Preformulation Characterization of Griffithsin, a Biopharmaceutical Candidate for HIV Prevention

Abstract

Griffithsin (GRFT) has shown potent anti-HIV activity, and it is being developed as a drug candidate for HIV prevention. Successful implementation requires thorough understanding of its preformulation characterization. In this work, preformulation assessments were conducted to characterize GRFT and identify its degradation pathways under selected conditions of temperature, light, pH, shear, ionic strength, and oxidation. Compatibility with vaginal fluid simulant, vaginal enzymes, Lactobacillus spp., and human cervicovaginal secretions was assessed. The purity, melting temperature, and HIV gp120-binding affinity of GRFT stored at 4°C and 25°C in phosphate-buffered saline (PBS) were assessed for 2 years. Chemical modifications were evaluated by intact mass analysis and peptide sequencing. Excised human ectocervical tissue permeability and localization of GRFT were evaluated. Our results demonstrated GRFT to be safe and stable under all the preformulation assessment conditions studied except oxidative stress. When GRFT was exposed to hydrogen peroxide or human cervicovaginal secretion, methionine 78 in the protein sequence underwent oxidation. GRFT did not permeate through human cervical tissue but adhered to the superficial epithelial tissue. The 2-year stability study revealed no significant change in GRFT’s aggregation, degradation, melting temperature, or gp120-binding affinity despite a slow increase in oxidation over time. These studies elucidated desirable safety and bioactivity profile for GRFT, showing promise as a potential drug candidate for HIV prevention. However, susceptibility to oxidative degradation was identified. Effective protection of GRFT from oxidation is required for further development.

A Highly Potent SARS-CoV-2 Blocking Lectin Protein

“…coli at a scale of tons . In addition, rGRFT is reported to be stable at room temperature in PBS for 2 years without formation of aggregation and degradation byproducts . Considering the board activity of rGRFT on different coronaviruses, the evolutionary cost of glycan loss on SARS-CoV-2, the available high-yield recombinant production strategies for rGRFT, and the good stability of rGRFT at room temperature, rGRFT may provide a possible solution for the emerging variants.…”

A Highly Potent SARS-CoV-2 Blocking Lectin Protein

“…48 In addition, rGRFT is reported to be stable at room temperature in PBS for 2 years without formation of aggregation and degradation byproducts. 49 Considering the board activity of rGRFT on different coronaviruses, evolutionary cost of glycan loss on SARS-CoV-2, available high-yield recombinant production strategies for rGRFT and good stability of rGRFT at room temperature, rGRFT may provide a possible solution for emerging variants.…”

Natural Products with Inhibitory Activity against Human Immunodeficiency Virus Type 1

“…In addition, GRFT synergizes with the HIV-1 drugs tenofovir, maraviroc, and enfuvirtide, which could be used for microbicide development [ 162 ]. Kramzer et al performed in vitro characterization of a preformulation of this compound and found physical, chemical, and biological stability profiles under stress conditions, except oxidative stress [ 163 ]. GRFT, being a lectin, is prone to oxidation; to overcome this property, an oxidation-resistant form of GRFT (Q-GRFT) has been developed and evaluated in rhesus macaque rectal tissue samples showing a nontoxic effect [ 164 ].…”