Benchmarking and optimization of a Next Generation Sequencing based
method for transgene Sequence Variant Analysis in Biotherapeutic Cell
In recent years Next-Generation Sequencing (NGS) based methods to detect
mutations in biotherapeutic transgene products have become a key quality
step deployed during the development of manufacturing cell line clones.
Previously we reported on a higher throughput, rapid mutation detection
method based on amplicon sequencing (targeting transgene RNA) and
detailed its implementation to facilitate cell line clone selection. By
gaining experience with our assay in a diverse set of cell line
development programs, we improved the computational analysis as well as
experimental protocols. Here we report on these improvements as well as
on a comprehensive benchmarking of our assay. We evaluated assay
performance by mixing amplicon samples of a verified mutated antibody
clone with a non-mutated antibody clone to generate spike-in mutations
from ~60% down to ~0.3% frequencies.
We subsequently tested the effect of 16 different sample and NGS library
preparation protocols on the assay’s ability to quantify mutations and
on the occurrence of false-positive background error mutations
(artifacts). Our evaluation confirmed assay robustness, established a
high confidence limit of detection of ~0.6%, and
identified protocols that reduce error levels thereby significantly
reducing a source of false positives that bottlenecked the
identification of low-level true mutations.