Rapid and Noninvasive Quantification of Capsid Gene Filling Level Using Water Proton Nuclear Magnetic Resonance

Abstract

The present work reports an enabling novel technology for quantifying the gene content in adeno-associated viral capsids. The method is based on the water proton nuclear magnetic resonance (wNMR) technique. Instead of analyzing the capsid directly, it utilizes water molecules to distinguish empty and full capsids, as water interacts with them differently. The transverse relaxation rate of water protons, R 2 ( 1 H 2 O), readily distinguishes empty and full capsids and is capable of quantifying the fraction of full capsids in a mixture of full and empty ones. It involves no sample preparation and no reagents. Measurement is rapid (data collection takes 1−2 min), noninvasive (the capsid sample can stay inside the original sealed and labeled container to be used in other studies or administered to a patient), and performed using a widebore benchtop NMR instrument. The method can be readily implemented at a production plant for product release as part of product quality control.

Decoupling Protein Concentration and Aggregate Content Using Diffusion and Water NMR

“…Furthermore, the turbidity of the sample does not influence the sample analysis. To date, it has been utilized in numerous biopharmaceutical-related studies, such as to investigate the gene filling of adeno-associated viral capsids, monitor vaccine adjuvant filling level, and sedimentation kinetics, in addition to the inspection of biologic product vials. − With respect to proteins, w NMR has been used to monitor the oxidation of a mAb in real time, as well as to obtain information about solution protein concentration under both static and flow conditions, with sensitivity shown to concentrations as low as 0.4 mg mL –1 . Taraban et al have also demonstrated that R 2 ( 1 H 2 O) is sensitive to protein aggregates with a size smaller than 0.45 μm and showed that it was capable of outperforming SEC, DLS, and microflow imaging .…”