Production of Therapeutic Antibody Fragment by Periplasmic E. coli ExpressionBy: Alan Schultz &Jack Bobzien

Project Objective

Our company has developed a Fab fragment product and needed the purification process verified.

Using E. Coli, propose a new purification train and determine cost evaluation.

Work within the given constraints.

Constraints

Affinity resins for Fab are too expensive for manufacturing scale.

Need to produce 50 kg/yr.

Expression levels are expected to reach 10% of total extracted protein in 10 years.

Achieve above a 99% purity in product.

https://www.venturacollege.edu/departments/academic/economics.shtml

Product

Fragment Antigen binding (Fab fragment)

Can be expressed in various hosts like E. Coli, yeast etc.

Periplasmic Expression

pI of 7.0

M.W. of 50 kDa

http://www.ruppweb.org/fab/fab_sketch_circled.gif

Host

E. Coli

Used Strain W3110

Widely used in the pharmaceutical industry.

http://scm-l3.technorati.com/11/06/03/44263/e-coli-.jpg?t=20110603144325

Overview

Upstream

Recovery

Purification

IMAC

Immobilized metal-ion affinity chromatography

His6-tagged Fab

Use of EDTA-Mg2+ and imidazole

92.4% purity in exit stream

Process Economics

Process Economics

Process Economics

Process Economics

Bottleneck

Overall: 64.74 hrs

V-105: 32.74 hrs

V-106: 26.21 hrs

Conclusion/Adaptation

Periplasmic Complications

For current system, an IMAC Column was major modification

$250 per g/ YFP

Questions