Bulk production of the antiviral lectin griffithsin

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

Competing technologies and potential economic impacts of PMPs in resource-poor settings GRFT's use as an HIV microbicide sets a target demographic of individuals who are at high risk of HIV exposure with many of those individuals in resource-poor settings. Therefore, in our analysis of bulk GRFT production, cost is paramount. Plantproduction systems have been touted as being economically advantageous as compared to more traditional cell culture production systems. Reasons for this include low upstream costs, ease of scalability and the lack of adventitious animal pathogens. Figure 2 GRFT Stability Profile. GRFT remains stable after 3 months stored at room temperature (RT) or 4°C. (a) SEC-HPLC. SEC was performed on a Beckman Coulter System Gold HPLC. An aliquot of 10 lL of the final formulation GRFT (4°C, RT and reference standard; 10 mg/mL) was applied, to an SEC column (TSKgel SuperSW3000, 4.6 mm I.D. 9 30 cm, 4 lm; TOSOH Biosciences, LLC; TSKgel guardcolumn SuperSW, 4.6 mm I.D. 9 30 cm, 4 lm; TOSOH Biosciences, LLC) equilibrated in running buffer (100 mM sodium phosphate [pH 7.2], 150 mM sodium chloride, 0.05% sodium azide). After injection, running buffer was applied to the column at flow rate of 0.2 mL/min for 30 min. The inset is a zoomed in picture of the GRFT peak. The large peak shows the dimer formation and purity of GRFT. GRFT is >99% pure and with no aggregation after 3 months. (b) gp120 ELISA. The 50% effective concentration (EC 50 ) for GRFT to gp120 was determined employing a gp120 ELISA as previously described (O'Keefe et al., 2009) . The log (agonist) vs. response -Variable slope (four parameters) was used to fit the curve and calculate EC 50 values in GraphPad Prism 5.0 software. The EC 50 values were determined to be 8.7 ng/mL, 10.1 ng/mL and 10.9 ng/mL for GRFT stored at 4°C, GRFT stored at RT and reference standard GRFT, respectively. (c) SPR. The equilibrium dissociation constant (K d ) of GRFT to gp120 was measured using a Biacore X100 2.0 instrument at ambient temperature. For each sample (4°C, RT and reference standard), the assay was performed in triplicate. A representative sensorgram is shown for the reference standard, which was sequestered from the initial GRFT production. Recombinant biotinylated gp120 (Du151, HIV1/Clade C, Immune-tech # IT-001-139p-Biotin) at a concentration of 1 lg/mL was immobilized on a SA sensor chip (Biacore #BR-1000-32) in 10 mM sodium acetate pH 5.5 with a flow rate of 5 lL/min and a contact time of 150 s. Serial dilutions of GRFT (125, 62.5, 31.3, 15.6, and 7.8 nM) were injected, at a flow rate of 5 lL/min, for a contact time of 60 s and a dissociation time of 600 s. Each set of data was analysed using the steady state affinity analysis (Inset) in the Biacore X100 2.0 evaluation software. The K d values (average AE SD, n = 3) were determined to be 32.2 AE 1.6 nM, 33.0 AE 1.3 nM and 31.9 AE 1.8 nM for GRFT stored at 4°C, GRFT stored at RT and reference standard GRFT, respectively. (d) DSF. The melting temperature (T m ) of GRFT, at a final concentration of 62.5 lM in PBS, was determined on a BioRad iQ5 multicolour real-time PCR system as previously described (Hamorsky et al., 2013) . The T m values (average AE SD, n = 3) were determined to be 74.2 AE 3.9°C, 76.8 AE 0.9°C and 77.5 AE 0.8°C for GRFT stored at 4°C, GRFT stored at RT and reference standard GRFT, respectively. Economic advantage, however, has not yet been proven in practice or in published studies to any appreciable exte