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.

Keywords: GRFT; HIV prevention; griffithsin; oxidation; preformulation.

Fig. 1

GRFT thermal stability. GRFT (450 μg/mL) in Milli-Q water was exposed to 5 °C, 25 °C/60%RH, 30 °C/65%RH, 40 °C/75%RH, and 65 °C in controlled temperature/humidity Carson 6010 environmental chambers for 28 days. Relative humidity (RH) control at 5 °C and 65 °C was not available. GRFT concentration was monitored via HPLC with fluorescence detection. The inset is included to better display the thermal stability trends at the four lower temperature conditions

Fig. 2

UV scans to determine the effect of pH and ionic strength on GRFT (250 μg/mL). GRFT solutions in water, pH 4, pH 7, and pH 10 buffers were analyzed by UV spectroscopy to determine the formation of insoluble aggregates. a GRFT solutions prepared in low ionic strength buffers (50 mOsm/kg). b GRFT solutions prepared in high ionic strength buffers (500 mOsm/kg). c Absorbance values at 280, 320, and 350 nm. The trend lines largely overlap due to the similar absorbance of the different samples at each pH

Fig. 3

GRFT stability in pH-buffered solutions. GRFT (450 μg/mL) stability in buffered solutions over the pH range of 3 to 9 was monitored via HPLC detection. The inset is included to better display the pH stability trends

Fig. 4

GRFT stability in a vaginal fluid simulant (VFS) (with an inset) and b enzymes commonly present in human cervicovaginal lavage (CVL) (with an inset). GRFT (450 μg/mL) in VFS and in each of the three enzymes (aminopeptidase 100 U/mL, lysozyme 100,000 U/mL, and proteinase K 100 U/mL) was monitored at 37 °C for 4 days and 6 h, respectively. GRFT concentration was monitored via HPLC fluorescence detection

Fig. 5

Cervical tissue microscopy of a GRFT-AlexaFluor488 and b unlabeled GRFT, and GRFT compatibility with lactobacilli (c). Representative H&E (first column) and fluorescence (FITC and DAPI filter overlay; second column) micrographs (a) or Antibody/DAB staining (second column) micrographs (b) are shown. GRFT tissue localization is represented by green fluorescence, as indicated by the white arrows (a) or by reddish-brown coloring, as indicated by the black arrow (b). Micrographs were taken with a × 20 objective for both a and b

Fig. 6

Oxidation of GRFT after exposure to 0.02% hydrogen peroxide (H2O2) and CVL. a Representative chromatograms and b–c representative intact masses observed after the exposure. HPLC chromatograms of GRFT exposed to H2O2 and human CVL (separately) are overlaid in order to visually compare the chromatographic similarity (a). The bottom (black line) chromatogram represents GRFT in 0.02% H2O2 at 5 h. The upper (blue line) chromatogram represents GRFT in human CVL at 2.5 h of exposure. Peak A is GRFT. Peaks B and C are oxidative products of GRFT. The extra peaks in CVL were also present in the CVL blank; therefore, they were not GRFT related. Mass spectrometric analysis of the H2O2 peak B identifies three species with intact masses corresponding to the heterodimer (b) and two monomers (c), respectively

Fig. 7

Representative SEC-HPLC chromatogram for long-term stability. GRFT was stored at 4 °C and 25 °C (RT) and compared to the reference standard. The 4 °C and 25 °C profiles overlap with each other, and they each overlap with the reference standard. The overlaid peaks represent the GRFT dimer mass of approximately 25 kDa