Insights into the purification of bispecific antibodies on multimodal systems: from chromatography to biophysics

Parasnavis, Siddharth, Sumant
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Bystroff, Christopher
Przybycien, Todd
Karande, Pankaj
Aspelund, Matthew
Cramer, Steven
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Chemical engineering
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This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.
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Bispecific antibodies (bsAb) have gained significant importance recently due to their ability to bind to multiple epitopes resulting in an increased efficacy towards their targeted indication as compared to monoclonal antibodies (mAb). While several techniques have been developed to steer the formation of the bsAb molecule, the efficient production of bsAbs is impacted by the inevitable formation of product related variants due to the mispairing of the light and heavy chains in the expression system. Due to similarities in structure and surface properties between the correctly formed bsAb and the mispaired variants, the effective removal of these impurities is extremely challenging with single mode resin systems. Multimodal resin systems can offer increased selectivity over single mode systems for similar molecules due to the unique combination of charge and hydrophobic moieties on the same ligand. The work presented in this thesis aims to shed light on the use of multimodal resins for the purification of bsAb from their impurities. To that end, a systematic workflow was developed and implemented to understand selectivity differences and preferred binding patches for bsAbs and their parental mAbs on a homologous set of multimodal cation exchange (MM CEX) resin systems. This workflow incorporated chromatographic screening of the parent mAbs and their fragments at various pH conditions followed by surface property mapping and protein footprinting using covalent labeling followed by LC/MS analysis.For the first set of molecules, linear gradient experiments on MM CEX resins showed that the bsAb molecule exhibited a unique transitory behavior on some of the resins as a function of pH. This lead to the hypothesis that specific domains of the molecule could be involved in preferential binding to the resin surface at different pH conditions. Domain contribution experiments indicated that the retention of both parental mAbs was likely driven by (Fab)2 interactions at higher pH conditions. Protein footprinting experiments performed at pH 7.5 using sulfo-nhs-acetate to identify the preferred binding patches for the parental mAbs and the bsAb confirmed that the Fc was indeed not involved at the binding interface and that residues in the variable regions of the Fabs were driving the interactions with the MM CEX resins. Parent mAb A containing more positively charged lysine residues in the variable region of the Fab as compared to Parent mAb B resulted in Parent mAb A being more retained on the MM CEX resins. While some of the same residues that were seen to be important for the parental mAbs were also found to be important for the bsAb, it is likely that different avidity contributions from the two Parental Fab arms of the bsAb played a key role in the observed chromatographic behavior. This workflow was then extended to study the chromatographic behavior of a product related variant found in the bsAb expression pool. This product related variant comprising of heterodimerized heavy chains originating from both the parent mAbs and light chains originating only from Parent mAb B was found to have similar chromatographic behavior as Parent mAb B on most MM CEX resins. Protein footprinting experiments at pH 7.5 using sulfo-nhs-acetate indicated that that the same residues in the variable regions of the light and heavy chains of the individual parental mAbs were also important to the binding of this product related variant. The relative contributions to binding from the two Fab arms was again found to have a significant impact on the observed chromatographic behavior. Results from this study demonstrated why the purification of the correctly formed bsAb from the mispaired product related variants is such a challenge in downstream processing. The workflow was then modified to include the use of a novel labeling chemistry, diethyl pyrocarbonate (DEPC). Unlike sulfo-nhs-acetate which required basic pH conditions limiting our studies, DEPC enabled us to perform the covalent labeling experiments at acidic pH conditions allowing for the study of preferred binding patches at lower pH conditions. Linear gradient experiments for the intact Parent mAb B and its fragments had indicated that at pH 5.5, the Fc may also be contributing to the chromatographic behavior of the mAb. Protein footprinting studies at pH 5.5 indicated that in addition to the contributions from the (Fab)2 domain, the Fc domain indeed played an important role in the retention of mAbs. This added avidity contribution from the Fc resulted in an increased retention of the molecule. In order to validate the chromatographic experiment for the constituent fragments, labeling studies were also performed for the individual fragments of mAb B at pH 5.5. Results indicated that while the residues found to be important for the binding of the intact mAb were also important for the retention of the fragments, additional residues that were sterically hindered on the intact mAb may be contributing to the binding interface of the fragments. Finally, this workflow was extended to study the chromatographic behavior for an additional set of industrially important bsAb and it’s parent mAbs. The two bsAb share a common parental mAb resulting in a unique opportunity to study how the common half between the two bsAb impacts their chromatographic behavior. The results indicated that subtle differences in the charge and hydrophobicity landscape of the protein surface resulted in unique chromatographic selectivities for the parental molecules giving rise to opportunities for their separation using MM CEX resins. Further, initial chromatographic screening for the bsAb indicated opportunities for their separation. The work presented in this this thesis lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules such as mAbs and bsAbs which can provide important insights into improved biomanufacturability and expedited downstream bioprocess development.
School of Engineering
Dept. of Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Troy, NY
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