Bulk production of the antiviral lectin griffithsin

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

ELISA to monitor the physical (degradation and aggregation) and functional stability (i.e. gp120-binding affinity), respectively. To be a viable microbicide product, GRFT must have a shelf life of at least 2 years. Therefore, we have designed a 2-year stability program (Table 3) to monitor the chemical, physical, thermal and functional stability of GRFT. Specifically, oxidation, aggregation, degradation, melting temperature and potency of GRFT will be evaluated. Currently, we have completed 3 months of stability including GRFT API stored at room temperature and 4°C. There was no detectable change in any measured aspect of the GRFT API. Figure 2 highlights some of the data generated at the 3 month stability time point. A SEC-HPLC method has been developed as a standard operating procedure (SOP) for the product release of GRFT as well as to monitor the physical stability. This SOP is being transferred to Kentucky Bioprocessing, Inc., a facility capable of large-scale cGMP production of GRFT, with the assay criteria that the GRFT native dimer must show >95% purity and the retention time of the dimer must be within 95% confidence interval of reference standard GRFT from three independent runs. SEC-HPLC is a dynamic method to show the purity of GRFT API as well as to monitor the aggregation and degradation of GRFT. Figure 2a shows that GRFT is >95% pure, and after 3 months of shelf life stability assessment, there is no detectible aggregation or degradation of GRFT. Another analytical assay we have developed for GRFT product release and to monitor stability is a potency validation assay, which will ensure the anti-HIV activity of GRFT. Specifically, GRFT's binding to a recombinant HIV-1 envelope glycoprotein gp120 (which is solely responsible for GRFT anti-HIV activity) will be verified by gp120 ELISA. The data in Figure 2b demonstrate that GRFT is functionally active, binding to gp120 in the nanomolar range. After 3 months, GRFT has not lost potency.