Improving Stepwise Assembly of a Bispecific F(ab') sub(2) from Two Different Fab' Molecules

An upstream process was developed for large-scale assembly of bispecific F(ab') sub(2) from two different Fab' molecules. We identified a novel product-related structural variant that is difficult to remove downstream and modified our process to decrease its generation during the assembly...

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Bibliographic Details
Published in:Industrial & engineering chemistry research 2017-02, Vol.56 (7), p.1713-1722
Main Authors: Williams, Ambrose J, Ovadia, Robert D, Giese, Glen S
Format: Article
Language:English
Subjects:
Assembling
Assembly
Cysteine
Efficiency
Hinges
Optimization
Reduction
Scaling up
Online Access:Get full text
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Summary:An upstream process was developed for large-scale assembly of bispecific F(ab') sub(2) from two different Fab' molecules. We identified a novel product-related structural variant that is difficult to remove downstream and modified our process to decrease its generation during the assembly step. The resulting multistep process includes affinity capture, an on-column cysteine uncapping step, batch reoxidation and pyridylation, and assembly of the two Fab' molecules into a F(ab') sub(2). Optimizations include selection of hinge domain-specific reducing agent, column chromatography conditions, and reaction pH. We selected L-reduced Glutathione to target hinge cysteines for reduction, uncapping the Fab' while minimizing off-target reduction. We scaled up the improved method for assembling bispecific F(ab') sub(2) with similar assembly efficiency but significantly improved product quality, generating a pool with bispecific F(ab') sub(2) for downstream processing.
ISSN:0888-5885
1520-5045
DOI:10.1021/acs.iecr.6b04265