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Early identification of unusually clustered mutations and root causes in therapeutic antibody development
Author(s) -
Qian Yueming,
Chen Zhiqiang,
Huang Xin,
Wang Xuning,
Xu Xuankuo,
Kirov Stefan,
Ludwig Richard,
Qian NanXin,
Ravi Kandasamy,
Tao Li,
Borys Michael C.,
Li Zheng Jian
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26728
Subject(s) - biology , dna sequencing , sequence (biology) , dna , plasmid , sequence analysis , microbiology and biotechnology , genetics , mutation , consensus sequence , computational biology , peptide sequence , nucleic acid sequence , gene , base sequence
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.