Antibody Binding to Recombinant Adeno Associated Virus Monitored by Charge Detection Mass Spectrometry

Abstract

Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector. However, neutralizing antibodies reduce its efficacy. Traditional methods used to investigate antibody binding provide limited information. Here, charge detection mass spectrometry (CD-MS) was used to investigate the binding of monoclonal antibody ADK8 to AAV serotype 8 (AAV8). CD-MS provides a label-free approach to antibody binding. Individual binding events can be monitored as each event is indicated by a shift of the antibody–antigen complex to a higher mass. Unlike other methods, the CD-MS approach reveals the distribution of antibodies bound on capsids, allowing AAV8 subpopulations with different affinities to be identified. The charge state generated by the electrospray of large ions is normally correlated with the structure, and the charge is expected to increase when an antibody binds to the capsid exterior. Surprisingly, binding of the first ADK8 to AAV8 causes a substantial decrease in the charge, suggesting that the first antibody binding event causes a significant structural change. The charge increases for subsequent binding events. Finally, high ADK8 concentrations cause agglutination, where ADK8 links AAV capsids to form dimers and higher order multimers.

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR

“…CD-MS has been used to analyze various AAV serotypes, genome truncations, and antibody binding to AAV. 35 , 36 , 37 As shown in Figure S1 , in cceAAV2-CB-GFP vectors, the ratio of full/partial particles was enhanced by ∼14%, compared to scAAV2-CB-GFP. In the case of cceAAV8-TTR-hFIX versus scAAV8-TTR-hFIX, the ratio of full/partial particles was enhanced by 6.5%.…”

Single-Ion Mass Spectrometry for Heterogeneous and High Molecular Weight Samples

“…AAV can elicit an immune response, and understanding antibody interactions with AAV is important. The binding of murine monoclonal antibody ADK8 to AAV8 was recently investigated using CD-MS. 67 Figure 5a shows a charge versus mass heat map measured for empty AAV8 where warmer colors indicate higher intensity. A scale is shown on the right of Figure 5a.…”

Adapting a trapped ion mobility spectrometry-Q-TOF for native mass spectrometry

“…30,33 Currently, nMS has been applied to measure macromolecules in the MDa mass range, such as adeno-associated virus capsids (3.5 -5 MDa), antibody-bound capsids species (5 -17 MDa), and oligomerized 20S proteasome (0.7 -9.6 MDa). [34][35][36][37] The Wysocki lab has demonstrated the application of SID, the only MS/MS that extensively dissociates MDa biomolecules, to investigate the structure of AAVs. 38 Here, we modified a commercial TIMS-Q-TOF mass spectrometer to study structures of high molecular weight (MW) protein complexes.…”