Development of a Serum-Free Chemically Defined Medium for Adherent and Suspension Culture

Abstract

The development of suspension cell lines is a sought after holy grail for bioprocess development. Suspension cell lines allow for easier scale up and better volumetric productivity for most biotherapeutics, including vaccine manufacturing. Various cell lines have readily adapted to suspension growth through the modification of the cell culture medium, but Vero cells have resisted the trend of becoming a suspension cell line. For Vero cells and other cell lines that remain anchorage-dependent, researchers have found methods such as roller bottles and microcarriers to scale up production of biotherapeutics. Although these technologies have enabled the use of anchorage-dependent cell lines for biomanufacturing, the industry prefers suspension cultures and will design new cell lines, or design their processes around existing suspension cell lines to avoid the extra complexity of using anchorage-dependent cell lines.

Medium development has been the main method to adapt cells from adherent culture to suspension. This was first done by reducing the amount of serum that is supplemented in cell culture media. Serum is animal derived and contains many essential nutrients, growth factors, and adhesion proteins that traditional basal mediums do not contain. While serum has been essential for the establishment of many cell lines, it has safety concerns when producing biotherapeutics. Since serums are animal-derived, they can contain viruses, prions, or be contaminated with chemicals that the animal has ingested. Not only do these safety concerns create problems for the biomanufacturing industry, but also the lot-to-lot variability of serums can lead to poor product consistency. To overcome these challenges, the industry has developed animal component-free medium supplements that can contain plant hydrolysates or peptones. These are undefined bioactive fractions of hydrolyzed plant proteins, and while they do not have the same safety concerns as animal derived materials, they also suffer for lot-to-lot variability since the raw materials can depend on soil conditions, weather patterns, and pesticide use. Nevertheless, industry has overcome these challenges by developing chemically defined media. Every component's concentration in this medium is known and therefore this reduces the lot-to-lot variability. Additionally, many of the compounds that are commonly used in chemically defined medium can be manufactured using animal origin-free materials.

As cell culture media have eliminated serum, cells have become more anchorage-independent and have been slowly adapted to suspension growth. Although this has not been historically true for Vero cells, which have been grown in tight cell aggregates in serum-free medium. More recently, however, Vero cells have been successfully grown as single cell suspensions using two different undisclosed medium formulations that contain undefined plant hydrolysates or peptones. While this was a major achievement, the medium formulations are unknown, and they contain undefined proteins which can possibly suffer lot-to-lot variability. This thesis seeks to develop a chemically defined medium to support suspension Vero cells and to identify the transcriptomic differences between adherent and suspension Vero cells.

This work began by creating a data set of the previously reported medium formulations for Vero, CHO, HEK293, and various other commercial cell lines for adherent and suspension cell culture. Classic basal mediums that require serum supplementation were first compared to serum-free medium formulations to identify compounds that were added to media to replace serum. These compounds included glucose, amino acids, vitamins, trace metals, lipids and fatty acids, along with growth factors and proteins. A second comparison was done within the serum-free medium formulations to compare adherent versus suspension mediums. Suspension media were found to be more enriched that the adherent media and contained higher concentrations of amino acids and fatty acids. This review resulted in theoretical chemically defined, serum-free medium formulations that could support the growth of adherent and suspension commercial cell lines.

From this data set, a design space was laid out for the creation of a chemically defined medium formulation that supports Vero cell suspension culture. Through this work, recombinant epidermal growth factor was found to be essential for Vero cell proliferation along with the addition of trace metals, lipids, amino acids, and vitamins. Even without any serum, Vero cells continued to adhere to non-tissue culture treated flasks, while it was found that other cell lines (CHO-K1, HEK293T and MDCK) could grow as single cell suspension in the same novel medium. In an effort to coerce the Vero cells to grow in suspension the concentration of calcium and magnesium was reduced 10x. While this did cause the Vero cells to detach and form a single cell suspension, the growth rate dramatically decreased. Nevertheless, this work demonstrated that a chemically defined medium can be developed for Vero cell suspension, although compounds need to be added to ensure that Vero cells continue to grow.

To further investigate the cause of the low growth rate of the suspension Vero cells, RNA-seq was performed to compare the suspension Vero cells to Vero cells grown adherently in the chemically defined medium, and Vero cells grown in DMEM/F12+10\% FBS. This data set demonstrated that the suspension Vero cells had down-regulated cell cycle genes, and had begun to express kidney-associated genes. Since Vero cells were originally isolated from a female Green African monkey's kidney, it is hypothesized that the suspension Vero cells were reverting back to kidney cells. Key genes that were found to be differentially expressed by the suspension Vero cells included \textit{tgfb1}, \textit{c-myc}, and genes that are associated with the epithelial-mesenchymal transition.

Finally, different methods for improving the medium formulation were compared in the final chapter. Comparing a literature search, NMR metabolite analysis and RNA-seq transcriptomics found that the transcriptomic data provided the most insight on the compounds that were beneficial for cell growth. The literature did not have enough specific information about Vero cell metabolism and the compounds that were identified using this method did not result in significant improvements in the medium formulation. NMR was more specific for Vero cells, but because it can only track relatively high concentrations (micromolar) of metabolites, changes in vitamins and growth factors could not be tracked. The metabolite analysis did show a disfunctional tricarboxylic acid cycle, and some compounds that were identified using the analysis did significantly improve the cell's growth rate. Overall, the compounds that were identified using the transcriptomic data had the largest effect on the growth rate. Through RNA-seq analysis, we identified retinyl acetate, progesterone, \textbeta{}-estradiol and prostaglandin E\textsubscript{2} as growth enhancing compounds. Using the compounds identified through RNA-seq, NMR and a literature review, the doubling time of Vero cells was reduced from 38 hours to 32.1 hours, which is better than the animal component-free commercially available media currently on the market today.