US 2020/0046847 A1 · Production of Immunotoxin D2C7 - (scdsFv) -PE38KDEL

Abstract

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 DE3). 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.

Claims10

Dependent claims

A method of producing clinical grade D2C7(scdsFv)-PE38KDEL immunotoxin, comprising the steps of:

a. culturing an E. coli lambda lysogen comprising a plasmid encoding said immunotoxin in a fermentor to produce a bacterial cell paste;
b. lysing bacteria of the bacterial cell paste in a buffer comprising MgSO4, 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;
g. storing the purified single-chain disulfide stabilized immunotoxin at a temperature of −70° to −90° C.

5 more elements

Dependencies are inferred from the claim text.

Production of Immunotoxin D2C7 - (scdsFv) -PE38KDEL

Life of this patent

Production of Immunotoxin D2C7 - (scdsFv) -PE38KDEL

Life of this patent

Abstract

Claims10