Direct identification and quantification of aspartyl succinimide in an IgG2 mAb by RapiGest assisted digestion.
A special tryptic digestion method has been developed to facilitate rapid identification and accurate quantification of site-specific aspartyl succinimide (Asu) formation in complex protein molecules, such as monoclonal antibodies (mAbs). This method replaces chaotropic reagents, such as urea and guanidine hydrochloride (GdnHCl) with an acid labile surfactant RapiGest (RG), eliminates alkylation and desalting steps, and accomplishes the reduced tryptic digestion of an IgG2 mAb in a mildly acidic condition (pH 6.0) with half the time required by conventional methods. The new digestion condition preserves the labile Asu during sample preparation and solves the problem that conventional method has been facing in detecting and quantifying Asu in complex proteins. The validity of this method was confirmed by subjecting a mixture of peptides containing a predetermined amount of Asu to the same digestion conditions. An excellent correlation was also observed for the Asu results from cation-exchange chromatography (CEX) and tryptic peptide maps generated with the new digestion method. This method is also applicable to other enzymatic digestions and used to monitor site-specific deamidation, isomerization, and other chemical modifications in complex proteins by LC/MS.
Huang
Holly Z
HZ
Analytical and Formulation Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA. hhuang@amgen.com
Nichols
Andrew
A
Liu
Dingjiang
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid / Q000032:analysis* / Q000378:metabolism*; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D012680:Sensitivity and Specificity; D013388:Succinimides / Q000032:analysis* / Q000378:metabolism*; D013501:Surface-Active Agents / Q000737:chemistry / Q000494:pharmacology*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010455:Peptides; D013388:Succinimides; D013501:Surface-Active Agents; D001224:Aspartic Acid; D014357:Trypsin