Characterization of serine-lysine cross-link in antibody high molecular weight species
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsYuetian Yan, Gaoyuan Liu, Shunhai Wang
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Abstract
The present invention generally pertains to methods of characterizing of a protein of interest. In particular, the present invention pertains to the use of post-column denaturation, size exclusion chromatography and mass spectrometry for detecting, identifying and characterizing crosslinking amino acid residues in a therapeutic antibody.
Claims33
A method for characterizing a covalent cross-link in a size variant of a protein of interest, comprising:
- (a) subjecting a sample including a protein of interest and a size variant thereof to nSEC to form an SEC eluate;
- (b) contacting said SEC eluate to a denaturing solution to form a denatured SEC eluate;
- (c) subjecting said denatured SEC eluate to mass spectrometry analysis to obtain at least one mass measurement of said protein of interest and at least one mass measurement of said size variant;
- and
- (d) comparing said mass measurements to characterize said covalent cross-link.
A method for identifying crosslinking amino acid residues in a protein of interest, comprising:
- (a) subjecting a sample including a protein of interest and a crosslinked variant thereof to chromatographic separation to form an enriched variant sample;
- (b) subjecting said enriched variant sample to digestion conditions to form a peptide digest;
- and
- (c) subjecting said peptide digest to LC-MS analysis to identify cross-linking amino acid residues.
A method for identifying a covalent cross-link in a HMW species of a therapeutic antibody, comprising:
- (a) subjecting a sample including a therapeutic antibody and at least one HMW species of said therapeutic antibody to SEC to form an enriched HMW sample;
- (b) subjecting said enriched HMW sample to complete or partial reduction and alkylation to form a reduced HMW sample;
- (c) subjecting said reduced HMW sample to dSEC to form a very enriched HMW sample;
- (d) subjecting said very enriched HMW sample to digestive conditions to form a peptide digest;
- (e) subjecting said peptide digest to stabilizing conditions to form a stabilized peptide digest;
- (f) subjecting said stabilized peptide digest to LC-MS/MS analysis to obtain peptide masses;
- and
- (g) comparing said peptide masses to predicted masses of crosslinked peptides to identify said covalent cross-link.
A method for selecting pH conditions for producing a therapeutic protein, comprising:
- (a) subjecting a sample including a therapeutic protein and at least one crosslinked variant thereof to LC-MS analysis to determine an abundance of said at least one crosslinked variant;
- (b) repeating step (a) using at least one alternative mobile phase buffered to at least one alternative pH;
- (c) comparing the abundances of step (a) and step (b) to determine pH conditions at which said at least one crosslinked variant is less abundant;
- and
- (d) using said determination to select pH conditions for producing said therapeutic protein.
A method for identifying a covalent cross-link in a crosslinked variant of a protein of interest, comprising:
- (a) contacting a sample including a crosslinked variant of a protein of interest to a first reducing agent to form a reduced sample;
- (b) subjecting said reduced sample to digestion conditions to form a peptide digest;
- (c) subjecting said peptide digest to LC-MS analysis to obtain a first set of peptide masses;
- (d) repeating steps (a)-(c) using a second reducing agent to obtain a second set of peptide masses, wherein said second reducing agent includes a heavy isotope that is incorporated into said covalent cross-link;
- and
- (e) comparing said first set of peptide masses and said second set of peptide masses to identify said covalent cross-link.
A method for detecting a covalently bound HMW species of a therapeutic antibody, comprising:
- (a) subjecting a sample including a therapeutic antibody and at least one covalently bound HMW species of said therapeutic antibody to SEC to form at least one fraction including said at least one HMW species;
- (b) collecting said at least one fraction to form an enriched HMW sample;
- (c) subjecting said enriched HMW sample to deglycosylating conditions to form a deglycosylated sample;
- and
- (d) subjecting said deglycosylated sample to post-column denaturation (PCD)-assisted nSEC-MS analysis to detect said covalently bound HMW species.
A method for characterizing a covalent cross-link in a HMW species of a therapeutic antibody, comprising:
- (a) subjecting a sample including a therapeutic antibody and at least one HMW species of said therapeutic antibody to SEC to form at least one fraction including said at least one HMW species;
- (b) collecting said at least one fraction to form an enriched HMW sample;
- (c) subjecting said enriched HMW sample to deglycosylating conditions to form a deglycosylated sample;
- (d) subjecting said deglycosylated sample to digestive conditions to form a fragmented sample;
- (e) subjecting said fragmented sample to complete or partial reduction and alkylation to form a reduced sample;
- (f) subjecting said reduced sample to PCD-assisted nSEC-MS analysis to obtain at least one mass measurement of at least one subunit dimer of said HMW species and at least one mass measurement of each of the component subunits of said at least one subunit dimer;
- and
- (g) comparing said mass measurements to characterize said covalent cross-link.
Dependencies are inferred from the claim text.