein the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance.

12. The method of claim 1, wherein the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis.

13. The method of claim 1, wherein the weak acid is formic acid or acetic acid.

14. The method of claim 1, wherein the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, or galacturonidation are in a deprotonated state.

15. The method of claim 1, wherein the weak acid mobile phase gradient has pH>3.

16. The method of claim 1, wherein the weak acid mobile phase gradient has a pKa value of about 4.5.

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

18. The method of claim 1, wherein the polypeptide is an antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts the structures of the naturally occurring glycuronic acids.

[0019] FIG. 2 depicts extracted ion chromatograms showing separation of glucuronidated (glua) and iduronidated (idoa) peptides. Antibody A was modified through forced glucuronidation/iduronidation. Samples were then digested by trypsin and analyzed by LC-MS/MS using a CSH C18 column. The IdoA and GluA peptides were eluted at 15.07 and 15.38 minute, respectively.

[0020] FIG. 3 depicts extracted ion chromatograms of the galacturonidated (glaa) peptide. Antibody A was modified through forced galacturonidation. Samples were then digested by trypsin and analyzed by LC-MS/MS using a CSH™ C18 column. The GlaA peptide was eluted at 15.16 minute.

[0021] FIG. 4 depicts the lack of stereoselective separation of candidate peptides on a BEH™ C18 column.

DETAILED DESCRIPTION

[0022] The following Detailed Description, which is given by way of example, is not intended to limit the presently disclosed subject matter to specific embodiments described and may be understood in conjunction with the accompanying drawings.

[0023] The present description relates to methods and compositions for determining the relative distribution of glucuronidation, iduronidation, and/or galacturonidation of polypeptides. Specifically, the present description relates to stereoselective LC-MS methods that achieve simultaneous separation and relative quantitation of glucuronidation, iduronidation, and/or galacturonidation of polypeptides.

1. Definitions

[0024] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0025] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, 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 references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0026] As used herein, the term “about” or “approximately” means within an acceptable error range for the 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 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0027] As used herein, “polypeptide” refers generally to peptides and proteins having more than about ten amino acids. The polypeptides can be homologous to the host cell, or preferably, can be exogenous, meaning that they are heterologous, i.e., foreign, to the host cell being utilized, such as a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell. In certain embodiments, mammalian polypeptides (polypeptides that were originally derived from a mammalian organism) are used, more preferably those which are directly secreted into the medium.

[0028] The term “protein” is meant to refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” or other small molecular weight drugs that do not have such structure. Typically, the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassed within the definition herein include all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds.

[0029] The term “antibody” is used herein in the broadest sense and encompasses various 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 multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

2. Methods for Determining the Relative Distribution of Glucuronidation, Iduronidation, and Galacturonidation of a Polypeptide

[0030] In certain embodiments, the methods for relative distribution and/or identification of glucuronidation, iduronidation, and/or galacturonidation of polypeptides described herein comprise: a) contacting a polypeptide with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; and d) analyzing the separated peptides to determine the relative distribution of glucuronidation, iduronidation, and/or galacturonidation of the peptides.

[0031] The methods outlined herein provide for the direct determination of the relative distribution of glucuronidation, iduronidation, and/or galacturonidation of polypeptides. These methods contrast with other approaches for investigating the glucuronidation, iduronidation, and/or galacturonidation of polypeptides. For example, approaches that measure the level of glucuronic acid in cell culture fluids do not necessarily correlate with the level of glucuronidation of polypeptides produced by such cultures, let alone provide any information concerning the distribution or identity of the modified amino acids of such polypeptides. The presently described approach also differs from methods requiring the release of glycuronic acids from peptides in the detection process, e.g., Lin et al., Eur. J Biochem, 106:341-351 (1980) and Spiro, JBC, 252(15): 5424-5430 (1977), as such approaches do not allow for the direct identification of the distribution or location of modified amino acids.

[0032] In certain embodiments, the protease is an aspartic, cysteine, metallo-, serine or threonine protease. Exemplary proteases useful in connection with the instant disclosure are known in the art, e.g., Verhamme et al. Proteases: Pivot Points in Functional Proteomics, Methods Mol Biol. 2019; 1871: 313-392, which is incorporated by reference in its entirety. 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.

