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Prokaryotic production systems have been widely used to manufacture recombinant therapeutic proteins. Economically, the prokaryotic production - especially of small therapeutic molecules - is advantageous compared to eukaryotic production strategies. However, due to the potential endotoxin and host cell protein contamination, the requirements for the purification process are disproportionately higher and therefore more expensive and elaborate to circumvent. For this reason, the goal of this work was to develop and establish a rapid, simple, inexpensive and 'up-scalable' production and purification process, using the therapeutic relevant protein anti-EGFR scFv hu225 as model molecule. Configuring high cell density cultivation of Escherichia coli - using the rha-BAD expression system as production platform - a specific product concentration up to 20 mgscFv/gCDW was obtained. By combining freeze-and-thaw, osmotic shock and pH induced host cell protein precipitation, almost 70% of the product was extracted from the biomass. In a novel approach a mixed mode chromatography was implemented as a capturing and desalting step, which allowed the direct application of further ion exchange chromatography steps for purification up to pharmaceutical grade. Thereby, 50% of the produced scFv could be purified within 10 h while maintaining the biological activity.
Single chain fragment variable; Mixed mode chromatography; Ion exchange chromatography; Periplasmic disruption
...Protein-L resin (GenScript USA Inc., Piscataway, NJ) was packed in Omnifit columns (6 mm x 20 mm, Sigma-Aldrich, St. Louis, MO) at pH7 (150 mM NaCl; 20 mM phosphate buffer). The scFv was be eluted upon pH shift to pH 2.5 (100 mM phosphate-citrate buffer)...
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