Improving the productivity of production cells, a major goal of biomanufacturers, involves the sequential application of cell-line engineering and optimization of media, feed, and process. Experts agree that this sometimes iterative process, like the development of higher human attributes, depends on both nature and nurture.
Crucell’s PER.C6® cell line illustrates the necessary balance between these two factors. The volumetric productivity of PER.C6 has become legendary, with the current titer record of 27 g/L at harvest and more than 40 g/L if you subtract the cell volume and count only the supernatant.
The success of PER.C6 is based on the cell itself, a human retina-derived cell that provides human-like glycosylation and all attendant benefits. “Processors don’t have to worry about undesirable post-translational modifications with PER.C6,” observes Kathryn Golden, a scientist in upstream processing at Percivia, a joint venture between Crucell and DSM that specializes in bioprocess applications of PER.C6.
But the role of post-cell-engineering optimization cannot be overstated either. Percivia’s mandate is to improve its parent company’s prolific cell line through media, feed, and process strategies. One such improvement is implementation of Refine Technology’s alternating tangential flow (ATF) technique, which perfuses fresh media through the culture while removing byproducts. ATF reportedly permits PER.C6 cells to grow to extremely high concentrations, up to 150 million cells/mL, in less than two weeks. Data shows that ATF generates viable biomass at ten times higher concentration than conventional cell culture.
Speed to Process Development
With its licensing of zinc finger nuclease (ZFN) technology from Sangamo, SAFC Biosciences gained the capability to knock genes in or out with high specificity. According to Bruce Lehr, director of development, ZFN generates clones within six months. SAFC works directly on customers’ cells or provides them with the ZFN kits to do the work themselves. SAFC expects to offer a full-spectrum service that combines cell-line engineering services with optimization based on media, feed, and processes within three years.
ZFNs are artificial restriction enzymes in which a zinc-dependent DNA-binding protein and a DNA-cleaving enzyme are fused, thus combining highly selective recognition with DNA splicing. ZFNs have received a lot of attention from medical researchers, who use the enzymes to knock out genes.
ZFNs work by removing a gene sequence, leaving behind a double-stranded break in the DNA backbone. In the absence of a suitable template the strands will often close perfectly, resulting in a clean deletion. When a homologous template strand is available it will insert into the break.
ZFN technology has been used to create knockout rats. In April 2010, a group from Singapore used the methodology to generate a zebrafish model for Parkinson disease. And recently, the Michael J. Fox Foundation awarded a one-year, $232,000 grant to Sigma Advanced Genetic Engineering (SAGE) Labs to develop a more convenient Parkinson model in rats. Researchers at SAGE are expected to use the company’s CompoZr ZFN technology to create the model.
Knockout/knockdown strategies can have a tremendous impact on cell-line optimization for biomanufacturing, an area in which SAFC has been active. Lehr notes the potential for inactivating genes that code for apoptosis, host-cell proteins, or critical post-translational modifications such as glycosylation, methylation, or phosphorylation. One SAFC customer asked the company to delete a marker on the production cell that was homologous to the target receptor in humans.
SAFC has thus far focused on CHO cells, the workhorses of monoclonal antibody manufacturing. As it works on these production cells, the company is simultaneously matching media and feeds, to “optimize productivity from a very early stage,” according to Lehr. The goal is to introduce platform production technologies that will assist small biotech companies and contract manufacturers to develop cells and move them into production rapidly.
“Large companies are already using platform technologies but the next tier down and many CMOs lack the time or expertise to develop them,” Lehr reports. “Having platform manufacturing methods will help these companies attract customers.” Another boost to platform methods, he says, will be the eventual approval of biosimilars.