Preparation of clinical grade proteins produced by recombinant DNA technologies.

Methods were developed for the production of clinical grade malaria vaccine candidates expressed in E. coli by recombinant DNA technologies. The essential features of the purification protocol consist of (1) mechanical breakage of host cells and solubilization of the recombinant proteins in 6 M guanidine hydrochloride; (2) ammonium sulfate fractionation; (3) affinity chromatography on a Ni(2+)-chelate gel in the presence of 6 M guanidine hydrochloride; and (4) ion exchange chromatograph on a Phospho Ultrogel column in the presence of 6 M urea. The use of undesirable chemicals (PMSF, DFP, TFA, acetonitrile, etc.) was avoided rather than demonstrating their complete removal after the purification steps. Testing of chromatographic fractions for host-cell proteins and the elimination of fractions with E. coli protein content was found necessary to obtain a final product that contained less than 0.01% of host derived proteins. The recombinant proteins were renatured either from 8 M urea or from 6 M guanidine hydrochloride by increasing the pH to 10.5 in the presence of glycine and EDTA, reduction with DTT, dilution to a protein concentration below 1 mg.ml-1, and dialysis against 0.9% NaCl. The method presented here can be tailor-fit, with minor modification, for the purification of almost any recombinant protein and the final product satisfies current regulations concerning the production of clinically acceptable therapeutic products.