An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-gamma produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-gamma, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process.

ABSTRACT:In radiation-cured acrylate coatings, a high conversion of monomer is generally desired to improve hardness, prevent further slow reaction upon aging, and avoid surface toxicity; however, shrinkage and, consequently, stress in coatings tend to increase with conversion. One route to achieve high conversion while maintaining low coating stress is to promote stress relaxation. In this study, acrylate coatings were cured with a UV lamp that was cycled on and off, varying the period from 2 to 60 min while keeping the total dose constant. All samples reached nearly the same conversion, between 46 and 48% conversion of functional groups, as measured by FTIR spectroscopy. The coating stress (measured with cantilever deflection) at the end of the exposure cycles, however, was lowered from 8 MPa (tension) to less than 3 MPa (tension) as the period was decreased from 60 to 2 min. The cycling had no significant effect on either the coating hardness or the modulus, as measured by a Hysitron triboscope. Stress relaxation and/or slower reaction during the dark periods are the likely causes for the lower coating stress. It was advantageous to introduce dark periods early in the processing.