Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain.
A unique, late-eluting "basic peak" (relative to the "main peak") was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a top-down characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversed-phase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or alpha-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and alpha-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal alpha-amidation in mAbs has not been reported previously.
Johnson
Keith A
KA
Wyeth BioPharma, Andover, MA 01810, USA. kxjohnson@wyeth.com
Paisley-Flango
Kari
K
Tangarone
Bruce S
BS
Porter
Thomas J
TJ
Rouse
Jason C
JC
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000577:Amides; D007074:Immunoglobulin G