Expedited Evaluation of Conformational Stability-Heterogeneity Associations for Crude Polyclonal Antibodies in Response to Conjugate Vaccines

Zhen Zheng,^1,2 Min Ma,⁴ Yifei Jia,¹ Yusi Cui,⁵ Rui Zhao,¹ Shuangshuang Li,² Cody Wenthur,^4,* Lingjun Li^4,5,6,* and Gongyu Li^1,3,*

^{Analytical Chemistry 2023, accepted.}

ABSTRACT: Conjugate vaccines have been demonstrated to be a promising strategy for immunotherapeutic intervention in substance use disorder, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. The antibodies generated following immunization with these species can provide long-lasting protection against overdose through sequestration of the abused drug in the periphery, which mitigates its ability to cross the blood-brain barrier. However, these antibodies exhibit a high degree of heterogeneity in structure. The resultant variations in chemical and structural compositions have not yet been clearly linked to the stability that directly affects their in vivo functional performance. In this work, we describe a rapid mass spectrometry-based analytical workflow capable of simultaneous and comprehensive interrogation of the carrier protein-dependent heterogeneity and stability of crude polyclonal antibodies in response to conjugate vaccines. Quantitative collision-induced unfolding-ion mobility-mass spectrometry with all-ion mode is adapted to rapidly assess the conformational heterogeneity and stability of crude serum antibodies collected from four different vaccine conditions, in an unprecedented manner. A series of bottom-up glycoproteomic experiments were performed to reveal the driving force underlying these observed heterogeneities. Overall, this study not only presents a generally applicable workflow for fast assessment of crude antibody conformational stability and heterogeneity at the intact protein level, but also leverages carrier protein optimization as a simple solution to antibody quality control.