Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

In Vitro Sialylation of Recombinant Alpha-1-Antitrypsin using ╬▒2,6 Sialyltransferase from Photobacterium Damselae Produces Disialylgalactose N-Glycans (#748)

Edward Pallister 1 2 , Matthew Choo 1 , Jien-Nee Tai 1 , Dawn Leong 1 , Wen-Qin Tang 1 , Kun Huang 2 , Andrea Marchesi 2 , Peter Both 2 , Christopher Gray 2 , Say Kong Ng 1 , Pauline Rudd 1 , Sabine Flitsch 2 , Terry Nguyen-Khuong 1
  1. Bioprocessing Technology Institute, Singapore, SINGAPORE
  2. School of Chemistry & Manchester Institute of Biotechnology, University of Manchester, Manchester, United Kingdom

The genetic disease AAT deficiency (AATD) is caused by a lack of Alpha-1-antitrypisn (AAT) in the body as a result of causes a number of complications ranging from chronic obstructive pulmonary disease to liver cirrhosis. Augmentation therapy of severe AATD sufferers involves human serum plasma AAT. Unfortunately, this treatment is expensive so a means of reducing the costs and improving the treatments efficiency are of significant interest. The half-life of alpha-1-antitrypsin is increased in vivo markedly by increasing the level of sialylation. Here we describe how the unique disialylgalactose sialylation (DSG) activity of a-2,6-sialyltransferase from Photobacterium Damselae. This disialylgalactose epitope is a unique a2,3 and a2,6 sialic acid sialylation of free end galactose residues. DSG can be used in tandem with CHO produced alpha-1-antitrypsin to markedly increase sialylation. We used numerous glycomic and glycoproteomic techniques such as permethylation, ethyl esterification  together with MS methods such as glycomic methods to such as MALDI-MS, Orbitrap-MS and ion-mobility mass spectrometry (IM-MS) to characterise the unique DSG epitope and identify the N-Glycans that contain the previously uncharacterised disialylgalactose motif. This is also the first example of producing the unique disialylgalactose sialylation on a CHO produced biotherapeutic glycoprotein.