Developing chemically defined (CD) media suited for multiple Chinese hamster ovary (CHO) cell lines can be a challenging and extensive process because of the complexity of cellular metabolism and the associated phenotypic variations observed in cell lines, even when derived from the same parental cell line. In this article, we present certain systematic approaches to develop CD cell culture media. Two feed media development applications have been explored, each with distinct goals. First, a systematic approach was used to develop a platform CD feed medium, which can be used for many antibody producing CHO cell lines without any major cell line-specific optimization. Second, an iterative optimization approach was applied to develop an enriched feed media tailored to individual cell lines, to achieve higher titers.
As cell culture technology has matured, platform processes have become the standard in the biotechnology industry, particularly in early-stage development, to enable the rapid production of toxicology and Phase 1 and 2 clinical material with acceptable product quality attributes. The cell culture platform often consists of a standard host cell, expression vector, transfection and selection methods, cell culture media and culture techniques with appropriate process control, and scale-up methodologies. An ideal cell culture platform medium not only enables rapid cell culture process development, but also could provide predictable process performance at different production scales and thereby aid in the development of downstream process platforms as well.
The majority of the biopharmaceutical industry currently is using fed-batch cultures of animal cells as the platform technology to synthesize biopharmaceutical proteins because it offers the benefits of high yields and operational simplicity.1 A fed-batch process requires a basal medium to support cell growth and productivity and a feed medium to prevent nutrient depletion, extend culture longevity, and maintain high volumetric productivity. A typical basal medium contains 50–70 chemical components, including carbon sources, amino acids, vitamins, salts, and trace elements. Feed media usually contain fewer components than the basal media, but at higher concentrations.2 Cell culture media have evolved over the years from serum-containing to chemically defined (CD) formulations.3 Recent advances in cell culture media optimization have resulted in higher volumetric productivities in fed-batch processes. Cell culture media optimization is an integral part of mammalian cell culture process development and optimization, which support all activities from cell line development and clone selection to process optimization and scale-up. Cell culture process development starts with cell line generation and selection, followed by process and media optimization in scale-down systems, including 96-well plates, shaker flasks, and bench-scale bioreactors. After the process conditions are defined, the process often is transferred to the pilot scale to test scalability and produce material for preclinical toxicology studies. Subsequently, the process is further scaled-up to generate clinical material under current good manufacturing practices (cGMP) regulations (Figure 1). The ideal cell culture medium should support robust cell growth, productivity, and product titer and quality goals, both during development and process transfer to different scales and manufacturing sites.
Figure 1. A platform cell culture medium, integrated from early cell line development activities to clinical production, streamlines process development to meet aggressive timeline
Historically, bovine serum had been a popular supplement in cell culture media because it improves cell growth and productivity. Regulatory agencies have encouraged the industry to avoid animal-derived supplements to mitigate the risk of introducing adventitious agents such as viruses and prions.4,5
Protein hydrolysates, which can provide similar benefits as serum, can be animal-derived from bovine milk or tissues, or non-animal–derived from yeast, cotton seed, soy, wheat, or other plant sources. Some disadvantages of hydrolysates include lot-to-lot variability leading to process and product inconsistencies. The undefined chemical composition of hydrolysates can lead to challenges in process understanding, making further medium optimization difficult.6 Therefore, many biopharmaceutical companies are transitioning to CD media formulations and a few vendors now also offer CD basal and feed media.7 We observed that the current version of our peptone-containing media, when used with most cell lines, lead to robust cell culture performance at different scales and generated titers greater than 1 g/L for most of our in-house cell lines. After removing the peptone from our platform feed medium, a systematic approach was applied to develop a suitable CD platform feed medium. An iterative optimization approach was then applied to develop enhanced feeds tailored to individual cell lines to achieve higher titers.