The impact of sialylation linkage‐type on the pharmacokinetics of recombinant butyrylcholinesterases

Abstract

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N‐glycans terminating in α‐2,3 sialylation. Human cells produce glycoproteins that include α‐2,3 and α‐2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α‐2,3 sialyltransferase genes followed by overexpression of the α‐2,6 sialyltransferase (26BChE) enzyme. The N‐glycan composition of 26BChE was compared to BChE with α‐2,3 sialylation (23BChE) derived from wild‐type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi‐antennary di‐sialylated glycans representing the most abundant glycoform. CD‐1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.

Therapeutic antibody glycosylation impacts antigen recognition and immunogenicity

“…In addition, they hypothesize that terminal β1‐4Gal‐linked glycans are cleared independent of ASGPR. Another study confirms that the linkage is relevant because a glycoprotein containing α2‐6 linked sialoglycans was cleared faster than that containing α2‐3 linked sialoglycans [20], thus exhibited a lower activity. Therefore, it may be possible that the reduced exposure of HySi detected in BALB/c mice accounts for a preferred recognition of α2‐6 linked sialic acids by ASGPR.…”

“…The antibodies were purified using Protein A chromatography, followed by size‐exclusion chromatography. For HySi, ConA purification was done to remove contaminations of high mannose glycans found after expression in the CHO HySi cell line which has also been observed by others [20].…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…For example, antibodies produced from mousederived animal cells have a high proportion of fucose modifications and a low proportion of galactosylation modifications (An, 2009). In contrast to human cells, CHO cells lack the expression of α-2,6-sialyltransferase, while only express α-2,3-sialyltransferase (Jenkins et al, 1996;Lin et al, 2015;Chung et al, 2020). Consequently, CHO cells inherently cannot produce glycoproteins with similar terminal sialic acid content as compared to human cells (Yin et al, 2017).…”