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.
MeSH terms
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Animals
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Antigens, Surface / genetics
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Antigens, Surface / immunology
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Antigens, Surface / isolation & purification*
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Bacterial Outer Membrane Proteins / genetics
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Bacterial Outer Membrane Proteins / immunology
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Bacterial Outer Membrane Proteins / isolation & purification*
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Bacterial Vaccines* / economics
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Cell Fractionation / methods*
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Centrifugation
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Chemical Precipitation
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Chromatography, Gel
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Chromatography, High Pressure Liquid
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Chromatography, Ion Exchange / methods
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Dogs
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Fermentation
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Filtration
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Lipoproteins*
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Lyme Disease / prevention & control*
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / isolation & purification*
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Technology, Pharmaceutical / economics
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Technology, Pharmaceutical / methods*
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / immunology
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Viral Nonstructural Proteins / isolation & purification*
Substances
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Antigens, Surface
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Bacterial Outer Membrane Proteins
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Bacterial Vaccines
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INS1 protein, influenza virus
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Lipoproteins
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OspA protein
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Recombinant Fusion Proteins
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Viral Nonstructural Proteins