Early identification of unusually clustered mutations and root causes in therapeutic antibody development

Biotechnology and Bioengineering
Yueming QianZheng Jian Li

Abstract

This study reports findings of an unusual cluster of mutations spanning 22 bp (base pairs) in a monoclonal antibody expression vector. It was identified by two orthogonal methods: mass spectrometry on expressed protein and next-generation sequencing (NGS) on the plasmid DNA. While the initial NGS analysis confirmed the designed sequence modification, intact mass analysis detected an additional mass of the antibody molecule expressed in CHO cells. The extra mass was eventually found to be associated with unmatched nucleotides in a distal region by checking full-length sequence alignment plots. Interestingly, the complementary sequence of the mutated sequence was a reverse sequence of the original sequence and flanked by two 10-bp reverse-complementary sequences, leading to an undesirable DNA recombination. The finding highlights the necessity of rigorous examination of expression vector design and early monitoring of molecule integrity at both DNA and protein levels to prevent clones from having sequence variants during cell line development.

References

Jan 7, 1998·Analytical Biochemistry·M M Ling, B H Robinson
May 1, 2007·Biomolecular Engineering·David Filpula
May 20, 2009·Bioinformatics·Heng Li, Richard Durbin
May 23, 2009·British Journal of Pharmacology·Patrick ChamesDaniel Baty
Feb 1, 2011·Bioinformatics·Robert Schmieder, Robert Edwards
Jun 12, 2012·MAbs·Frédéric Ducancel, Bruno H Muller
Oct 31, 2012·Biotechnology and Bioengineering·Lauren FeeneyMichael W Laird
Nov 6, 2012·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Jinmei FuReb J Russell
Jul 10, 2014·Current Opinion in Biotechnology·Michael Butler, Maureen Spearman
Oct 10, 2014·Nature Biotechnology·Gary Walsh
Aug 8, 2015·MAbs·Yoonjoo ChoiChris Bailey-Kellogg
Jun 1, 2016·Nature Methods·Vivien Marx
Jun 21, 2016·Current Opinion in Structural Biology·Karl E Griswold, Chris Bailey-Kellogg

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Citations

Dec 19, 2020·Computational and Structural Biotechnology Journal·Yuanli SongZheng Jian Li

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