1. 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 ( 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:
$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