Berkowitz, S. | Search bioRxiv

bioRxiv2023

Rapid high-resolution size distribution analysis for adeno-associated virus using high speed SV-AUC

When optimized, sedimentation velocity analytical ultracentrifugation (SV-AUC) provides the most-accurate, broadest-range, and highest-resolution size distribution analysis of any method. Generating simulated data for an adeno-associated virus (AAV) sample consisting of four species differing only in their DNA content and having closely spaced sedimentation coefficients, allows manipulation of the SV-AUC experimental protocol to optimize the size distribution resolution. In developing this high speed SV-AUC (hs-SV-AUC) protocol several experimental challenges must be overcome: 1) the need for rapid data acquisition, 2) avoiding optical artifacts from steep boundaries and 3) overcoming the increased potential for convection. A protocol, hs-SV-AUC, has been developed that uses high rotor speeds, interference detection and low temperatures to overcome these challenges. By confining data analysis to a limited radial-time window and using a very short run time (< 20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed, making interference detection is as simple to employ as absorbance detection. Experimental size distributions from the same AAV sample by hs-SV-AUC at 45K rpm and 10 {degrees}C versus low-speed SV-AUC at 15K rpm, and 10 {degrees}C illustrates the improved size distribution resolution offered by the hs-SV-AUC protocol.

biophysics↗

bioRxiv2021

Boundary convection during velocity sedimentation in the Optima analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units used in conducting characterization work on adeno-associated virus (AAV) has shown evidence of sample convection. Further investigation reveals that this problem arises from the temperature control system design, which is prone to producing destabilizing temperature induced density gradients that can lead to density inversions. The observed convection impacts both the qualitative and quantitative data generated by the Optima. The problem is intermittent and variable in severity within a given Optima unit and between Optima units. This convection appears to be mainly associated with low rotor speeds and dilute samples in dilute solvents, such as AAV samples in formulation buffers containing relatively low concentrations of salts, sugars, etc. Under these conditions it is found that a sufficiently robust stabilizing density gradient is not always present during sedimentation, making the sample susceptible to convection by localized density inversions. Because SV-AUC is used as an analytical tool in making critical decisions in the development and quality control of biotherapeutics, it is imperative to alert users about this potential problem. In general special attention to data quality needs to be made by those researchers working with very large biopharmaceutical particles (e.g. gene therapy products that involve viral vectors or nanoparticles), where the conditions leading to convection are most likely to occur. It is important to note that the XL series analytical ultracentrifuges do not suffer from this problem, indicating that this problem is unique to the Optima. Attributes that reveal the presence of this problem and strategies for its elimination or minimization are provided.

biophysics↗