Design of resin-bound C/S-glycoside sialidase inhibitors for use in therapeutic recombinant glycoprotein production

CARB 74

Stephen R. Houghton, srhought@syr.edu and CNC. Boddy, cnboddy@syr.edu. Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, NY 13244-4100
Maintaining high sialic acid content on glycoproteins intended for therapeutic uses is important for the biotechnology industry because desialyated glycoproteins exhibit reduced activity, stability, serum half-life, and immunogenicity. In addition, variably sialyated glycoproteins complicate purification and formulation of the glycoprotein product. During recombinant protein expression in Chinese Hamster Ovary (CHO) cells, extracellular sialidases are responsible for cleaving the O-glycosidic bond between sialic acid and other sugars. This enzymatic cleavage can be prevented by preparation of carbon or sulfur glycoside competitive inhibitors. We aim to synthesize and screen a library of carbon and sulfur glycosidic sialidase inhibitors bound to a water compatible resin such as PEGA. Hydrophobic interactions of the resin should increase binding affinity to the sialidase. Attachment of a resin will simplify removal of the inhibitor from the glycoprotein product, prevent uptake into the cell during protein expression, and allow for possible multivalent interactions.

Existing inhibitors, commonly used in treatment of influenza, are not cost effective for large scale operations, therefore we will utilize inexpensive metabolically engineered sialic acid as starting material to synthesize resin bound glycosides. Ki values for inhibitors will be determined for bacterial sialidases in vitro followed by CHO cell sialidases for evaluation of application in CHO cell protein expression cultures. Implementation of this on an industrial scale could significantly decrease cost of production as well as improve the protein product giving pharmacokinetic profile with reduced side effects.

Currently, we have synthesized two heterobifunctional PEG linkers in 9 steps, prepared for attachment of sialic acid to PEGA. A resin bound negative control for each linker length has also been prepared, as well as the initial steps for C and S-glycoside inhibitors. Another literature preparation for an irreversible inhibitor is in process. New methodology for preparation of an allyl C-glycoside of sialic acid is also being investigated.

 

General Posters
6:00 PM-8:00 PM, Tuesday, August 21, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Carbohydrate Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007