Art and Science
Despite recent advances, particularly high-performance media that support 10 million cells per mL, culture medium development still relies on trial and error, according to Aziz Cayli, Ph.D., CEO of Cellca. The company specializes in cell-culture platform technologies, including its own CHO cell line specifically developed for large-scale manufacturing, and cell-culture media.
This is somewhat of a paradox. Due to safety and regulatory concerns, and the desire to standardize, modern media for large-scale production lack complex ingredients like serum and hydrolysates. Achieving the activity of these ingredients requires adding many more discrete compounds, particularly trace elements, that were present naturally in serum and hydrolysates.
The purity of modern reagents may be to blame at some level. Twenty years ago additives contained trace quantities of other substances that apparently benefited cells.
“Today, chemicals are nearly 100 percent pure, and we need to add those trace elements back,” Dr. Cayli observed. “Natural components contain a large number of chemicals. Eliminating one may remove from 10 to 50 individual components, including growth factors, trace elements, and lipids.”
Thus, the “magic dust” of several undefined ingredients gives way to many more scientifically characterized components.
An associated trend, ever-increasing cell density, has led to the use of feeds and supplements that also contain these nutrients. Off the shelf some of these preparations hold individual components at or near their solubility limits.
“We still don’t have a good understanding of what specific ingredients cells need to grow and produce,” Dr. Cayli said.
The challenges are to gain knowledge of cellular demand, and use it to tune in desired characteristics and performance. These objectives will require “a more rational approach to understanding factors affecting cell metabolism, growth, protein production and protein quality.”
Despite difficulties, rising biomass concentrations tell us that today’s cell culture media are much improved over those a decade ago. When hydrolysates or sera were widely used, cell counts ranged from about two to three million cells/mL.
“Today, by improving media quality, we have pushed viable cell concentrations to 30 million/mL in simple fed-batch cultures,” reported Dr. Cayli, adding that this is primarily due to media improvements.
Cellca has two goals in addition to raising biomass concentrations even further: suppressing metabolic waste (e.g., lactate), and improving product quality. Both objectives, Dr. Cayli believes, can be achieved by improving and fine-tuning media. One aspect of quality improvements is control over glycosylation. “Modern media contain as many as 70 components that may affect glycosylation.”
Recapitulating the art-and-science dichotomy, Thomas Noll, Ph.D., professor of cell-culture technology at the University of Bielefeld, noted that animal cell culture development itself was still “an empirical procedure. The application of proteomics and metabolomics remains hampered by the unavailability of a CHO protein database and, in case of metabolomics, by the cells’ mechanical instability and their compartmentalization.”
Dr. Noll’s group is developing new strategies for rational process development based on process characterization and identifying the influence of process conditions, at the cellular and molecular levels, through application of differential proteomic and (intracellular) metabolomic analysis. Proteomics and metabolomics allow the most direct measurement of a cell’s physiological activity and, according to Dr. Noll, “have proven their potential for microbial cell-line and process development.”