Customized CHO Cell Line Development Solutions for Every Protein of Interest: A Case Study
Tailored cell line development solutions are essential to overcome challenges in the production of complex and difficult-to-express proteins. This blog post dives into a recent project in which our experts created a highly customized CHO Cell Line Development Service program, achieving scalable results.
Tackling Complexity with a Tailored Approach
Developing stable and scalable production cell lines for complex biologics presents unique challenges, particularly when timelines are tight and molecule behavior is unpredictable.
Recently a new client approached Sartorius with a request to generate, a new CHO cell line for a difficult-to-express therapeutic protein. With compressed timelines and a clear need for rapid progress, they were looking for a cell line development partner who could both anticipate technical risks and implement a strategy that would deliver a new production cell line ready to scale for clinical manufacturing.
The project launched in April 2024 at Sartorius’ Cell Line Development Center of Excellence in Ulm, Germany. Given the non-standard nature of the target molecule, we expected some challenges related to expression (titer) and product quality. To support candidate selection and to de-risk development early in the project, we implemented an optimized mini-pool approach which would also support extensive product quality attribute (PQA) assessments.
De-Risk Early With an Optimized Pool Generation
Our approach began with refining the pool generation strategy. We adjusted key parameters to boost success rates and uncover the most promising production candidates early on:
- Increased the number of mini-pools screened to broaden the diversity of expression profiles
- Adjusted the seeding density in a 96-well format to support robust outgrowth
- Performed extensive screening in small-scale fed-batch mode
- Advanced top mini-pools to single-cell cloning
These modifications allowed us to quickly deliver pool material early in the project timeline, giving our client a head start with formulation development and analytical testing. These insights reduced project risk and improved confidence in the emerging process.
Single-Cell Cloning
Following mini-pool generation, we carried out single-cell cloning using the high-throughput automation capabilities of our CellCelector™ platform.
The CellCelector™ allows us to combine and analyze thousands of cell growth parameters and expression data points with our proprietary multivariate data analysis (MVDA) tool. Combined with advanced machine learning algorithms, we were able to identify and individually select high-producing monoclonal cell lines during single-cell cloning, approximately 14 days after cell thaw of the selected top mini-pools.
High-producing clones were subsequently transferred to a 384-well plate, expanded, and further evaluated in a high-throughput, small-scale fed-batch run using a Tecan robot.
The top-performing clones identified in the small-scale fed-batch experiment were then further evaluated in fed-batch mode on the Ambr® 15 for cell growth parameters (including viability and viable cell density), titer, and product quality. The top 4 clones identified on the Ambr® 15 were further characterized for stability (over 70 population doublings) and manufacturability.
From Lab Scale to Manufacturing Readiness: Surpassing Titer Expectations
In parallel to the stability study, we sought to further boost the productivity of the leading clone candidates (Figure 1) by performing process optimization using the Ambr® 250. This approach proved highly effective: following process optimization, the lead clone delivered a titer exceeding 0.9 g/L, which was nearly double the client’s minimum success criterion of 0.5 g/L.
The next step after the process optimization was to verify the scalability or manufacturability of the leading clone candidates at benchtop scale. As such, a research cell bank (RCB) vial was thawed, and cells were seeded into a 5 L Univessel® bioreactor using the optimized parameters. After the 14-day fed-batch culture, cell growth parameters (including viability, viable cell density, and lactate levels), titer, and product quality were evaluated and confirmed to be properly maintained upon scale-up (Figure 1).
RCBs of the lead clones and the scale-up process protocol were then transferred to the client’s contract development and manufacturing organization (CDMO) partner. The CDMO partner was able to successfully complete a 50 L scale bioreactor run, confirming both scalability and process robustness while achieving a final titer of 1.5 g/L; the titer achieved at 50 L scale was >50% greater than the 0.9 g/L achieved on Ambr®250 during process optimization.
From DNA to Research Cell Bank in 28 Weeks
Using our integrated cell line development platform, Sartorius advanced the project from DNA synthesis to RCB in just 28 weeks.
This timeline included the following stages:
- Transfection and mini-pool generation, screening, and selection
- Single-cell cloning and clone evaluation on Ambr® 15
- RCB preparation and stability studies
- Process optimization on Ambr®250 with scale-up confirmation
- Delivery of early pool and clone materials for analytical development
- Process and RCB transfer to CDMO
Ongoing Success and Long-Term Partnership
With master cell bank preparation and characterization ongoing at our Newhouse site in the UK, the foundation for clinical manufacturing is now in place.
The success of this customized CHO cell line development project was the result of proactive planning, flexible execution, and close collaboration with the client. By combining our technical expertise and the use of advanced cell culture platforms like the Ambr® 15 and Ambr® 250 with our modular cell line development toolbox, Sartorius enabled the client to move quickly from early development phase to pilot-scale readiness.
Whether you’re facing tight timelines, working with complex modalities, or simply need a more scalable solution for your protein of interest, Sartorius can support your journey from DNA to RCB and beyond.
Learn more about our CHO Cell Line Development Services