Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.

Thomas Linke,Xiangyang Wang,Alan K Hunter,Andrew Fulton,Kripa Ram,Christopher Thompson,Michaela Wendeler,Guoling Xi,Matthew T Aspelund,William K Wang,Timothy M Pabst

doi:10.1002/btpr.1983

Abstract

We describe the development and scale-up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1380–1389, 2014

Highlights

Recombinant immunotoxins represent an important class of anticancer drugs typically composed of truncated protein toxins fused to an antibody fragment.[1,2,3,4] The antibody fragment replaces the cell-binding domain of the native toxin, allowing the toxin molecule to be directed against an oncology target of interest
Toxins evaluated in the clinic as part of immunotoxin therapy trials include those derived from ricin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A (PE).[3]
Moxetumomab pasudotox (m. pasudotox, CAT-8015) is a recombinant immunotoxin composed of the VH and VL portions of an anti-CD22 antibody connected by a disulfide bond and fused to a truncated form of Pseudomonas exotoxin (PE38) by a peptide bond to VH

Summary

Introduction

Recombinant immunotoxins represent an important class of anticancer drugs typically composed of truncated protein toxins fused to an antibody fragment.[1,2,3,4] The antibody fragment replaces the cell-binding domain of the native toxin, allowing the toxin molecule to be directed against an oncology target of interest. Toxins evaluated in the clinic as part of immunotoxin therapy trials include those derived from ricin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A (PE).[3]. Pasudotox, CAT-8015) is a recombinant immunotoxin composed of the VH and VL portions of an anti-CD22 antibody connected by a disulfide bond and fused to a truncated form of Pseudomonas exotoxin (PE38) by a peptide bond to VH. M. pasudotox is currently in clinical trials for the treatment of B-cell malignancies.[5,6,7,8,9,10,11,12] The immunoglobulin variable domain is composed of affinity matured VH and VL chains of an anti-CD22 monoclonal antibody, while PE38 contains the PE toxin domains II and III. Domain II has translocation activity while domain III catalyzes the ADP-ribosylation of elongation factor 2, leading to the inhibition of protein synthesis and cellular death.[13,14] The calculated molecular weight is 63,398 Da and the pI is approxi-

Methods

Results

Conclusion