Evaluation of “Difficult-to-Express” Monoclonal Antibodies in a CHO-Based Hybrid Site-Specific Integration System Under Industrially Relevant Conditions
Date
2025-08-17
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Biotechnology Journal
Abstract
Variation in the primary sequence of monoclonal antibodies (mAbs) can negatively affect their behavior in biopharmaceutical
manufacturing platforms, and efforts to identify mAbs with poor “developability” characteristics lack robust methods for assessing
mAb expression from an industrially relevant platform. Recent advancements in site-specific integration-based (SSI) platforms in
Chinese hamster ovary (CHO) cells can mitigate the high transcriptional variation observed with random integration and the
low industrial relevance of transient expression by providing a flexible platform for mAb expression from a consistent clonal
background. This work applies a novel SSI-based expression system capable of generating isogenic cell pools in less than 1 month
to systematically compare the expression of ten sequence variants of two therapeutically relevant mAbs from two genomic loci
under industrially relevant culture conditions. Eight single amino acid mutations in trastuzumab resulted in reduced productivity
compared to the wild-type mAb in batch cultures, and three mutations maintained a low-expressing phenotype in fed-batch
cultures. The mutations resulted in variant-specific patterns of decreased domain stability and increased ER stress. The application
of industrially relevant SSI systems in developability workflows could strengthen the understanding of the sequence determinants
of mAb expression to improve mAb design, candidate selection, and process development decisions.
Description
This article was originally published in Biotechnology Journal. The version of record is available at: https://doi.org/10.1002/biot.70102
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work
is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2025 The Author(s). Biotechnology Journal published by Wiley-VCH GmbH.
Keywords
difficult-to-express, recombinase-mediated cassette exchange, site-specific integration
Citation
Giancola S, Stevenson JF, Philibert I. Meta-Evaluative Practices of Clinical and Translational Research Evaluators. Journal of Clinical and Translational Science. Published online 2025:1-21 https://doi:10.1017/cts.2025.10121