This study focused on modulating the glycosylation patterns via genetic engineering approaches to modulate biotherapeutics’ sialic acid content. The sialylation level on biologics is one of the most critical quality attributes in that the sialylation level on glycoproteins influences their pharmacokinetic characteristic. We have sought to improve the sialylation level by overexpressing α-2,6 sialyltransferase 1 (ST6GAL1) and two N-acetylglucosaminyltransferases (GNT-IV and GNT-V). The resultant CHO cells showed high ST6GAL1, GnT-IV and GnT-V gene expression. N-glycans profiling of erythropoietin (EPO) expressed from the glycoengineered CHO indicated that approximately 92% of the N-glycans were tri- and tetraantennary N-glycans. EPO sialic acid quantification of this glycoengineered CHO cells increased the sialylation level 45% as compared with EPO from wild type CHO cells. Meanwhile, to understand the endogenous expression level of sialyltrasnferases in CHO and their role in glycoprotein sialylation, we used siRNA to evaluate role of three sialyltransferases, ST3GAL3, ST3GAL4 and ST3GAL6 in glycoprotein sialylation. By performing single, double and triple siRNA transfection into CHO cells, we measured the change in sialyation level by MAL II enzyme linked lectin absorption assay and HPLC sialic acid quantification. The results indicated all three sialyltrasnferases contributed to protein sialylation. What’s more, a triple siRNA knockdown (combining ST3GAL3, ST3GAL4 and ST3GAL6 siRNA) rendered the lowest sialylation level in contrast to single and double ST3GALs siRNA gene knockdown. Furthermore, ST3GAL4 was observed to play a role in protein and N-linked sialylation as indicated by EPO sialic acid quantification. The overexpression of three glycosyltransferases and the knockdown of sialyltransferases will allow researchers to fine tune the sialylation level on EPO and other biologics in order to meet medical needs.

Glycoengineering to improve biotherapeutic quality from CHO cells

253rd National Meeting (2017)

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