Early identification of unusually clustered mutations and root causes in therapeutic antibody development.
Biotechnol Bioeng
; 115(9): 2377-2382, 2018 09.
Article
em En
| MEDLINE
| ID: mdl-29777592
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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Vetores Genéticos
/
Fatores Imunológicos
/
Anticorpos
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Mutação
Tipo de estudo:
Diagnostic_studies
/
Etiology_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Biotechnol Bioeng
Ano de publicação:
2018
Tipo de documento:
Article