Production of immunotoxin D2C7—(scdsFv)—PE38KDEL
D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a Phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE31). D2C7-IT was produced by a 10 L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein.
1. A method of producing clinical grade D2C7(scdsFv)-PE38KDEL immunotoxin, comprising the steps of:
a. culturing an E. coli BLR (λ, DE3) lambda lysogen comprising a plasmid encoding said immunotoxin in a fermentor to produce a bacterial cell paste, the expression of said immunotoxin was under the control of the T7 promoter and induced with Isopropyl β-D-1-thiogalactopyranoside;
b. lysing bacteria of the bacterial cell paste in a buffer comprising MgSO 4 , DNaseI, and lysozyme;
c. collecting inclusion bodies from the lysed bacteria;
d. solubilizing the inclusion bodies and reducing proteins of the solubilized inclusion bodies to form reduced proteins of the inclusion bodies;
e. refolding the reduced proteins of the inclusion bodies in the presence of a protease inhibitor to form single-chain disulfide stabilized immunotoxin;
f. purifying the single-chain disulfide stabilized immunotoxin to remove the protease inhibitor and endotoxin; to form purified, single-chain disulfide stabilized immunotoxin; and
g. storing the purified single-chain disulfide stabilized immunotoxin at a temperature of −70° to −90° C.
2. The method of claim 1 wherein the E. coli lambda lysogen is made by transforming competent cells of an E. coli lambda lysogen with a plasmid encoding said immunotoxin, and recovering a transformant.
3. The method of claim 1 wherein the step of purifying comprises anion-exchange chromatography.
4. The method of claim 1 wherein the step of purifying comprises size exclusion chromatography.
5. The method of claim 1 wherein the step of purifying comprises anion-exchange chromatography and size exclusion chromatography.
6. The method of claim 1 wherein intermediate purification products are stored at −70° to −90° C.