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

Conjugate vaccines have been demonstrated to be a promisingstrategyfor immunotherapeutic intervention in substance use disorder, whereina hapten structurally similar to the target drug is conjugated toan immunogenic carrier protein. The antibodies generated followingimmunization with these species can provide long-lasting protectionagainst 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 instructure. The resultant variations in chemical and structural compositionshave not yet been clearly linked to the stability that directly affectstheir in vivo functional performance. In this work,we describe a rapid mass-spectrometry-based analytical workflow capableof simultaneous and comprehensive interrogation of the carrier protein-dependentheterogeneity and stability of crude polyclonal antibodies in responseto conjugate vaccines. Quantitative collision-induced unfolding-ionmobility-mass spectrometry with an all-ion mode is adapted to rapidlyassess the conformational heterogeneity and stability of crude serumantibodies collected from four different vaccine conditions, in anunprecedented manner. A series of bottom-up glycoproteomic experimentswas performed to reveal the driving force underlying these observedheterogeneities. Overall, this study not only presents a generallyapplicable workflow for fast assessment of crude antibody conformationalstability and heterogeneity at the intact protein level but also leveragescarrier protein optimization as a simple solution to antibody qualitycontrol.

Automated summary

Conjugate vaccines are a promising strategy for immunotherapeutic intervention in substance use disorder. These vaccines utilize a hapten similar to the target drug, which is conjugated to an immunogenic carrier protein. The antibodies produced provide long-lasting protection against drug overdose by sequestering the drug in the periphery and preventing it from crossing the blood-brain barrier. However, these antibodies exhibit structural heterogeneity, and the relationship between their variations and stability remains unclear. This study presents a rapid mass-spectrometry-based workflow that can assess the conformational heterogeneity and stability of crude antibodies in response to conjugate vaccines.