Leveraging in-silico mediated approaches for the implementation of multimodal chromatography in mab purification

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Authors
Jang, Dongyoun
Issue Date
2024-12
Type
Electronic thesis
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en_US
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Chemical engineering
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Abstract
The ever-expanding mAb therapeutic market, the concomitant diversification of new modalities derived from the mAb structure as well as the emergence of novel chromatographic materials continue to challenge the adaptability of the mAb platform processes. Additionally, while multimodal chromatography has been demonstrated to offer unique selectivity to address key purification challenges, they are seldom employed for mAb processes due to their complex behavior. Advancements in high-throughput techniques have greatly intensified the exploration of a large experimental design space, but the expansive combinations of protein diversity and available chromatographic material can ultimately complicate identification of versatile purification processes. This thesis work addresses this challenge through the development of an in-silico mediated workflow to efficiently evaluate and compare the extent of synergistic impurity separation across all possible resin sequences, with a focus on the implementation of multimodal chromatography. This approach was characterized by extensive selectivity screens to generate product and impurity retention databases, which then became inputs to an in-silico process ranking tool to rapidly and quantitatively identify highly separable processes based on novel separability indexes. The first application of this approach was for the expedited development of non-protein A 3-step processes for the effective removal of CHO HCPs for a mAb therapeutic. The resulting processes were determined to be highly separable as well as orthogonal, resulting in HCPs levels to below 100 ppm while maintaining good mAb recovery. To further demonstrate the utility of this approach, the in-silico mediated workflow was adapted for the rapid development of a 2-step polishing sequence for the removal of challenging product-related impurities for a mAb and Fc-containing therapeutic. The resulting databases revealed an unexpected ability of multimodal resins to resolve differently glycosylated variants (glycoforms), enabling the development axviii straight flowthrough and integrated polishing sequence using only multimodal resins for the orthogonal removal of aggregates and glycoforms. Both applications of this workflow demonstrated its versatility to efficiently address industrially relevant purification challenges for mAb-based therapeutics while also providing new insights into purification opportunities afforded by multimodal chromatography. Building upon these insights, the application of multimodal cation exchange (MMCEX) resins was extended towards their development as potential alternatives to affinity solutions for the removal of LC impurity from a Fab therapeutic. The results revealed that selectivity was determined by a complex interplay of global protein biophysical properties and the relative electrostatic and hydrophobic properties of the multimodal ligands. Though complex, the additional levers to modulate selectivity revealed several MMCEX bind-elute opportunities for the purification of a Fab therapeutic containing a very challenging LC impurity load. Finally, the insights on multimodal chromatography learned throughout this work inspired the high-throughput evaluation of a library of novel MMCEX and MMAEX membrane adsorbers for the removal of CHO HCPs. The results showcased the potential of these novel polymer grafted membrane adsorbers to replace resin-based chromatography and enabled a systematic investigation into the impact of ligand chemistry on selectivity, thereby informing the rational design of next-generation multimodal ligands. As a whole, this thesis work demonstrates a platform workflow that is highly applicable across various industrial purification challenges, establishes the broad preparative utility of multimodal chromatography, and provides valuable implications towards development of next-generation chromatographic materials.
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December2024
School of Engineering
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Rensselaer Polytechnic Institute, Troy, NY
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