Chinese hamster ovary (CHO) cells are used extensively in the biopharmaceutical industry to produce recombinant proteins that require post-translation modification for full biological functionality. Optimization of culture conditions for recombinant CHO cell lines presents challenges in light of the diverse nutritional requirements observed with different clonally derived cell lines. To address this challenge we have taken advantage of new technologies (high throughput screening and state-of-the-art cell line engineering), advances in medium development (a library of diverse CHO media formulations containing both animal-component free and chemically defined formulations), and the application of sophisticated Design of Experiment (DOE) experimental design and analysis techniques to develop a new strategic approach to medium development. This article describes this new strategic process and documents its use to develop several optimized formulations for a humanized IgG-producing CHO cell line.
Cell culture medium development is a complex process, involving the adjustment of numerous interacting components to their optimal concentrations with a goal of strong consistent support of cell growth and protein productivity. When optimizing medium for a specific CHO clone, the traditional approach of testing one medium component at a time to determine its optimal level is frequently used.1 While keeping all other components constant at their original levels, this method uses a large series of titrations of each individual medium component. At the end of the first round of titration, the working concentration exhibiting the highest activity is set as the optimal concentration. A second round of titration is performed for the next component, while keeping the concentration of the first component at its previously identified optimum. After selecting the working optimum concentration of the second medium component, subsequent rounds of titration are performed for the other medium components. This strategy is labor intensive, costly, time consuming, and fails to recognize synergistic interactions of components. To reduce costs and decrease media development time, a new streamlined strategic approach has been created to accentuate media development by applied DOE experimental techniques for screening cells for growth in a variety of media formulations (CHO Media Library), media mixing screening, and factorial designs combined with spent medium analysis to improve efficiency.
While the use of DOE techniques can improve the efficiency of media screening and subsequent optimization starting with any appropriate collection of media formulations, one of the cornerstones to this new approach is the availability of the proprietary CHO Media Library. The CHO Media Library was constructed from more than 30 different animal-component free (ACF) or chemically defined (CD) CHO formulations that were screened for cell growth and protein productivity using multiple CHO cell lines (Table 1). The top 18 ACF formulations (including eight CD formulations) have been ranked and put into the Library. To increase the high throughput capacity, a scaled down culture system based on 50-mL culture vessels with 25-mL working volumes was used to perform media screening. In developing a new medium formulation for a specific CHO cell line, the cell line is first screened with the formulations contained in the collection of media formulations selected for screening (e.g., CHO Media Library). The top three to five formulations are selected for further analysis using DOE mixing designs to identify the best formulation mixtures supporting this cell line, followed by spent medium analysis and DOE-based optimization of individual components using factorial designs. This strategy of combining medium mixing with individual component optimization allows multiple criteria to be analyzed simultaneously to identify synergistic responses and accelerate the desired user outcomes. Additionally, cell culture conditions can be optimized with the use of feeding strategies and modification of culture parameters (e.g., temperature shift) to best support recombinant protein production. The overall approach of optimization of medium and cell culture conditions can significantly improve pharmaceutical protein production in an efficient manner. Meanwhile, continued analysis of new media formulations and information from ongoing state-of-the-art studies in genomics/proteomics/metabolomics allows enhancement of the contents of the CHO Media Library (Figure 1).