[0033] In certain embodiments, the chromatographic support comprises a support material with a positively charged surface modifier. In certain embodiments, the surface modifier can be attached to a chromatographic support base material via derivatization, coating, and/or crosslinking, thereby imparting the chemical character of the surface modifier to the base material of the chromatographic support. Exemplary surface modifiers are identified in U.S. Pat. No. 7,223,473, which is hereby incorporated by reference in it is entirety. In certain embodiments, the surface modifier may be an organotrihalosilane, such as octyltrichlorosilane or octadecyltrichlorosilane. In certain embodiments, the chromatographic support comprises a hydrophobic stationary phase. In certain embodiments the hydrophobic stationary phase is linked, covalently or non-covalently, to the surface modifier. For example, but not by way of limitation, the surface modifier can comprise one or more alkyl, alkenyl, alkynyl, aryl functional groups as the hydrophobic stationary phase. In certain embodiments, the functional groups include, but are not limited to C1-C30 alkyl, such as octyl (C8), octadecyl (C18), and triacontyl (C 30); alkaryl, e.g., C1-C4-phenyl groups. In certain embodiments, the chromatographic support is a Charged Surface Hybrid (CSH™) stationary phase. In certain embodiments, the chromatographic support is CSH™ C18 stationary phase.

[0034] In certain embodiments, the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, and/or galacturonidation are in a deprotonated state. In certain embodiments, the weak acid is formic acid or acetic acid. In certain embodiments, the weak acid mobile phase gradient has pH>3. In certain embodiments, the weak acid mobile phase gradient has a pKa value of about 4.5.

[0035] In certain embodiments, the separated peptides are analyzed by using mass spectrometry. In certain embodiments, the mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS/MS).

[0036] In certain embodiments, the methods of presently disclosed subject matter further comprise the identification of glucuronidated, iduronidated, and/or galacturonidated peptides. In certain embodiments, the peptides are identified by their respective chromatographic retention time and/or mass spectra. In certain embodiments, the relative distribution of glucuronidation, iduronidation, and/or galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments, the relative distribution of glucuronidation, iduronidation, and/or galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis.

[0037] 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, e.g., a bispecific or a trispecific antibody.

3. Exemplary Embodiments

[0038] A-1. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of glucuronidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of glucuronidation of the peptides.

[0039] A-2. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of iduronidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of iduronidation of the peptides.

[0040] A-3. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of galacturonidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of galacturonidation of the peptides.

[0041] A-4. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of glucuronidation and iduronidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides; b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of glucuronidation and iduronidation of the peptides.

[0042] A-5. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of glucuronidation and galacturonidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides: b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge: c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of glucuronidation and galacturonidation of the peptides.

[0043] A-6. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of iduronidation, and galacturonidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides: b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge: c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of iduronidation, and galacturonidation of the peptides.

[0044] A-7. In certain embodiments the instant disclosure is directed to methods for determining the relative distribution of glucuronidation, iduronidation, and galacturonidation of a population of polypeptides, the method comprising: a) contacting the population of polypeptides with a protease to generate peptides: b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge; c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides; d) analyzing the separated peptides to determine the relative distribution of glucuronidation, iduronidation, and galacturonidation of the peptides.

[0045] B-1. In certain of embodiments A-1 to A-7, the protease is selected from the group consisting of trypsin, lys-C, Glu-C, and Asp-N. In certain of embodiments A-1 to A-7, the protease is trypsin. In certain of embodiments A-1 to A-7, the protease is lys-C. In certain of embodiments A-1 to A-7, the protease is Glu-C. In certain of embodiments A-1 to A-7, the protease is Asp-N.

[0046] C-1. In certain of embodiments A-1 to A-7, the chromatographic support comprises a support material with a positively charged surface modifier.

[0047] C-2. In certain of embodiments A-1 to A-7, B-1, and C-1, the hydrophobic stationary phase is linked to the surface modifier.

[0048] C-3. In certain embodiments of C-2, the hydrophobic stationary phase comprises an alkyl, alkenyl, alkynyl or aryl functional group.

[0049] C-4. In certain embodiments of C-3, the hydrophobic stationary phase comprises a C18 functional group.

[0050] C-5. In certain embodiments of C-3, the chromatographic support comprises a CSH™ C18 stationary phase.

[0051] D-1. In certain embodiments of A-1 to A-7, B-1, and C-1 to C-5, the separated peptides are analyzed by mass spectrometry. In certain embodiments of D-1, the mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS/MS). In certain embodiments of D-1, the relative distribution of glucuronidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of iduronidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of glucuronidation and iduronidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of glucuronidation and galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of iduronidation and galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis. In certain embodiments of D-1, the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis.

