Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels

Sialylation of N-glycans associated with recombinant human acetylcholineste rase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their abilit y to sialylate overexpressed rHuAChE. High-resolution N-glycan structural a nalysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associat ed with rHuAChE produced in HEK-293 cells belong mainly to the complex-bian tennary class and are only partly sialylated, with approx. 60% of the glyca ns being monosialylated. This partial sialylation characterizes rHuAChE pro duced by cells selected for high-level expression of the recombinant protei n. In low-level producer lines, the enzyme exhibits a higher sialic acid co ntent, suggesting that undersialylation of rHuAChE in high-level producer l ines stems from a limited endogenous glycosyltransferase activity. To impro ve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialy ltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantl y higher proportion of fully sialylated glycans as shown by sialic acid inc orporation assays, direct measurement of sialic acid, and HPAEC glycan prof iling. Genetically modified sialylated rHuAChE exhibited increased circulat ory retention (the slow-phase half-life, t(1/2)beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increa se in circulatory residence was observed when rHuAChE was subjected to exte nsive sialylation in vitro. The engineered HEK-293 cells in which the glyco sylation machinery was modified might represent a valuable tool for the hig h level of expression of recombinant glycoproteins whose sialic acid conten t is important for their function or for pharmacokinetic behaviour.