Volker Sandig, Ph.D., CSO at ProBioGen, highlighted the importance of production platforms for manufacturing recombinant proteins, antibodies, and viruses. He noted that even under the best conditions, expressed proteins produced may interfere with host cell metabolism, be modified or degraded by the cell, or misfold or aggregate.
Dr. Sandig referred to these as “difficult proteins,” and described ways to deal with them by enhancing the folding apparatus, providing alternative cell substrates, or through selection and screening approaches.
Protein misfolding may occur despite intracellular mechanisms to prevent it, for example the glucosidase II system, the calnexin calreticulin system, chaperones, and enzymes such as peptididyl-prolyl-cis-trans isomerases and protein-disulfide isomerases. Misfolded proteins recognized by chaperones within the endoplasmatic reticulum may trigger the unfolded protein response—a crosstalk mechanism between the endoplasmic reticulum and nucleus, which affects proteins subsequently produced.
“Metabolic limitations of the specific producer cell can induce the unfolded response as well,” Dr. Sandig said, resulting in a shut-down of transgene transcription of difficult proteins, and limited expression of even well-expressed products. Molecular engineering can help cells overcome these challenges while maintaining expression.
What does selection mean in the context of dealing with difficult proteins? As an integral element generating high-producing clones, ProBiogen scientists use drugs that identify cells that carry the desired transgene and marker gene conferring resistance to the drug. High drug concentrations are more easily tolerated by cells that express high marker and transgene levels.
Enhancing the folding apparatus is another hallmark of this approach. Cells are equipped either for rapid proliferation or for production of secreted proteins. Changes occurring in a mature plasma cell are the most prominent examples of the latter. While introduction of individual chaperones has mostly failed to improve the yield of secreted proteins, the co-expression of specific modulators affecting secretion have shown benefit.
“We have evaluated a number of such candidate modulators identifying ones with prominent effects. However, the benefit of the specific regulator is closely linked to the individual starter cell and requires extensive fine-tuning,” explained Dr. Sandig.
Currently, CHO are the workhorse cells for manufacturing biologics. Expression of some proteins is compromised in CHO as a result of signal transduction or CHO-specific degradation or modification. In these instances other cell types may overcome this hurdle in much less time than it takes to optimize a CHO line.
“We repeatedly noted this benefit with designed human and duck cell lines of the AGE1 platform. Of course, alternative cell substrates require documentation for origin and history, and possess the usual characteristics of top-performing pharmaceutical producer cell lines, including suspension growth in chemically defined media.”
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.”