title: Large scale purification process for recombinant NS1-OspA as a candidate vaccine for Lyme disease.
abstract: A purification process was developed for the manufacture of NS1-OspA as a candidate vaccine for Lyme disease. NS1-OspA was expressed as a soluble recombinant protein in E. coli from 50 and 200 liter fermentations. A multistep bench scale procedure, including detergent treatment, sonication, Q-Sepharose chromatography, dialysis, and SP-Sepharose chromatography, was used to effectively purify NS1-OspA from E. coli to > 90% purity with a 35% yield. This procedure was subsequently modified and specific steps eliminated to create a manufacturing scale process for the purification of NS1-OspA. Continuous flow centrifugation, mechanical lysis, QA-52 anion exchange chromatography, acid induced precipitation, and cross flow filtration gave a final purity of > 75%, a recovery of > 70%, and measured endotoxin levels of < 5 micrograms/mg of protein. Unique features of this process include using unclarified lysates and a single use (disposable) resin for the batch chromatography step. This simple, cost effective production process provides a recombinant protein with the yield and purity suitable for use as a subunit veterinary vaccine.
authors.affiliation: BioProcess Sciences, Pfizer Central Research, Lincoln, NE 68501, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Caldwell
authors.forename: J
authors.initials: J
authors.lastname: Varghese
authors.forename: N K
authors.initials: NK
authors.lastname: Puri
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001428:Bacterial Vaccines / Q000191:economics; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D005374:Filtration; D008074:Lipoproteins; D008193:Lyme Disease / Q000517:prevention & control*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*; D013678:Technology, Pharmaceutical / Q000191:economics / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; C085820:INS1 protein, influenza virus; D008074:Lipoproteins; C072045:OspA protein; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins