[1S06m] 15. Advanced Cell and Cell Culture Engineering for Biopharmaceuticals and Functional Food - Part 1

10:50 AM - 11:10 AM JST(1:50 AM - 2:10 AM UTC)

[1ACCE-03] Development of a co-culture system of multiple recombinant CHO cell lines for oligoclonal antibodies production

○JIE REN¹, Noriko Yamano- Adachi¹, Takeshi Omasa¹ (1. Department of Biotechnology, Graduate School of Engineering, the University of OSAKA (Japan))

Keywords:

site-specific integration,cell line development,Oligoclonal antibodies

Oligoclonal antibodies are defined as mixtures of a few monoclonal antibodies that target multiple epitopes or antigens to enhance therapeutic efficacy against complex diseases, such as cancer and viral infections, which involve multiple targets or escape pathways. The first FDA approval of co-formulated monoclonal antibodies in 2020 highlighted the clinical potential of such approaches. However, reproducible and scalable manufacturing remains a major challenge. Among different methods, single-batch manufacturing through co-culturing multiple CHO cell lines in a single bioreactor offers cost-effectiveness and scalability, but requires strict control of cell ratios and antibody composition to ensure product consistency.In this study, we investigated to develop a stable co-culture platform for oligoclonal antibody production. CHO cell lines expressing several fluorescent proteins were constructed to evaluate the cell ratio during co-culture. Meanwhile, recombinase-mediated cassette exchange (RMCE) was employed to achieve targeted integration of transgenes into a predefined chromosomal locus, enabling more predictable gene expression and minimizing the variation caused by random integration events. Cre/loxP landing pad flanking GFP was integrated into the LOC113836906 locus of CHO-K1 cells. After passaging to PDL60, cell growth, mRNA levels, and gene methylation status were evaluated, leading to the selection of the optimal clone G1. To further validate the RMCE system, a new plasmid carrying a Cre/loxP landing pad flanking mCherry was constructed to replace GFP at the same locus, confirming efficient site-specific replacement. The integration of two antibodies into selected genomic loci was successfully achieved and confirmed by DNA sequencing. Multiple clones were evaluated in fed-batch culture, and those with similar expression and production profiles were selected for coculture experiments. A novel ELISA was developed to independently quantify each antibody, enabling monitoring of their ratio in the supernatant. High-density seeding parameters were determined based on coculture of two fluorescent protein-expressing cell lines to ensure similar growth during protein production. Coculture of the two antibody-expressing cell lines resulted in an almost 1:1 titer ratio at harvest, demonstrating the feasibility of balanced multi-antibody production. Further optimization and stability assessment are being conducted to support robust single-batch manufacturing.