Building a Stronger Foundation for Protein Therapy Development: A New Genetically Engineered CHO Host Cell Line
Success in biopharmaceutical manufacturing is strongly influenced by early development decisions, including the choice of host cell line. While advances in cell line development have boosted product yield and quality, therapeutic pipelines are expanding to include increasingly complex molecules — requiring further improvements.
Our cell line development team leveraged their extensive experience in developing commercial cell lines and their knowledge in protein expression and cellular machinery to genetically engineer a next-generation CHO host cell line, providing a stronger starting point for cell line development projects.
How We Engineered a New CHO Host Cell Line
Proteomic profiling
We hypothesized that by eliminating genes expressing energy-intensive host cell proteins, our CHO cell line would have more resources available for expressing the desired therapeutic protein. Profiling a representative panel of producer clones identified 67 highly expressed, energy-intensive proteins for further investigation.
Knockout study
CRISPR-mediated gene editing was used to individually knock out each of the 67 targets. Stable knockout pools were generated, transfected with a model monoclonal antibody (mAb) expression construct, and evaluated in fed-batch runs. This approach enabled systematic assessment of each gene’s impact on titer and productivity.
The outcomes are listed below:
- 35 targets showed no impact on titer
- 23 targets had a negative impact on titer or a lethal phenotype
- 8 targets were excluded during evaluation
- 1 target showed significantly increased protein expression and higher productivity
Generating a new host cell line
The top target was knocked out in our CHO host and single-cell cloning was performed using the Cellcelector™ to ensure monoclonality of the isolated cells. Successful gene knockout in the clonal cell lines was confirmed by genotypic analysis.
Thirty-five clones were expanded, transfected with a mAb expression vector, and evaluated for their performance in fed-batch runs. Up to 2-fold titer increases were observed in the top four clones.
The highest-performing clone was selected as the lead engineered host cell line, and its performance comprehensively characterized.
Performance of the New Engineered CHO Host Cell Line
Multiple cell line development campaigns were performed using our new CHO host cell line to evaluate its performance across different protein types.
Higher productivity
The genetically engineered CHO host cell line achieved up to 80% higher titers than the wild type host (Figure 1) while cell-specific productivities increased by up to 280%.
Productivity gains were achieved without compromising critical quality attributes. Analytical characterization revealed comparable N-glycan and charge variant profiles across mAbs and Fc-fusion proteins.
Stability
Long-term cultures showed high stability, with > 90% of clones tested maintaining fed-batch titers after 8 weeks, representing a >10% improvement compared to clones derived from the wild type host. Recombinant gene copy numbers were at low levels (7 – 29 copies) and remained unchanged over the entire cultivation period, demonstrating genetic stability.
Scalability
mAb- and Fc-fusion-expressing clones were scaled into the Ambr® 250 and 5 L stirred-tank bioreactors to evaluate their scalability. Comparable growth and titer profiles were observed for each molecule across scales, supporting seamless tech transfer.
Process Intensification
The engineered CHO host cell line also supports higher titers and long culture duration in continuous perfusion. In a 28-day process run on the Ambr® 250 High Throughput, the target viable cell density of 60 × 106 cells/mL was achieved, with perfusion maintained using 4Cell® SmartCHO media at 1.5 vessel volumes per day. These results show that the new cell line is ideally suited to intensified processing, providing a cost-effective platform for therapeutic protein production.
Biosimilar development
The engineered CHO host cell line was also tested in biosimilar production under both fed-batch and perfusion modes.
- In fed-batch mode, titers increased from 4 g/L in the wild type to 8 g/L in the engineered CHO host cell line.
- In perfusion mode, productivity reached 2 g/L/day.
The data demonstrate that the host’s productivity gains make it ideal for supporting more efficient biosimilar processes, with higher yields per batch.
Summary
Our rationally designed, genetically engineered CHO host cell line provides a robust foundation for the production of protein therapeutics. By strategically removing energy-intensive host cell proteins, we achieved higher titers without compromising stability, scalability, or critical quality attributes, helping you accelerate your journey from DNA to IND.
Learn more about our CHO Cell Line Development Service or talk to an expert about your project.
