Systematic Approaches to Develop Chemically Defined Cell Culture Feed Media - It is important to ensure that the transition from peptone-containing to CD media doesn't affect product quality. - BioPha

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Systematic Approaches to Develop Chemically Defined Cell Culture Feed Media
It is important to ensure that the transition from peptone-containing to CD media doesn't affect product quality.


BioPharm International
Volume 23, Issue 11

A SYSTEMATIC APPROACH TO MEDIUM DEVELOPMENT

Establishing Criteria for Platform Medium and Model Cell Lines


Figure 2. Considerations for developing a robust platform chemically defined medium
Several criteria need to be considered before developing a CD platform medium. Robustness of the medium must be demonstrated for cell line development applications, clone selection, seed and inoculum trains, and in production cultures using representative cell lines. These model cell lines should be identified to include diversity in properties such as host cell, expression plasmid, selection and amplification methods, growth, metabolism, and specific productivity (Qp). In addition, manufacturability and scalability criteria must be met, including consistent preparation, filtration methods, and stable shelf life. Moreover, titers should be comparable to that achieved in the peptone-containing platform medium, with acceptable product quality profiles (Figure 2).

Platform Medium Development


Figure 3. A systematic medium development strategy
Because of the complexity of cell culture media formulations, it was not practical to use Design of Experiment (DOE) approaches to simultaneously optimize all compositions. Instead, we applied a step-by-step optimization strategy by starting with media screening, followed by subgroup titration, single and multiple component titration, and finally conducting confirmation studies (Figure 3).

Starting point: An in-house chemically-defined feed (CDF), previously used in combination with peptone, was chosen as the starting point after wide-ranging screening experiments that compared this with other in-house and commercially available feed media. This medium had a simple proprietary formulation, which made optimization less complex. The overall strategy involved enriching this feed with essential nutrients previously shown to improve performance for many cell lines. The same CD basal medium, which was chosen after extensive small-scale screening studies in batch mode, was used for all experiments conducted for feed optimization.


Figure 4. For two different model cell lines tested in shake flasks, vitamin subgroup titration studies demonstrated similar growth compared to the peptone-containing process
Identifiying key subgroups: To quickly optimize the current CDF, we used an optimization strategy that involved enriching the feed medium with subgroups of media components. Approximately 30 media components were divided into three different subgroups, namely amino acids, vitamins, and an "others" sub-group that included nucleosides, fatty acids, amongst other components. Instead of working with individual components, these subgroups were titrated into the CDF in shaker flasks to reduce experimental complexity. Traditionally, components have been individually titrated at various concentrations and optimized. Our subgroups strategy was less labor intensive, demonstrated the importance of the vitamin subgroup concentration, and helped identify the optimal vitamin subgroup concentration. The newly adjusted concentration of vitamins resulted in improved cell growth and extended viability, even for a cell line that exhibited poor growth and high lactate synthesis (Figure 4).


Figure 5. For 10 different model cell lines tested in 2-L bioreactors, the average titer in the chemically defined (CD) medium was 30% higher than the peptone-containing process
Identification and optimization of key components: To complement shaker flask studies, a modified version of the CDF was evaluated in 2-L scale-down bioreactors using a cell line that exhibited robust growth and productivity. Although shaker flasks or high-throughput systems are more powerful for screening a larger number of medium components or cell lines, small-scale bioreactors with pH and dissolved oxygen control capabilities are more representative of conditions encountered in large-scale processes used in clinical or commercial manufacturing. The bioreactor process using the vitamin-enriched feed medium was conducted for 14 days, and supernatant samples were collected daily to quantify specific components in the spent medium and to determine the specific amino acid consumption and production rates. Based on specific amino acid consumption rates, key amino acid concentrations were adjusted in the feed medium. Supplementing CDF medium with vitamins and amino acids resulted in higher cell density, specific productivity, and overall final titers. Key amino acids asparagine, tryptophan, and valine were not depleted during production.


Figure 6. Scale-up of the chemically defined medium from 2 to 400 L (data shown for 2 2 L and 1 400 L runs)
Confirmation: Multiple cell lines previously used in early-stage and late-stage projects were tested in 2-L scale-down bioreactors for confirmation that the newly developed CDF was optimal for a variety of cell lines. Compared to the peptone containing medium, the CD medium resulted in an average titer improvement of 30%. Among the 10 cell lines tested, only three had lower titers in CD media (Figure 5). It was worth pointing out that the cell lines shown in Figure 5 also were preselected in a peptone-containing medium. To implement the new CD medium in a manufacturing facility, there are many process scalability items to be tested, such as large-scale medium preparation and cell culture process performance. An example of such a scale-up run using a late-stage cell line at a 400-L scale is shown in Figure 6. In this case, titer, cell growth, and metabolism profiles were comparable between the 2-L and 400-L scales.


Table 1. For five different model cell lines tested for product quality, aggregate levels, glycosylation patterns, and charge variants have minor and acceptable differences
Product quality: Changing medium formulations may introduce challenges in comparability of product quality. It is necessary to ensure that the transition from peptone-containing to CD media did not cause major product quality changes, especially for the molecules already in the clinical development stage. Our results show that the product quality differences were acceptable in terms of protein aggregation level, glycosylation pattern, and charge variants (Table 1). Furthermore, we have demonstrated that the CD medium gave consistent product quality profiles at different scales throughout cell line development, process development, and manufacturing stages.


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