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Downstream processing of a plant-derived malaria transmission-blocking vaccine candidate

: Menzel, S.; Holland, T.; Boes, A.; Spiegel, H.; Fischer, R.; Buyel, J.F.

Fulltext ()

Protein expression and purification 152 (2018), pp.122-130
ISSN: 1046-5928
Journal Article, Electronic Publication
Fraunhofer IME ()

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30–75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg−1 plant material after purification based on an initial yield of ∼49 mg kg−1 biomass after blanching.