Analytical tools for improving cell line development

Cell line development (CLD) involves screening thousands of clones to identify the most stable and productive candidate for an upstream manufacturing process (Figure 1). Such an evaluation requires considerable time and resources for the preparation and execution of multiple cultures and analytical tests. In March 2022, Lukas Klein and Dirk Müller (Scientist and Head of Media and Process Development at Sartorius, respectively) hosted a BPI Ask the Expert webinar on the integration of their company’s Ambr 15 cell culture bioreactor and the Byte label-free biolayer interferometry (BLI) technology to accelerate CLD studies. Presenters highlighted applications for media selection and platform optimization.

Figure 1: Cell line development (CLD) includes several activities that require a lot of time, resources and analytical skills. The BPI Ask the Expert presentation summarized in this article focused on technologies for streamlining studies in stages two, three and four of the CLD process, as shown below.

Critical Abilities
The Ambr 15 Cell Culture Bioreactor includes a workstation that can be installed in a biosafety cabinet, with single-use reactors and process control software. Four culture stations run up to 48 cultures in parallel, each with a working volume of 10-15ml. A liquid handling robot automatically collects culture samples for on-line or off-line analysis of pH, metabolites, dissolved oxygen (DO), viable cell count (VCC), viable cell density ( VCD) and other important settings. The system provides standard controls for reactor temperature and stirring speed as well as advanced controls for adjusting the pH and DO levels of each reaction vessel.

Such controls, Klein explained, allow the microbioreactors to simulate the culture dynamics of benchtop systems, giving analysts insight into how clones might grow at laboratory scale. The system can perform batch, batch, and even perfusion mimic processes using adherent or suspension-adapted cells from mammalian and insect lines.

The Octet system performs real-time, label-free characterization of biomolecular interactions using BLI. The instrument’s sensors are coated with a ligand (eg, protein A) to immobilize a desired analyte (eg, a monoclonal antibody, MAb). This allows the system to measure its concentration and binding characteristics. The Octet system analyzes multiple samples per cycle and its sensors can be regenerated for additional applications.

Combined with the instrument’s “dip and read” method, these capabilities enable high-throughput evaluation of CLD preparations. This could reduce analytical costs and experimental effort, Klein explained. In terms of labor costs, high performance liquid chromatography (HPLC) and enzyme immunoassays (ELISA) are respectively three and 15 times more expensive to run than an Octet workflow.

Case studies
Media Screening: Müller explained that integrating the two technologies could streamline the measurement of MAb titles during CLD. Both presenters gave examples of such integration. In one case, Sartorius scientists evaluated the performance of two CHO-DG44 clones in five culture media, including vendor formulations and in-house developed variants. Since filtering media can be time-consuming and labor-intensive, Klein explained, the team needed a method that could reliably generate data quickly and at multiple times, but with minimal labor and hands-on time. The team used the Ambr 15 cell culture bioreactor to grow clones in candidate media, programming the instrument to collect and analyze samples daily throughout the culture process. These materials have been uploaded to the Octet platform for title analysis.

Using this information and results from other experiments, the team calculated the time-resolved cell-specific productivity of the clones (qP) in each medium. The first clone featured similar VCDs and qP values ​​in most media, although the option with the lowest viability was excluded from further evaluation. For the second clone, two media generated a high number of cells, while the other three candidates showed a decrease in viability around the eighth day of the culture process. Ultimately, the built-in workflow guided media selection by highlighting potential title and productivity limitations.

The integration of the Ambr 15 cell culture bioreactor and Octet technology has also greatly accelerated screening activities. Klein pointed out that operators typically need two hours to prepare 48 samples for HPLC analysis and three hours for 72 samples. Run times average 4.6 hours and 7 hours, respectively, and related buffer preparation takes three hours, regardless of the number of samples. In contrast, operators need 30 minutes and 40 minutes to prepare 48 and 72 samples for Octet system analysis. These values ​​represent time savings of 75% and 80%. The Octet system can process up to 72 samples in 20-30 minutes, a time saving of 92-94%. The platform also eliminates the need for buffer preparation and consumables are significantly reduced.

Process optimization: Optimizing a platform process for CLD involves extensive screening of multiple clones and conditions. Klein described how his team used the Ambr 15 cell culture bioreactor and Octet technology to streamline process optimization.

The team needed to establish a new process focused on high qP as the most important target value. Parameters that have a significant impact on qP scalability included stirring speed, ballast gassing rate, and starting volume. Thus, Klein and his colleagues focused on optimizing these conditions. Modde software was used to define a Design of Experiments (DoE) workspace for these parameters. The program identified 12 conditions for testing, and clones were cultured in duplicate accordingly in an Ambr 15 system, which automatically collected and analyzed culture samples. Next, the team uploaded these materials to the Octet platform for title determination. All data, including subsequent data qP calculations, were reintroduced in the Modde program to identify the optimal conditions.

The software showed that increases in starting volume tended to decrease qP. Meanwhile, increasing the stirring speed had positive effects on qP up to a certain value, after which productivity decreased. Multiparameter analyzes revealed that increasing ballast gassing rates improved qP values ​​when stirring speeds were kept low, but these gains decreased and then reversed as stirring speed increased. The team also observed that cell viability decreased slightly at high ballast agitation and gassing rates, but DO levels showed no corresponding decline. Thus, shear stress would be an important consideration for the clone in question, while oxygen limitation would not be an issue. Balancing all of these concerns, the team decided to perform low-volume cultures at moderately high shaking speeds and with minimal ballast gassing.

Significant gains: Müller reiterated that using the automation capability of the Ambr 15 cell culture bioreactor with the high-throughput analyzes of the Octet platform could significantly streamline the execution of CLD studies. Adding Modde software to a workflow could help further optimize microculture settings.

Questions and answers
Can the Ambr 15 Cell Culture bioreactor serve as a good small-scale model for process characterization? It can serve this purpose, although it is mainly used to filter and optimize process parameters. Sartorius recommends using the Ambr 250 Cell Culture System for process characterization because it features advanced process controls and operates to represent pilot-scale culture performance.

What are the benefits of the Octet system’s sensor regeneration capability? Sartorius leverages this feature to process 72 samples simultaneously. The regenerative capability also significantly reduces analysis and consumable costs.

find more online
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Brian Gazaille, PhD, is Associate Editor at BioProcess International, part of Informa Connect; [email protected]