A fully automated plate-based optimization of fed‑batch culture conditions for monoclonal antibody‑producing CHO cell line

Immunoglobulin-based (IgG) therapies such as monoclonal antibodies (mAb) are increasingly used in the treatment of multiple human diseases. Process development for mAb entails laborious workflows that optimize medium and determine key cell culture conditions needed for producing high-antibody titers. Fed‑batch processes are applied in cell culture production to supply critical nutrients at physiological conditions. A combination of high‑performing basal medium with feed solution and the optimal feed regimen are often combined for a given cell line to boost high-antibody titers. Given the unique combinations needed to establish culture conditions, design of experiments (DoE) can be used to statistically determine the optimal fed-batch combinations based on randomization.

An automated and miniaturized 96-well plate-based system for suspension cell culture was designed to match the high demand for high-throughput applications in biopharmaceutical process development. Unique combinations of batch feeds were generated using full factorial (2ⁿ) DoE and evaluated across various basal cell culture media for IgG1-producing CHO cell line. Next, an automated workflow was designed for media optimization experiments that reduces hands-on time to minimize unintended human errors yet increase walk-away time while decreasing the total time needed for product development.

We thus demonstrate a broadly applicable and efficient automated workflow for establishing optimal feed combination for best performing fed-batch processes using a 96-deepwell plate. The workflow contains the use of an integrated liquid handler - Biomek i7 hybrid workstation – automated cell counter – Vi-CELL BLU cell viability analyzer – a SpectraMax i3 plate reader and a Kuhner shaker incubator. These systems allow for automation of an entire media optimization workflow, including transfer of cells and cell culture medium, fed-batch screening, and feed combinations, automated viable cell quantification, and quantification of IgG production from the viable cells using the ValitaTiter system. Moreover, automated data tracing is accomplished using the Biomek data acquisition and reporting tool (DART), which gathers data and synthesizes runtime information from Biomek log files, capturing each manipulation of the sample across integrated systems during the Biomek method run.

The IgG1-producing CHO cell line was cultured in Forti-CHO medium, and individual HyClone Cell Boost™ feed supplements were evaluated in spiked batch and fed-batch experiments. Basal medium was spiked with feed combinations using cell boost HyClone reagents starting on day 0 of batch cultures, followed by frequent analysis for cell viability and IgG quantity for 240 hours. Candidate cell boost supplements were thereafter applied in fed-batch cultures establishing their relative ratios. The selected fed-batch culture conditions supported mAb titers ranging from 1- 4 µg/µL, which can be scaled up in large culture conditions. This workflow provides opportunity for high-throughput analysis of DoE-based screening of fed-batch conditions for process development of monoclonal antibody production in a hands-free environment.