Engineering mammalian cell line to improve sialylation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Includes bibliographical references (p. 209-231).One of the key problems faced by many biotechnology companies is the cleavage of terminal sialic acid on the glycans of the therapeutic glycoproteins. This is caused by the degradative action of sialidase released to supernatant when the cell starts to die. This phenomenon is undesirable because the loss of terminal sialic acid results in a product which is rapidly removed from the plasma by the interaction with asialoglycoprotein receptors in the liver. Many studies have been done in this area for decades and no general approach has been produced thus far. In this study, RNA interference is utilized as a genetic approach to knock down the activity of sialidase which is responsible for cleaving terminal sialic acid. At the first stage of the studies, 21-nt double stranded siRNA sequences capable of knocking down sialidase are identified. The best sialidase siRNA sequence transiently knocks down sialidase mRNA by 9 folds and accompanied by a 4 fold reduction in sialidase activity. The most potent sialidase siRNA was located in UTR region and did not follow the widely-used Tuschl's rule. At the second stage of the studies, a siRNA sequence is integrated into CHO cells using a plasmid with a drug selection marker to produce stable cell lines. It is found that the Pol III promoter is not strong enough to generate sialidase siRNAs. The modified CMV promoter is more appropriate for knocking down sialidase activity as clones with over 50% sialidase activity reduction can be isolated. We have isolated stable clones with over 60% sialidase knock down during the course of the cell cultivation. Growth rate and glycoprotein specific productivity of stable clones with reduced sialidase activity are not affected by siRNA activity or reduced sialidase expression. Glycan site occupancy of IFN[gamma] produced by stable clones remains relatively unchanged. Two of the stable clones successfully maintain constant sialic acid content of IFN[gamma] during prolonged cell culture even though cells are dying during these periods while the parent cell line loses the sialic acid at the rate of 0.05 mole sialic acid /mole IFN[gamma]/day. This result is comparable to when sialidase inhibitor is used to deactivate sialidase. Microheterogeneity analysis reconfirms the consistent fraction of asialo, monosialyl, and bisialyl form of IFN[gamma] for cell lines with reduced sialidase level during prolonged cell culture. On the other hand, parent cells are found to have more asialo and monosialyl form of IFN[gamma] as the cells dies demonstrating the effect of sialidase release on glycoproteins during prolonged cell culture. Maximal sialic acid content during growth phase is found to be slightly altered by sialidase knock down. This could be due to clonal variation of parent cells or due to sialic acid salvage pathway disruption by reduced sialidase activity. At the third stage of the studies, we develop a GFP-based method to rapidly and effectively isolate cells which express high amounts of sialidase siRNA. Subpopulations of CHO cells with a high level of mean fluorescence intensity have lower sialidase mRNA level and activity. This implies a positive correlation between GFP fluorescence intensity and siRNA generated to silence sialidase. For similar fluorescent intensity, cells transfected with GFP-based Pol II-driven plasmid exhibits sialidase knock down 1-3 folds stronger than those transfected with GFP-based Pol III-driven plasmid. We have successfully knocked down sialidase using a siRNA approach and produced not only stable cell line, but also functional and viable cell where cell growth is not affected by sialidase knock down. This method is a generic method that can be adopted by any biotech companies to reduce the sialidase degradative activity, producing a more consistent protein quality over the cell cultivation.by Frederyk Anthonius Ngantung.Ph.D