Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from <i>Photobacterium damselae</i> To Generate a Highly Sialylated Recombinant α-1-Antitrypsin

Abstract

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from <i>Photobacterium damselae</i> can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the <i>P. damselae</i> in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the <i>in vitro</i> activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.