Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

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ABSTRACT:The effect of different cell culture conditions on N-glycosylation site-occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t-PA) and a recombinant enzyme (glycoprotein 2-GP2). Both molecules contain a N-glycosylation site that is variably occupied. Different environmental factors that affect the site-occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t-PA (type I t-PA) by approximately 2.5-4%. Decreasing the cultivation temperature from 37 to 338C or 318C gradually increased site-occupancy of t-PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site-occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site-occupancy of t-PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N-glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site-occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N-glycan site-occupancy within a specific range. An increase in culture pH correlated with a decrease in site-occupancy. Similarly, delaying the timing for butyrate addition also decreased site-occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures.Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N-glycan site-occupancy, within a specific range when applied appropriately.

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B-vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed.

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CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) Genentech responded to a virus contamination in its biologics manufacturing facility by developing and implementing a series of barriers specifically designed to prevent recurrence of this significant and impactful event. The barriers included steps to inactivate or remove potential virus particles from the many raw materials used in cell culture processing. Additionally, analytical testing barriers provided protection of the downstream processing areas should a culture contamination occur, and robust virus clearance capability provided further assurance of virus safety should a low level contamination go undetected. This conference proceeding will review Genentech's approach, and lessons learned, in minimizing virus contamination risk in cell culture processes through multiple layers of targeted barriers designed to deliver biologics products with high success rates.

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