Abstract
Plants provide an advantageous expression platform for biopharmaceutical proteins because of their low pathogen burden and potential for inexpensive, large-scale production. However, the purification of target proteins can be challenging due to issues with extraction, the removal of host cell proteins (HCPs), and low expression levels. The heat treatment of crude extracts can reduce the quantity of HCPs by precipitation thus increasing the purity of the target protein and streamlining downstream purification. In the overall context of downstream process (DSP) development for plant-derived malaria vaccine candidates, we applied a design-of-experiments approach to enhance HCP precipitation from Nicotiana benthamiana extracts generated after transient expression, using temperatures in the 20–80°C range, pH values of 3.0–8.0 and incubation times of 0–60 min. We also investigated the recovery of two protein-based malaria vaccine candidates under these conditions and determined their stability in the heat-treated extract while it was maintained at room temperature for 24 h. The heat precipitation of HCPs was also carried out by blanching intact plants in water or buffer prior to extraction in a blender. Our data show that all the heat precipitation methods reduced the amount of HCP in the crude plant extracts by more than 80%, simplifying the subsequent DSP steps. Furthermore, when the heat treatment was performed at 80°C rather than 65°C, both malaria vaccine candidates were more stable after extraction and the recovery of both proteins increased by more than 30%.
Highlights
Plants have been used to produce biopharmaceutical proteins for more than 25 years (Hiatt et al, 1989)
The establishment of plantbased systems as a common production platform for vaccines is still hampered by issues with yields, downstream processing (DSP) efficiency and regulatory compliance (Fischer et al, 2013). In this design of experiments (DoE) study we describe the optimization of DSP for a new pre-erythrocytic malaria vaccine candidate fusion protein (C9S) and a transmission-blocking vaccine candidate (FQS)
The amount of target protein per gram biomass was used as the response and normalized to the amount of target protein measured after heat precipitation at 80◦C, with an incubation time of 5 min and a storage time of 0 h to compensate for batch-to-batch variations
Summary
Plants have been used to produce biopharmaceutical proteins for more than 25 years (Hiatt et al, 1989). The recombinant protein expression levels that can be achieved in plants were initially low, but have reached the gram product per kilogram biomass range (Bendandi et al, 2010; Buyel, 2015). Combined with these improvements in USP, the often challenging purification of target proteins from plant extracts (Buyel and Fischer, 2014d) means that DSP can account for up to 80% of the total production costs (Wilken and Nikolov, 2012). Process-based purification approaches solely exploiting the different intrinsic properties of the products and HCPs are more appealing and should offer a higher success rate
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