[0052] E-1. In certain embodiments of A-1 to A-7, B-1, and C-1 to C-5, the separated peptides are analyzed by respective chromatographic retention time. In certain embodiments of E-1, the separated peptides are analyzed by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of glucuronidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of iduronidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of glucuronidation and iduronidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of iduronidation and galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of glucuronidation and galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance. In certain embodiments of E-1, the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides as monitored by UV absorbance.

[0053] F-1. In certain embodiments of A-1 to A-7, B-1, C-1 to C-5, D-1, and E-1, The method comprises the identification of glucuronidated, iduronidated, or galacturonidated peptides. In certain embodiments of F-1, the method comprises the identification of glucuronidated peptides. In certain embodiments of F-1, the method comprises the identification of iduronidated peptides. In certain embodiments of F-1, the method comprises the identification of galacturonidated peptides. In certain embodiments of F-1, the method comprises the identification of glucuronidated and iduronidated peptides. In certain embodiments of F-1, the method comprises the identification of iduronidated and galacturonidated peptides. In certain embodiments of F-1, the method comprises the identification of glucuronidated and galacturonidated peptides. In certain embodiments of F-1, the method comprises the identification of glucuronidated, iduronidated, and galacturonidated peptides.

[0054] F-2. In certain embodiments of F-1, the peptides are identified by their respective chromatographic retention time. In certain embodiments of F-1, the peptides are identified by their respective mass spectra. In certain embodiments of F-1, the peptides are identified by their respective chromatographic retention time and mass spectra.

[0055] G-1. In certain embodiments of 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.

[0056] G-2. In certain embodiments of 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 where greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, or galacturonidation are in a deprotonated state. In certain embodiments, the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation are in a deprotonated state. the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with iduronidation are in a deprotonated state. the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with galacturonidation are in a deprotonated state the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation and iduronidation are in a deprotonated state. the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation and galacturonidation are in a deprotonated state. the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, and galacturonidation are in a deprotonated state. In certain embodiments of G-2, the weak acid mobile phase gradient has pH>3. In certain embodiments of G-2, the weak acid mobile phase gradient has a pKa value of about 4.5.

[0057] H-1. In certain embodiments of 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

Example 1: Assay for Determining the Relative Distribution of Glucuronidation, Iduronidation, and Galacturonidation of Polypeptides

[0058] Antibody A polypeptides were digested using a protease (such as trypsin, lys-C, Glu-C, or Asp-N). The resulted peptides were separated by using a CSH™ C18 column. The stereoselective separation was based on a charge-charge interaction between the negative charge from glycuronic acid and the positive surface charge from the stationary phases and a hydrophobic interaction between the glycuronidated peptides and the hydrophobic stationary phase. A mobile phase gradient using weak acids (such as formic or acetic acid, but not trifluoroacetic acid) in the mobile phases was employed to achieve separation of glucuronidated, iduronidated, and galacturonidated peptides. The weak acids were selected, since, at pH>3, majority of the carboxylic groups (pKa=˜4.5) from the glycuronic acid moiety is negatively charged, which promotes a stereoselective charge-charge interaction between the glycuronic acid moiety and the positively charged stationary phase.

[0059] Antibody A polypeptides were digested using a protease (such as trypsin, lys-C, Glu-C, or Asp-N). The resulted peptides were analyzed by using a BEH™ C18 column, whose stationary phase is not positively charged. A mobile phase gradient using trifluoroacetic acid in the mobile phases (pH<3) was employed, where trifluoroacetic acid is a commonly used ion pair reagent to improve peak shape for improved chromatographic separation. As shown in FIG. 4, the stereoselective separation cannot be achieved on the BEH™ C18 column.

[0060] The eluted peptides were subsequently analyzed by ESI-MS/MS. The identity of glucuronidated, iduronidated, and galacturonidated peptides was confirmed by their respective retention time from the chromatographic separation (FIGS. 2 and 3) and by their mass spectra collected from the ESI-MS/MS analysis. The relative distribution of glucuronidation, iduronidation, and galacturonidation was determined by integrating the respective peaks on the liquid chromatograms (monitored by UV absorbance) or on the extracted ion chromatograms (based on the molecular mass, FIGS. 2 and 3).