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30 | OCTOBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News See Protein Expression on page 32 explained, "are inherently adaptable and easy to scale up to industrial bioreactors" However, for early-phase drug discovery of complex biopharmaceuticals, companies of- ten rely on HEK293 transient expression plat- forms. "This can increase the risk of surprises when molecules are advanced and eventually expressed in CHO," warned Dr. Rajendra. "To reduce this risk, we developed a transient CHO platform that is based on the same cell line, media package, and DNA expression cas- sette used for our stable CHO platform." According to Dr. Rajendra, harmonization of transient and stable expression approaches can provide better predictability of protein quality and expression when transitioning the molecules from discovery to development and ultimately manufacturing. "This transient CHO system can rapidly (within seven days) generate high titers," asserted Dr. Rajendra. "The system is scalable to 10 L." Dr. Rajendra's group introduced another modification: the use of stable CHO pools (instead of master wells or clones) to gener- ate gram quantities of therapeutic protein. "It takes 2–4 weeks to generate a stable CHO pool," noted Dr. Rajendra. "In contrast, it usually takes several months to generate clon- al CHO cell lines. Hence, speed is one major advantage of stable pools. "Additionally, advantages over transient expression include use of smaller amounts of plasmid DNA for transfection, ease of volu- metric scale-up, and the flexibility of perform- ing multiple production runs over an extend- ed period of time using a frozen cell bank. We recently published CHO pool titers ranging from 2 to 7.6 g/L." Dr. Rajendra indicated that at present, stable pools are primarily used for preclinical material generation due to concerns of clonal heterogeneity, genetic and expression stabil- ity, and product quality consistency. In the future, however, the use of stable pools may extend to the generation of toxicology lots and the first human dose studies. "Although this is going to take some time," Dr. Rajendra concluded, "given the advancements in host cell engineering, trans- poson-mediated approaches, and analytical tools that allow for in-depth product qual- ity assessment, I am hopeful that we will see a shift from the 'clonality' paradigm to a "product quality consistency' paradigm." Glycoengineering in CHO Although they are widely used, CHO ex- pression systems present challenges similar to those posed by other mammalian cells. In particular, CHO cells produce recombinant proteins that are heterogeneous in complex- type N-glycans. "Glycosylation is one the most impor- tant post-translational modifications of pro- teins," stated Andrew (Cheng-Yu) Chung, researcher, department of chemical and biomolecular engineering, Johns Hopkins University (JHU). "Glycans on proteins play a key role, particularly in protein folding. Thus, they significantly impact protein sta- bility and protein-protein interactions." Chung, who is in the laboratory of Michael J. Betenbaugh, Ph.D., is studying sialylation of engineered proteins. "Because of the nega- tive charge, size, and hydrophilic characteris- tics of sialic acid groups, sialylation is one of the very critical modifications on the glycan terminus," Chung explained. These proper- ties allowed sialic acid to have a substantial influence on protein-protein interactions. Further, sialylation can affect biotherapeu- tics' efficiencies." Chung and colleagues examined the ap- plication of genetic engineering strategies to modulate the level of sialylation content on various potential biotherapeutics. "One common way to manipulate the glycan struc- ture is via genetic engineering tools such as CRISPR/Cas9 to knockin/knockout or over- express certain glycoenzymes," he noted. "For example, overexpressing glycoenzymes involved in the sialylation pathway is a strat- egy to produce biologics with increased sialic acid content that may affect circulation reten- tion time (CRT). Increased CRT may mean lower doses for patients." Another strategy to make glycosylation in CHO cells more uniform and similar to the analogous process in human cells is to add specific chemical supplements to the cell culture medium. Protein Expression Continued from page 26 BIOPROCESSING Industry's Leading BSA Manufacturer Quality Control Consistency Our facilities manufacture in accordance with Current Good Manufacturing Practice (cGMP) as per US FDA and the New Zealand Ministry for Primary Industries (MPI) regulations and guidance. Our vertically integrated process ensures complete control from the time the product is collected to the moment it is a finished and tested product. It is truly a "closed loop" system. We provide unparalleled lot to lot consistency. 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