Purification of bispecific antibodies by multimodal chromatography and identification of salt-tolerant resin-binding interfaces
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Authors
DiSpirito, Cameron, Kevin
Issue Date
2025-12
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
IgG-like bispecific antibodies (bsAbs) are an emerging class of biotherapeutic molecules that can simultaneously bind two distinct antigens. This dual specificity provides medical advantages over monospecific antibodies for certain indications, like cancer and hemophilia. Despite their therapeutic advantages, bsAbs pose a challenging separations problem; during synthesis a variety of product related impurities are produced with similar biophysical properties to the bsAb product. Conventional methods for impurity removal, such as single mode chromatography, are less effective at removing these product related impurities. As the prevalence of bsAbs increases, so does the demand for efficient purification procedures. Multimodal chromatography demonstrates improved selectivity over its single-mode counterparts. Leveraging both hydrophobic and electrostatic interactions, it is capable of resolving product related impurities where single mode chromatography fails. Although the utility of these resins has been well documented for a variety of different applications, the fundamental interactions governing the behavior of retained proteins is not fully understood. First, we demonstrated the utility of a MMCEX resin, Capto MMC ImpRes, for removing ½ IgG, homodimer, and LC-mispaired product related impurities from an asymmetric IgG-like bsAb. After demonstrating this multimodal resin’s superior selectivity over single mode resins, we performed linear gradient screens at both pH 6.5 and pH 8 to elucidate the impact of pH on selectivity. The results from these experiments informed a second round of screening experiments at high (8.5, 9, 9.5) and low pH (6, 5.5, 5) to identify conditions for complete product related impurity removal. These results were used to develop a single column process which employed a series of pH and salt steps to remove the product related impurities. This process increased the purity of the bsAb from 67.3% to 94.5%, with a 72% recovery.
Next, we implemented high-throughput screening techniques to examine the utility of novel prototype MMCEX resins and conditions for product related impurity removal from the same bsAb. Partition screening experiments were conducted from pH 4.5-6 with 0.5 M or 1 M NaCl to determine the optimal conditions for LC-mispair removal with each unique resin. Conditions were identified that demonstrated high selectivity while maintaining strong retention of the bsAb with several different prototype resins. Partition screening experiments were also performed from pH 8-9.5 with no NaCl or 0.1 M NaCl to determine conditions for removal of the homodimer and ½ IgG impurities. Several resin-condition pairs were identified that displayed high selectivity factors between the bsAb AB and parent A mAb while simultaneously providing high retention of the bsAb. In particular, prototype multimodal resins P2, P3, P4, and 442 demonstrated good performance for removal of both impurities, which suggested that these resins might be useful for a single column polishing process.
After exploring different candidates for product related impurity removal, we probed the fundamental interactions between bsAbs and Capto MMC with covalent labeling-mass spectrometry. These studies used two different covalent labeling chemistries, diethylpyrocarbonate (DEPC) and p-hydroxyphenylglyoxal (HPG), in the presence and absence of NaCl to determine both preferred binding regions and salt tolerances of these binding interfaces for two bsAbs. In both cases, the bsAbs displayed more salt tolerance around their hydrophobic complementarity determining regions (CDRs). In addition, the fragment crystallizable (FC) region displayed significantly less salt tolerance, which suggested that this domain is less involved at elevated salt conditions. A comparison between the retention and binding interfaces of both bsAbs was also conducted.
In the final section of this thesis, covalent labeling was employed to study the interaction of the bsAb at the processing conditions developed for Capto MMC ImpRes. This included studying the pH dependence of the bsAb’s binding interface from pH 7.5 – 9.5, which showed interaction sites on both halves of the bsAb and several differences between the two pHs. In addition, the salt-tolerant binding patches of the bsAb and LC-mispair impurity were probed with DEPC at pH 5.5 in the presence of NaCl. Despite some limitations in labelling at this condition, several key difference were observed between the bsAb and LC-mispair, which shed light on their different retention behavior.
The work described in this thesis helps to advance the state of the art of bsAb downstream bioprocessing using multimodal chromatography and sheds light into the resin binding regions on these complex biomolecules.
Description
December2025
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY