Exploiting the Di-Sialyl Galactose Activity of α2,6 Sialyltransferase from Photobacterium Damselae to Generate a Highly Sialylated Recombinant Alpha-1-Antitrypsin.

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 alpha-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced alpha-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected alpha-2,6-sialylation of galactose, a second disialyl galactose motif NeuSAc-alpha-2,3(NeuSAc-alpha-2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated alpha-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of alpha-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.