title: 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.
authors.affiliation: Owensboro Cancer Research Program, 1020 Breckenridge St., Suite 201, Owensboro, KY, 42303, USA. j.fuqua@louisville.edu.|University of Louisville School of Medicine, James Graham Brown Cancer Center, 529 S Jackson Street, Louisville, KY, 40202, USA. j.fuqua@louisville.edu.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Fuqua
authors.affiliation: Institute for Health Metrics and Evaluation, University of Washington, 2301 5th Ave, Suite 600, Seattle, WA, USA. wangav@uw.edu.
authors.forename: Valentine
authors.initials: V
authors.lastname: Wanga
authors.affiliation: Owensboro Cancer Research Program, 1020 Breckenridge St., Suite 201, Owensboro, KY, 42303, USA. kenneth.palmer@louisville.edu.|University of Louisville School of Medicine, James Graham Brown Cancer Center, 529 S Jackson Street, Louisville, KY, 40202, USA. kenneth.palmer@louisville.edu.
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Palmer
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001546:Bentonite; D001709:Biotechnology; D015636:Magnesium Chloride; D037121:Plant Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; D037121:Plant Lectins; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D001546:Bentonite; C499654:griffithsin protein, Griffithsia