Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry.
OBJECTIVE
The aim of this study was to perform structural characterization of a recombinant monoclonal antibody (MAb), huN901, by electrospray time-of-flight mass spectrometry (ESI-TOFMS) using both "top-down" and "bottom-up" approaches.


METHODS
In the top-down approach, the molecular masses of the deglycosylated huN901 and the light and heavy chains of the antibody were measured by direct infusion MS and liquid chromatography-mass spectrometry (LC-MS). In the bottom-up approach, trypsin and Asp-N protease were used to digest the separated, reduced and alkylated light and heavy chains followed by LC-MS analysis of the digests.


RESULTS
The primary structure and post-translational modifications of huN901 were characterized by both top-down and bottom-up MS approaches. Modifications of N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glycosylation, and deamidation were identified in the antibody heavy chain by both protein mass measurement and peptide mapping. No modifications were found in the complementarity determining regions (CDRs) of both chains. Both trypsin and Asp-N protease digestion had an average sequence recovery of 97%, and generated complimentary mapping results with complete sequence recovery.


CONCLUSIONS
ESI-TOFMS is a superior tool to characterize MAb and other complex protein pharmaceuticals.
Wang
Lintao
L
ImmunoGen, Inc., Cambridge, Maryland 02139, USA.
Amphlett
Godfrey
G
Lambert
John M
JM
Blättler
Walter
W
Zhang
Wei
W
D000478:Alkylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D019002:CD56 Antigen / Q000737:chemistry / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D019002:CD56 Antigen; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; D014357:Trypsin; D016282:Aspartic Acid Endopeptidases