Bulk production of the antiviral lectin griffithsin

Fuqua, Joshua L.; Hamorsky, Krystal; Khalsa, Guruatma; Matoba, Nobuyuki; Palmer, Kenneth E.

Development of GRFT as an HIV topical microbicide requires a production system for making bulk quantities of high-quality GRFT available for formulation and characterization. GRFT has been expressed using multiple recombinant systems including E. coli and plant systems Hahn et al., 2015; O'Keefe et al., 2009; Vafaee et al., 2014) . Expression of GRFT using E. coli systems has been performed with modest success, where cultures grown in shake flasks were able to produce 12 mg of soluble GRFT per litre, but the majority of the protein formed insoluble inclusion bodies. Optimization of E. coli expression systems using fermentors allowed production of >500 mg of GRFT per litre of media . Initial investment in fermentor systems represents a significant upfront manufacturing cost and necessitates removal of bacterial endotoxin. As an algal protein, GRFT is an ideal candidate for plant-based expression. Recently, GRFT was demonstrated to be stably expressible in chloroplasts of Nicotiana tabacum (Vafaee et al., 2014) , but more commonly GRFT has been expressed transiently in Nicotiana benthamiana using Agrobacterium-mediated expression systems or recombinant tobacco mosaic virus (rTMV)-based expression systems (Fuqua et al., 2015; Hahn et al., 2015; O'Keefe et al., 2009; Vafaee et al., 2014) . The largest body of published work on manufacturing-scale GRFT production has been performed using rTMV in N. benthamiana with reported in planta expression levels ranging from of 0.5-1.0 g/kg fresh weight of plant material, and purification methods yielding 30-60% of total expressed GRFT. We recently reported our efforts to optimize the original purification process for bulk manufacturing of GRFT from laminar tissues of N. benthamiana using rTMVbased expression vectors; our new process was able to drastically improve the recovery of GRFT yielding 60-90% of total expressed GRFT (Fuqua et al., 2015; O'Keefe et al., 2009) . The updated purification method was piloted at kilogram quantities of plant biomass to assure a robust and relatively simple production methodology was established for GRFT prior to applying cGMP methods for eventual clinical application (Fuqua et al., 2015) . In combination, the yield, stability and established purification and analytical protocols applied for manufacturing GRFT provide a valuable example for understanding the complexities and challenges of PMPs at industrial scale, towards developing a plantmade HIV microbicide.