Pharmaceutical Research

, Volume 22, Issue 8, pp 1338–1349

Structural Characterization of a Recombinant Monoclonal Antibody by Electrospray Time-of-Flight Mass Spectrometry

  • Lintao Wang
  • Godfrey Amphlett
  • John M. Lambert
  • Walter Blättler
  • Wei Zhang
Research Paper

DOI: 10.1007/s11095-005-5267-7

Cite this article as:
Wang, L., Amphlett, G., Lambert, J.M. et al. Pharm Res (2005) 22: 1338. doi:10.1007/s11095-005-5267-7

Purpose

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.

Key Words

electrospray time-of-flight mass spectrometry monoclonal antibody protein characterization structural post-translational modifications 

Abbreviations

Asn

asparagine

Asp

aspartate

CDR

complementarity determining region

CHO cells

Chinese hamster ovary cells

ESI-TOFMS

electrospray ionization time-of-flight mass spectrometry

GudHCl

guanidine hydrochloride

HPLC

high-performance liquid chromatography

isoAsp

isoaspartate

LC–MS

liquid chromatography–mass spectrometry

MAb

monoclonal antibody

MW

molecular weight

SEC

size-exclusion chromatography

TFA

trifluoroacetic acid

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Lintao Wang
    • 1
  • Godfrey Amphlett
    • 1
  • John M. Lambert
    • 1
  • Walter Blättler
    • 1
  • Wei Zhang
    • 1
  1. 1.ImmunoGen, Inc.CambridgeUSA

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