Enhancing Therapeutic Protein Production Extracellular Sialidase Inhibition in CHO cells Stephen Houghton and Christopher N. Boddy Department of Chemistry, Syracuse University Syracuse, NY 13244 Therapeutic Glycoproteins Key Considerations Scheme 2 – sulfone (SmI2 coupling) Important class of pharmaceuticals: 2005 sales Neupogen® Neulasta ® : anaemia, $3.5 B (EPO) erythropoietin: cancer , $2.5 B Procrit®/Eprex®: anaemia, $3.3 B Remicade®: rheumatoid arthritis, $3.5 B Intron® and Peg-Intron®: hepatitis, $1.4 B Cerezyme® : Gaucher’s disease, $1 B C/S-GLYCOSIDE Resist hydrolysis by enzymes Stable RESIN-BOUND Increased hydrophobic interactions – better Ki Not cell permeable – should not interfere with cell function and protein export Readily separable - facilitates protein purification process, low impact on process TYPE OF RESIN PEGA - Water compatible (swells in aqueous media) COST EFFECTIVE < $1/L cell culture Need large quantities of inhibitor (5000-10000-40000 L) bioreactors Use metabolically engineered sialic acid (1g/L) Sialic acid on glycans Expressed Protein EXTRACELLULAR MATRIX CYTOSOL Sialic Acid Important biological molecule: Present on cell surfaces and macromolecules Protein glycosylation terminal residue Molecular recognition, cell signaling Mammals, viruses, some bacteria Cleaved by sialidases Scheme 3 – S-glycoside Resin-Bound C-glycoside inhibitor Sialic Acid Affects Therapeutic Proteins Removal of sialic acid affects: Serum half-life Solubility Protein function Immunogenicity Purification process Dosing: more required more frequently Production cost Incidence of side effects Enzyme Assay Why Design and Inhibitor? PEGA-loaded with sialic acid bound to a linker by a C-glycosidic linkage 4-MU-NeuAc Biotechnology Industry Problem: Extracellular sialidases are released into cell culture fluid by cell lysis Expressed protein becomes desialylated Must carefully monitor sialic acid content Standard inhibitors can increase sialic acid content by 20-30% (DANA) Glycoprotein market: 2003 - $10 billion 2010 – $53 billion (estimated) 50% of new FDA submissions are biologics 100+ therapeutic proteins licensed since 1982 Scheme 1 – Allyl C-glycoside Impact Provide better protein product: Better pharmacokinetic profile More tolerated by patients (less side-effects) Significantly decrease costs: Purification Dosage Acknowledgements: The Boddy Lab, Syracuse University Department of Chemistry