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

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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.

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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

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Acknowledgments

We would like to thank Drs. Alex Lazar and John Thomas for many helpful discussions.

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Authors and Affiliations

  1. ImmunoGen, Inc., Cambridge, Maryland, 02139, USA

    Lintao Wang, Godfrey Amphlett, John M. Lambert, Walter Blättler & Wei Zhang

Authors
  1. Lintao Wang
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  2. Godfrey Amphlett
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  3. John M. Lambert
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  4. Walter Blättler
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  5. Wei Zhang
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Correspondence to Wei Zhang.

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Wang, L., Amphlett, G., Lambert, J.M. et al. Structural Characterization of a Recombinant Monoclonal Antibody by Electrospray Time-of-Flight Mass Spectrometry. Pharm Res 22, 1338–1349 (2005). https://doi.org/10.1007/s11095-005-5267-7

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  • DOI: https://doi.org/10.1007/s11095-005-5267-7

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