Downstream processing of alkaloids from whole-cell biotransformation: The application of membrane filtration and solvent extraction

Biosynthesis of alkaloid pharmaceuticals has been widely studied but with a focus on increasing the yield and productivity within the upstream bioreactor. The recovery and purification of the product alkaloid, however, has not largely been investigated. This study examines the potential application of ultrafiltration, nanofiltration and liquid-liquid extraction in the downstream processing of a clarified yeast fermentation broth containing the natural benzylisoquinoline alkaloid oripavine and the de-methylated product, nororipavine, a precursor that can be used to synthesize the FDA-approved medicine buprenorphine. A coupled liquid-liquid extraction (with toluene and isoamyl alcohol at 60:40 ( v /v) as extractant) followed by a nanofiltration step proved the most promising of the five processes assessed, achieving a concentration factor of ~3 in each step. This coupled process concentrated nororipavine from ~3.5 g/L to 32 g/L while removing impurities from the fermentation broth. The concentrated stream generated by this process was directly used in a downstream chemical reaction to produce 3,17-dibenzylnororipavine, a key intermediate in buprenorphine manufacture, with a yield of 93% and purity of 92%. This study provides useful insights into the processing of benzylisoquinoline alkaloids downstream of fermentation, which is pivotal for their industrial production. • Options for processing nororipavine following biotransformation in yeast were examined. • The recovery and concentration of nororipavine by solvent extraction and membrane filtration was assessed. • Liquid-liquid extraction coupled with nanofiltration was the most effective process. • A highly concentrated (~ 32 g/L) nororipavine stream was obtained. • Chemical benzylation produced dibenzylnororipavine with 93% yield and 92% purity, a key step in buprenorphine manufacture.