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

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.

Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics

“…Site-directed mutagenesis and antibody transient expression were performed as previously described. 23 In brief, the template DNA of mAb12 plasmid was methylated prior to the mutagenesis reaction, in which two overlapping primers were used and one of them contained the target mutation. The mutagenesis products were transformed into E. coli competent cells, where unmethylated linear mutated DNA was circularized and replicated.…”