Affiliation:
1. Downstream Process Development (DSPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone,
Shanghai200131, China
Abstract
Background:
Protein A resins have been widely used for product capture during mAb,
bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly
bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification,
certain truncated byproducts may contain the same Fc region as the product but fewer numbers of
the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for
byproduct separation based on the difference in VH3-binding valency. As the ligands of different
VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would
be interesting to know whether they possess comparable capabilities for separating species with
the same Fc region but different numbers of VH3 domain.
Objective:
This study aims to explore the potential of different VH3-binding Protein A resins for
separating antibody species with the same Fc region but different numbers of VH3 domain.
Methods:
The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion,
the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect
SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess
the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3,
Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein
A resins for separating species having the same Fc region but different numbers of VH3 domain
was evaluated using an artificial mixture composed of the product and a truncated byproduct,
which contained one and zero VH3 domain, respectively (both species contained the same Fc
region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
monitor Fab purification and separation of species containing the same Fc region but different
numbers of VH3 domain.
Results:
When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed
varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct,
which contained the Fc region only without any VH3 domain, from the product, which included
one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating
these two species.
Conclusion:
Although different VH3-binding Protein A resins showed varied DBCs towards a
VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region
but different numbers of VH3 domain.
Publisher
Bentham Science Publishers Ltd.