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

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 an 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 was 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.

Enhanced Stability Differentiation of Therapeutic Polyclonal Antibodies with All Ion Unfolding-Ion Mobility-Mass Spectrometry

“…Data in Figure S2 clearly show that the full width at half-maximum (fwhm) values of pAbs are significantly higher than that of mAb, suggesting much higher heterogeneity for pAbs. The observed increment in microheterogeneity of pAbs should be linked to the elevated sequence variation in the antigen-binding domain and the glycosylation at the CH2 domain during antibody production . Overall, native IM-MS measurements initially reveal varied degrees of difference in chemical composition, or molecular microheterogeneity, for different therapeutic pAbs.…”

“…The observed increment in microheterogeneity of pAbs should be linked to the elevated sequence variation in the antigenbinding domain and the glycosylation at the CH2 domain during antibody production. 7 Overall, native IM-MS measurements initially reveal varied degrees of difference in chemical composition, or molecular microheterogeneity, for different therapeutic pAbs.…”

“…However, pAbs are produced by a large number of B cell clones each generating antibodies to a specific epitope, so polyclonal sera are a composite of antibodies with unique specificities . Our recent study on pAbs revealed the major driving force for its heterogeneity is the sequence variation of complementarity determining region, although the glycosylation of the constant region can also increase the microheterogeneity compared to mAbs . As a result, pAbs are more heterogeneous than mAbs, which makes it difficult to quantify the composition of pAbs, especially stability analysis and comparison between different species.…”

“…6 Our recent study on pAbs revealed the major driving force for its heterogeneity is the sequence variation of complementarity determining region, although the glycosylation of the constant region can also increase the microheterogeneity compared to mAbs. 7 As a result, pAbs are more heterogeneous than mAbs, which makes it difficult to quantify the composition of pAbs, especially stability analysis and comparison between different species. Therefore, due to the inherent heterogeneity of polyclonal antibodies and associated challenges in analyzing a set of antibody mixtures, the toolbox for direct analysis of the conformational stability of pAbs with acceptable molecular weight information preserved is still under development with growing needs.…”

Vaccines to Treat Substance Use Disorders: Current Status and Future Directions

“…An analysis indicated that both the sequence variations in the antigen-binding domain (Fab) and glycosylation at the CH2 domain (Fc) played simultaneous roles in the observed molecular microheterogeneity. These variations may have significant implications for the conformational stability and binding efficacy of the antibodies towards haptens [ 39 ]. In summary, this methodology facilitates the development of vaccines capable of eliciting an immune response specifically tailored to the target drug.…”