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20 | APRIL 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News duce development time," Dr Freeman con- tinues. "Application of our CRISPR/Cas9 screening expertise to engineer product-spe- cific solutions, particularly for difficult-to- manufacture proteins, will open commercial- ly viable pathways for this class of formerly inaccessible targets." Analysis and Characterization Engineered cell lines undergo constant evaluation during their development. Where genomics defines the success of transfection or gene editing, proteomic analysis uncovers a cell's actual activity. For production cells, the obvious measure of success is productiv- ity, but other quality- and productivity-relat- ed proteomic indicators can signal viability, growth characteristics, or product quality. A proteomics platform has been devel- oped by Biognosys. The platform, known as Hyper Reaction Monitoring (HRM-MS™), quantifies up to 9,000 proteins per sample, thus enabling monitoring of altered protein expression in genetically modified cells. HRM is a label-free proteomics work- flow based on data-independent acquisition (DIA). It uses high-resolution LC-MS/MS, most notably the Thermo Fisher Scientific Orbitrap platforms (QExactive or Orbitrap Fusion series) or the Sciex TripleTOF series. It seeks to answer two questions related to protein profiling of engineered cells. First, what happens to the proteome (for discovery proteomics)? Second, what happens to pro- teins of interest (targeted proteomics)? "Discovery of unbiased proteomics aims to understand global proteomic dynamics in a cell, tissue, or organism," states Stephan van Sint Fiet, Ph.D., chief commercial officer at Biognosys, "and is applied in biomarker research, drug and target discovery, pathway modeling, mechanism-of-action studies, and many other areas." HRM allows investigation of a cell line's global expression pattern, and how expres- sion changes during cell-line development and during the production of biotherapeu- tics. Global protein abundance is an impor- tant piece of data. "However, one should keep in mind that most proteins rely on post-translational modifications (PTMs) like N-glycosylation for their stability and proper solubility," clarifies Dr. van Sint Fiets. "PTMs containing peptides can be enriched and quantified on a global scale using HRM-MS." Protein folding is another quality attribute of interest. "To investigate folding," says Dr. van Sint Fiets, "we have recently acquired an exclusive license that combines HRM-MS with technology that enables the global inves- tigation of protein conformational changes in native cell lysates, with no need to purify the protein of interest. The Final Word? Horizon Discovery's Dr. Freeman notes that in the 30 years that therapeutic proteins have been produced in CHO cells, the only significant parental cell-line improvement with broad commercial uptake has been the single-gene GS knockout. Given Horizon Dis- covery's expertise in this area, this represents a significant opportunity for further develop- ment of CHO lines through genome engineer- ing, which has not yet been extensively utilized to optimize performance. But not all experts are sold on the idea that genetic manipulation can achieve tangible benefits in CHO cells. Florian Wurm, Ph.D., emeritus professor at the Swiss Federal Institute of Technology and founder and CSO of the protein expression services company ExcellGene, has long main- tained that improvements in upstream titers have resulted almost entirely from improved understanding of culture media and feeds. The problem with the CHO cell, as a substantial body of research demonstrates, is its inherent genetic instability and heterogeneity. All cell lines undergo genetic changes over time that result in significant phenotypic dif- ferences. The CHO cell, moreover, under- goes more subtle genomic drifting whose ef- fects may not readily be anticipated. "Cell engineering by genomic interac- tion is a very tough issue in CHO, a cell host that is constantly changing," comments Dr. Wurm. "This is why I am critical of the over- stated expectations from genomic sequenc- ing and its applications. At ExcellGene, we routinely generate 5–10 g/L using entirely unmodified CHO cells." The vaccine industry is challenged to produce large quantities of vaccines in a rapid and cost-effective way. Major changes to current bioprocesses are both diffi cult and very expensive to implement. Using HEK293 it has been demonstrated that addition of recombinant human insulin to commercially available chemically defi ned media, can be used as a supplement to increase VCD and specifi c viral yield. To learn more visit www.novonordiskpharmatech.com Increase specifi c Infl uenza production with recombinant human insulin *Data kindly supplied by Aziza Manceur, National Research Council Canada. Hemagglutinin (HA) assay is used for quantifi cation of Infl uenza (H1N1). Insulin Human AF used in the experiments is supplied by Novo Nordisk Pharmatech. CD 293 media is trademark of Thermo Fisher Scientifi c. Consistency. Proven Insulin concentration (mg/L) 40 HA concentration (ug/mL) Infl uenza production in HEK293 30 20 10 0 0 5 10 25 50 100 Insulin concentration (mg/L) 10 Total cell count (E05 cells/mL) HEK293 cells in CD 293 media * 8 6 4 2 0 0 5 10 25 50 100 Cell-Line Development Continued from page 19 Bioprocessing