Improving the large scale purification of the HIV microbicide, griffithsin

Abstract

Background: Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery.

Results: We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained.

Conclusions: The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.

Figure 1

Removal of plant protein contaminants from GRFT Extract with Heat. Plants expressing GRFT through a TMV-based expression system were harvested at pH4 in sodium acetate buffer and the extract was divided into 7 aliquots. Each aliquot was incubated for 15 minutes at temperatures ranging from room temperature (24°C) to 90°C and centrifuged to remove any precipitant. Samples of the initial extract and all assessed temperatures were analyzed by SDS-PAGE with coomassie staining. The initial extract contains multiple contaminating proteins, which remain after incubation at room temperature. Once the extracts are heated to at least 50°C the majority of contaminating proteins precipitate except for the target protein GRFT and the TMV coat protein. The composition of the extract remains similar with temperature treatments ranging from 50 – 80°C, but at 90°C incubation nearly all proteins are precipitated. Table: Densitometry readings corresponding to SDS-PAGE shown in Figure 1.

Figure 2

Effects of pH on the activity of MgCl ₂ and bentonite to remove the TMV coat protein. Extracts were harvested in sodium acetate (pH 4–5) or tris (pH7-9) buffer depending on the desired pH range and were visualized using coomassie stained SDS-PAGE. Sample 1 shows the proteins in the initial extract and sample 2 represents the proteins after the extract is heated to 55°C and centrifuged. Sample 3 represents the extract after it was adjusted to its corresponding pH a) pH 4.0 and b) pH 9.0. Sample 3 extracts were centrifuged to remove additional precipitate and represented in sample 4. Samples 5–12 represent various temperature, bentonite, or MgCl₂ treatments of sample 4 performed in duplicate. MgCl₂ (M), bentonite (B), and bentonite & MgCl₂ (MB) Samples 5 & 6 were incubated overnight at 42°C. Samples 7 & 8 were incubated overnight at 42°C after treatment with 0.01 M MgCl₂. Samples 9 & 10 were incubated overnight at 4°C with the addition of 1 mg/mL bentonite. Samples 11 & 12 were incubated overnight at 4°C after the addition of 1 mg/mL bentonite & 0.01 M MgCl₂. Table: Densitometry readings of SDS-PAGE represented in Figure 2 with values from three additional gels representing additional pH values that were explored (pH 6.5, 7.0, 8.0). The shaded area represents an extract condition achieving greater than 80% purity of GRFT.

Figure 3

Effects of MgCl ₂ (0, 0.001 M, 0.01 M, 0.1 M) on the protein content of GRFT extracts treated with bentonite. Coomassie stained SDS-PAGE was used to see gross changes in purity of the GRFT extracts in both a and b. The initial GRFT extract is represented in sample 1 and subsequent heat & centrifugation step at sample 2. The supernatant was treated with multiple MgCl₂ concentrations (samples 5–12) in duplicate with the corresponding bentonite concentration 0 mg/mL bentonite (a) or 10 mg/mL bentonite (b). The resulting supernatants of overnight treatment with MgCl₂ and/or bentonite are as follows; (3&4) 0 MgCl₂ & 0 bentonite, (5&6) 0 MgCl₂, (7&8) 0.001 M MgCl₂, (9&10) 0.01 M MgCl₂, (11&12) 0.1 M MgCl₂. Samples 11 and 12 both show a dramatic reduction in coat protein after treatment with 0.1 M MgCl₂ irrespective of the bentonite concentration. Table: Densitometry readings of SDS-PAGE represented in a and b including two additional bentonite concentrations not shown. The shaded area represents an extract condition achieving greater than 80% purity of GRFT.

Figure 4

Summary of the effects of 0.1 M MgCl ₂ and 10 mg/mL bentonite on GRFT extract purity. a) Coomassie stained SDS-PAGE showing the initial extraction of GRFT (1), subsequent pH adjustment to pH4 (2) and heating to 55°C followed by centrifugation (3). The resulting supernatant was untreated (control) or treated with MgCl₂ and/or bentonite and stirred overnight at 4°C. The following samples are the resulting supernatants; (4) Control −4°C, (5) 0.1 M MgCl₂, (6) 10 mg/mL Bentonite, (7) 0.1 M MgCl₂ & 10 mg/mL Bentonite . The following samples are the resulting pellets after treatment; (8) Pellet - 4°C, (9) Pellet - 0.1 M MgCl₂, (10) Pellet - 10 mg/mL Bentonite, (11) Pellet - 0.1 M MgCl₂ & 10 mg/mL Bentonite. b) Comparison of gp-120 binding GRFT concentrations relative to the control sample (4). Determined through a gp-120 binding ELISA and represented as a percentage of functional GRFT in the control extract. c) Comparison of the TMV coat protein concentration relative to the control extract. Determined through densitometry measures of TMV specific westerns and represented as a percentage of TMV in the control extract. b and c both analyzed by a one-way ANOVA with Bonferroni’s multiple comparison test comparing all groups. * represents a post-hoc test with a p-value < 0.05. Table: Densitometry readings of SDS-PAGE represented in a. The shaded area represents an extract condition achieving greater than 80% purity of GRFT.

Figure 5

Purity and TMV CP content of GRFT extracts after Capto ™ MMC purification. SDS –PAGE (a) and TMV western blots (b) were used to visualize purity and TMV contamination. The initial extract (1) contains both GRFT and TMV CP as well as other plant protein contaminants. Lanes 2–4 were loaded with 15 μg of chromatography purified protein from the extracts that had previously been heated and treated with: control – no treatment (2), 0.1 M MgCl₂ (3), 0.1 M MgCl₂ & 10 mg/mL bentonite (4). Included for comparison was 15 μg of GRFT purified by the previously published methodology O’Keefe et al., [4] (5). Above each lane is the densitometry determined GRFT purity.

Figure 6

Analysis of industrial pilot scale purification of GRFT. Representation of the purity, by SDS –PAGE a, and TMV CP content, by western blot b, of GRFT during industrial pilot scale purification. Sample volumes loaded on the gel were normalized to the initial extraction volume. The initial extract (1) contains both GRFT and TMV CP as well as other plant protein contaminants. The extract was pH adjusted to pH4 (2)heated (3) and filter pressed(4). After which the extract was incubated overnight with MgCl₂ and bentonite and simultaneousely filter pressed and sterile filtered (5). Samples 6–7 are the GRFT final product At 1x volume, 2xvolume and 15 μg of protein. Included for comparison was 1.5 μg and 3.0 μg of GRFT purified by the previously published methodology O’Keefe et al., [4] (5). The table represents the densitometric measures of GRFT and TMV protein content in the extracts.