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TUTORIAL Bioprocessing ated in formats ranging from 1 mL cultures in 24-well plates to 1 L cultures in shaker flasks with successful protein expression also obtained in 96-well plate formats. Protein Functionality and Glycosylation Pattern Assessment Although ultra-high protein yields are de- sirable, the resultant protein is less valuable if it is aggregated, misfolded, degraded, or im- properly glycosylated, thus requiring signifi- cant additional purification efforts to yield a final protein product with high purity. The quality of the proteins expressed in the high- density Expi293 system were compared to the quality of the same proteins expressed in the FreeStyle 293 expression system in terms of biological activity, binding, and glycosyl- ation patterns. Recombinant erythropoietin (EPO) was tested for biological activity using a TF-1 cell-based proliferation assay. The biological activity of EPO was highly similar for both expression systems, with an EC50 value of 0.45 ng/mL for EPO made in the Expi293 system vs. 0.58 ng/mL for the FreeStyle 293 system (Figure 3A). The ligand-binding activity of `-2 adren- ergic receptor was equivalent for both ex- pression systems, with Kd values of 1.069 vs. 1.057 nM and Bmax values of 30.1 vs. 29.1 pmol ligand/mg for `-2 adrenergic receptor produced in the Expi293 and FreeStyle 293 systems, respectively. Additionally, the binding of an anti-GFP antibody for its target protein in an immu- noblot was demonstrated to be equivalent for antibody produced in both expression systems (data not shown). Analysis of the N-linked carbohydrates for a recombinant human IgG produced in both the Expi293 and FreeStyle 293 systems showed that the glycosylation profiles were highly comparable for antibody made in ei- ther system (Figure 3B). For both proteins, the G0F structure was the most prevalent, followed by G1F, with these two structures accounting for approximately 85% of the total N-glycans in each case. Antibody fucosylation levels have the po- tential to impact antibody Fc-related effector functions; no differences in the levels of GO (nonfucosylated) variants were observed be- tween the systems. These data indicate that the quality of the protein expressed in the Expi293 expression system, as determined via biological *Meredith B. Jones, Ph.D., *Chao Yan Liu, *Sanjay Vasu, Ph.D., and *Isabel Cisneros are scientists, Henry Chiou, Ph.D. (Henry. Chiou@lifetech.com), is a senior product manager, and Jonathan F. Zmuda, Ph.D., is an associate director, R&D; at Life Tech- nologies. The authors would like to thank Yolanda Tennico for her expert assistance with glycosylation profiling. The Expi293 Expression System and FreeStyle 293 Ex- pression System are for Research Use Only. Not for use in diagnostic procedures. *These authors contributed equally to this work. Genetic Engineering & Biotechnology News | genengnews.com | October 1, 2012 | 51 activity, binding, and glycosylation analysis, is equivalent to the quality of protein expressed in the FreeStyle 293 expression system. Together, the results demonstrate that sig- nificant improvements in functional protein yields can be attained using a novel transient mammalian expression system that incorpo- rates multiple advances in protein expression technology into a single, easy-to-use format. High transfection efficiencies at increased cell densities, along with the incorporation of transfection enhancers, were vital in achieving multifold increases in recombinant protein expression levels. Using the Expi293 system, protein expres- sion levels that rival those achieved in some sta- bly transfected cell lines were obtained in a sim ple and rapid transient expression system. A B Figure 3. Assessment of biological activity and glycosylation profiles of proteins generated using the Expi293 System and the FreeStyle 293 System. (A) Human erythropoietin was expressed using the Expi293 and FreeStyle 293 Expression Systems and tested for biological activity using a TF-1 cell-based proliferation assay. (B) Highly similar N-linked glycosylation profiles were obtained for human IgG expressed in the Expi293 and FreeStyle 293 Expression Systems.