Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation.
Protein Expr Purif
; 78(1): 27-37, 2011 Jul.
Article
in En
| MEDLINE
| ID: mdl-21421055
ABSTRACT
VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of â¼ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biotechnology
/
Recombinant Fusion Proteins
/
Immunotoxins
/
Escherichia coli
Country/Region as subject:
America do norte
Language:
En
Journal:
Protein Expr Purif
Journal subject:
BIOLOGIA MOLECULAR
Year:
2011
Document type:
Article
Affiliation country: