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18 | APRIL 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News growth prospects. According to market research firm Global Market Insights, cell-line development should become a $7.5 billion market by 2023, up from $2.8 billion in 2015, reflect- ing an annual growth rate of 13%. Such figures mean opportunity to market participants, who are already positioning themselves in the competitive cell-line development landscape. For example, in February, GE Healthcare entered a three-year collaboration with the Austrian Centre of Industrial Biotechnology for identifying new techniques for optimizing CHO cell-line performance. The collaboration's initial emphasis is on CHO cell-line growth and expression. In its original announcement of the deal, GE Healthcare noted that CHO cells had not traditionally been the object of optimization due to limitations of existing optimization technologies and a demanding regulatory climate. Improving CHO Cells "The recent productivity gains are mainly due to optimi- zation of media, feed, and processes plus improved expres- sion vector and selection workflows," comments Daniel Ivansson, staff research engineer, GE Healthcare. He adds that GE Healthcare intends to bring about future productiv- ity gains through the "engineering and design of the cell it- self." The company is emphasizing the development of CHO starter cells with enhanced phenotypes. Current optimization methods involve empirical, trial- and-error methods for selecting clones from suboptimal starter cell lines. Modern gene-editing and analysis tools have changed the picture to the point where more detailed characterization of cell performance is possible with im- proved starting cells. Transcriptomics, in particular, provides insights into the regulation of cellular performance. "Among the tools we will employ are high-throughput formats for assessing product secretion and cellular growth of desired phenotypes," Ivansson details. "We will also use RNA-Seq, global epigenetic analysis, genome sequencing, and metabolomics to characterize phenotypes. We will also use proteomics as needed." Difficult Proteins Early in 2017, Selexis announced that it was applying its SUREtechnology Platform™ to cell lines for three Sanofi bio- therapeutics: a "naked" antibody, a bispecific antibody, and a complex recombinant vaccine. Selexis CEO Igor Fisch, Ph.D., explains how one platform cell-improvement technology can serve the production of such disparate products. He begins by noting that generating clini- cally and commercially viable therapeutic proteins involves well-known transfection, transcription, translation, and trans- port of products via the cell's secretion pathways, whereby it is folded, modified, and secreted into the media. "However, parameters for maximal secretion vary signifi- cantly between proteins," he points out. "Transcriptional, metabolic, folding, post-translational modifications, and transport requirements can be quite different, even among monoclonal antibodies." By employing appropriate epigenetic elements, Selexis ob- tains high transcriptional levels of mRNA, thus bypassing the need for strong selection processes based on drugs such as methotrexate (MTX) or l-methionine sulfoximine (MSX). Next, applying whole-genome sequencing to the compa- ny's proprietary suspension-adapted CHO-K1 cell line with unique bioinformatics analysis tools, Selexis can address po- tential secretion issues for bispecifics, large multimeric pro- teins, or vaccines. "Using these techniques," Dr. Fisch explains, "we can en- gineer CHO-M to overcome expression issues by generating CHO-M libraries engineered in defined pathways to allow proper folding, glycosylation, and transport to the mem- brane for secretion." Through this approach, Selexis has re- cently demonstrated a 30-fold increase in expression for a difficult-to-express vaccine protein. Selexis also uses whole-genome sequencing for clonal as- sessment of cell lines, including the integrity of the transgene copies at all locations in the genome and the site of integra- tion of the transgene in the host cell genome. This approach ensures that no bias exists in the clonality assessment, com- pared to mRNA or partially digested gDNA sequencing. Selexis realized several years ago that novel therapeutic proteins would entail complex manufacturing. At the time, the company identified bottlenecks that affected secretion, folding, and productivity, particularly for difficult-to-express proteins. This was the justification for developing the SURE CHOM-Mplus Libraries. Selexis was the first company, in collaboration with the University of Lausanne and the Swiss Institute of Bioinformat- ics, to sequence and annotate its CHO-K1 line. It used these data to improve the cell's production characteristics across the transcription, translation, and secretion continuum. Selexis works with a variety of cell lines, including CHO, BHK, HEK-293, B cell, and C2C12 cell lines. Most recently, Selexis launched a novel metabolic selec- tion method based on the co-expression of a vitamin B5 transporter. The method exploits the dependence on vitamin B5 evidenced by mammalian cells, which use the essential nutrient to sustain efficient energy production. "This method yields smaller polyclonal cell populations and produces higher yields of the recombinant proteins," as- serts Dr. Fisch. And it can be used to identify and isolate cell lines that grow even when they produce recombinant proteins that are toxic for CHO-M. Cell lines that grow under such circumstances do so because of positive metabolic selection. Cell-Line Development Poised for Growth Bioprocessing Feature Selexis reports that it has used its SUREtechnology Platform to generate nearly 80 clonal research cell banks (RCBs) that have reached clinical and commercial manufacturing. The platform has made it possible to generate stable and high-performing manufacturing cell lines in approximately three months from the time of transfection, with productivity levels reaching 1–7 g/L for monoclonal antibodies. The company also asserts that its SUREdevelopment Process improves productivity and jump- starts the scale-up process for CMOs. Hyper Reaction Monitoring (HRM), a label-free discovery proteomics workflow developed by Biognosys, allows investigation of a cell line's global expression pattern. HRM was used to carry out single-shot proteome profiling of HeLa, Jurkat, and HEK-293 cell lines and generate this heatmap, which identifies and quantifies thousands of proteins across the cell lines. Distinctive differences in proteome expression can be observed. Continued from page 1