Stability of plasmid and viral banks supporting the cGMP manufacture of Q-Griffithsin from a TMV-based viral vector

Abstract

The "whole genome" TMV-based expression system, Geneware®, was used in the cGMP production of the plant-made pharmaceutical Q-Griffithsin and demonstrates stable expression for up to a two-year period. Virion and plasmid banks which contained viral cDNA and a Q-Griffithsin sequence were able to produce >200 g of Q-Griffithsin. Data assessing the quality and stability of the product banks were measured through functional assessments of visual symptomology and product expression.

Keywords: Microbicide; Plant-made pharmaceuticals; Q-Griffithsin; Stability; TMV; Virion.

Figure 1. Qualification and Stability of Virion and Plasmid Banks.

A. Figure A describes the qualification and stability testing process for both virion and plasmid banks. Dots indicate tests performed under various time points. The MPB was double strand sequenced in its entirety (>10kb) at time point zero. After the initial sequencing, only the Q-GRFT insert (366 bp) was sequence verified (at time points two, eight, twelve, and twenty-four months); and transcribed into RNA (at time points two, twelve, and twenty-four months). Expression levels of Q-GRFT from plasmid transcripts were measured at all time points using densitometry analyzed SDS-PAGE of clarified plant extracts. The virion qualification process differed in that there was no need to sequence or transcribe, and only a functional assessment of expression was performed to evaluate the quality of the virion. B. Figure B displays the quantified results of the functional assessment of Q-GRFT expression performed on transcripts prepared from the MPB. Dots represent four samples taken from plants at each time point transcribing and expressing Q-GRFT from the same MPB. Q-GRFT expression levels (mg/kg) were quantified via SDS-PAGE using densitometry on clarified plant extracts. The average expression from plasmid at 0, 2, 12, and 24-month data points was; 804.1 ± 44.9 mg/kg, 1142.078 ± 73.7 mg/kg, 526.08 ± 25.4 mg/kg, 697.2 ± 41.96 mg/kg, respectively. Q-GRFT expression was analyzed by a repeated measures one-way ANOVA (p < 0.0001) with Bonferroni’s post-hoc tests comparing results to time 0. The MBP expressed Q-GRFT with significant variation from time 0 at all time points tested, but all were above the 200 mg/kg target. * p < 0.05, **p < 0.01. C. Figure C displays the quantified results of the functional assessment of Q-GRFT expression performed on virions. Dots represent four samples taken from plants at each time point expressing Q-GRFT from the same MVB. Q-GRFT expression levels (mg/kg) were quantified via SDS-PAGE using densitometry on clarified plant extracts. The average expression from virion at 0, 2, 8, 12, and 24-month data points was; 672.1± 54.8, 796.4 ± 49.9, 697.98 ± 29.03, 723.9 ± 67.4, 626.4 ± 48.4, respectively. Q-GRFT expression was analyzed by a repeated measures one-way ANOVA (p = 0.0455) with Bonferroni’s post-hoc tests comparing results at each time point to time 0. Q-GRFT expression levels showed a significant difference at the two-month time point. However, all other time points appear to show consistent expression over the two-year observation period and are well above the 200 mg/kg qualification standard. * p < 0.05.