A sensitive mutation screening method supporting cell line development for biotherapeutics.
Random genetic mutations, which can occur during cell line development, can lead to sequence variants that comprise pharmaceutical product quality generated by recombinant technology. Mutation screening can minimize the probability of selecting clones harboring sequence variants. Here we report a polymerase chain reaction (PCR)-based mutation screening approach using high-resolution melting (HRM) analysis combined with a mutation enrichment step using limiting dilution to detect low-level mutations at 0.5%. The method allows unknown mutation discovery regardless of its location in a transgene as well as independent of its position in an HRM fragment, ranging from approximately 200 to 300 bp in size.
Protein Analytical Chemistry Department, Genentech, South San Francisco, CA 94080, USA.
Ildana
I
Valisheva
Pharmaceutical Technology Development, Genentech, South San Francisco, CA 94080, USA.
Reed J
RJ
Harris
Protein Analytical Chemistry Department, Genentech, South San Francisco, CA 94080, USA. Electronic address: judithz@gene.com.
Judith
J
Zhu-Shimoni
D001691:Biological Therapy; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
Enrichment; High-resolution melt (HRM); Limiting dilution; Mutation; Nucleic acid; Sequence variant (SV)