Development of Native Ion Mobility-Mass Spectrometry Approaches for the Structural Characterization of Antibody-Based Therapeutics
Villafuerte-Vega, Rosendo
2024
Abstract
Monoclonal antibody (mAb)-based therapeutics have revolutionized the landscape of contemporary medicine. By harnessing the power of the immune system in a controlled manner, these therapeutics have drastically improved patient outcomes in the clinic, delivering more personalized, effective treatment options compared to conventional small molecule drugs. These therapeutics, however, possess complex higher order structure (HOS) features that can be altered by post-translational modifications and degradation. Although high-resolution biophysical tools exist for HOS characterization, they often require long timescales not conducive for the rapid screening of candidate molecules. This limitation necessitates new analytical technologies that can probe HOS with minimal sample preparation and purification. To fill this technology gap, this dissertation presents an ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU)-based framework for evaluating the structures and stabilities of various emerging mAb-based therapeutic modalities. In Chapter 2, we combine IM-MS with CIU to probe the gas-phase structures and unfolding pathways of a knob-into-hole (KiH) bispecific antibody (bsAb) and its parent mAbs. By measuring the stabilities of mAb fragments and deglycosylated constructs, we provide a general mechanism for the gas-phase unfolding of the KiH bsAb, where low- and high-energy CIU transitions correspond to the unfolding of Fab and Fc domains, respectively. Specifically, our data indicate that the low-energy Fab unfolding event is driven by the stability of the hole Fab domain, while high-energy transitions are associated with the unfolding of the knob portion of the Fc. Together, these results provide a unique framework for evaluating the domain-level stabilities of both KiH bsAbs and mAbs using CIU. Chapter 3 leverages the sensitivity of CIU responses to alterations in hinge disulfide bond configurations to build an assay that accurately predicts mAb flexibility for a series of anti-CD40 human(h) IgG2 cysteine to serine (C/S) variants in a manner that correlates with receptor agonism. We find that rigid, agonistic variants, which feature a disulfide crossover within their hinges, experience less gas-phase structural collapse and possess lower gas-phase stabilities than flexible, nonagonistic variants. By using a CIU-based classification approach, we accurately identify hIgG2 variants that exhibit optimal immunostimulatory activity, thus demonstrating the ability of CIU to predict mAb structure-function relationships for the first time. In Chapter 4, we shift our focus to probing the HOS of an Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine (Gly-Ser) linkers. We demonstrate that Fc-IL-10 is highly dynamic in the gas-phase, generating more structural transitions during CIU and broader IM profiles compared to proteins of similar size. Moreover, we elucidate the gas-phase unfolding pathway of Fc-IL-10 using similar approaches presented in Chapter 2, where we find that low- and high-energy transitions are associated with the unfolding of IL-10 and Fc domains, respectively. Importantly, we observe that an increase in Gly-Ser linker length stabilizes IL-10 dimers, highlighting the potential of CIU to inform the engineering of stable Fc-fusion protein therapeutics. Lastly, Chapter 5 presents a novel in vitro serum stability assay that incorporates stable mAb-based internal standards. Our results indicate that the use of NISTmAb and its Fc fragment as internal standards can improve the accuracy and precision of sample recovery calculations, enabling a more confident stability assessment of mAb therapeutics in serum. Collectively, the methodologies presented in this dissertation underscore the utility of IM-MS and CIU in probing the domain-level stabilities of mAb-based therapeutics and establishing connections between mAb HOS and function.Deep Blue DOI
Subjects
Ion Mobility Mass Spectrometry Monoclonal Antibodies Collision Induced Unfolding Higher Order Structure Fusion Proteins
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