Extraction and purification of malaria vaccine candidate CLCT produced by transient expression in Nicotiana benthamiana plants

Plasmodium falciparum is a malaria parasite that infects more than 220 million people every year. Various non-vaccine campaigns have failed to eradicate malaria and the World Health Organization has recommended only a single malaria vaccine, but this has mediocre efficacy. Additional efforts are therefore required to design and produce medicines that trigger a highly protective immune response. Importantly, the production of such medicines must be cost-efficient to ensure availability in developing countries of the global south, especially sub-Saharan Africa, where they are most needed. Here, we report the development of downstream processing for the novel malaria vaccine candidate CLCT. This fusion protein combines four P. falciparum antigens and was produced by transient expression in Nicotiana benthamiana plants using Rhizobium radiobacter (formerly Agrobacterium tumefaciens). The protein accumulated to levels of 118.8 mg kg− 1 and the blanching of plant biomass enabled the efficient precipitation of > 90% of host cell proteins, thereby achieving a ~ 25-fold increase in the purity of the heat-stable target protein in the homogenate. Plant extracts were clarified by a series of bag, depth and sterile filtration steps with an overall recovery of 36.6%. An ultrafiltration/diafiltration step was then used for buffer exchange and to concentrate the product 2.6-fold, with a step recovery of ~ 65%. We optimized a hydrophobic interaction chromatography capture step, achieving a CLCT recovery of ~ 40%. The overall process yield of purified CLCT was ~ 12 mg kg− 1 biomass with a final purity of ~ 83%, which is sufficient for initial activity testing in preclinical animal model immunization studies.