A Novel Process for the Large-Scale Purification of Recombinant Tick Anticoagulant Peptide Using Perfusion Chromatography

Abstract

Tick anticoagulant peptide (TAP) is a 60 amino acid peptide (Mr=6977, pI=4.9) found in the saliva of the soft tick Ornithodorous moubata that specifically inhibits blood coagulation factor Xa (fXa). A recombinant form of TAP (rTAP) secreted by Saccharomyces cerevisiae was purified from 200 liters of fermentation broth with POROS™, strong cation exchange (SCX) and reversed-phase high performance liquid perfusion chromatography (RP-HPLC) media (20 μm nominal particle diameter). The higher linear flow rates and dynamic capacities, as well as low backpressures, obtained with perfusion chromatography media permitted 37.5 g of rTAP to be efficiently captured and significantly enriched from 400 liters of diafiltered fermentation broth (24.3 g yield) with 1.25 liter of SCX media using a low pressure column and peristaltic pump in 4.5 hours. Subsequently, 16.7 g of rTAP obtained from the capture step was purified in a high-resolution mode with the same SCX media, after the media was cleaned and repacked into an 800 ml high-pressure column. By doing multiple rapid cycles at high linear flow rates, preparative-scale high-resolution purification of multi-gram amounts of the peptide was done on this relatively small column in only 10.5 hours. Finally, the peptide was desalted and decolorized on a 200 ml RP-HPLC column of perfusion chromatography media by doing multiple rapid cycles. After lyophilization, 12 g of peptide (46.9% yield) was obtained that was >96% homogeneous by several analytical criteria and fully active in inhibiting blood coagulation factor Xa (fXa). The process reduced the purification time one-half, increased the yield from 32% to 47%, and eliminated the need for cold-chain isolation facilities.