What resources does Wyatt offer to quantify AAV quality attributes with
SEC-MALS?
Introduction
ASTRA Viral Vector Analysis (VVA) module includes key functionality for concentration, full-to-total ratio (Vg/Cp) and molar mass of adeno-associated virus (AAV) by SEC-MALS or FFF-MALS. In ASTRA 8.1, the VVA module further streamlines your AAV workflow to produce results more quickly. In this article, we introduce key improvements to maximize productivity and help validate and transfer your AAV method across your organization.
AAV SOP Guidance Manual
Starting with ASTRA 8.1, we integrated the AAV SOP Guidance Manual into ASTRA. Simply select Help -> AAV SOP Guidance… and voilà! This comprehensive guide is suitable for all labs—from academic research to GMP-level product release. The manual is a go-to reference for your entire organization to reference for experimental design, method development, and data analysis. We describe our platform method in full detail to support development of a Standard Operating Procedure, and we provide expert guidance on data interpretation, analysis, and troubleshooting.
Embedded within ASTRA, the entire text is searchable, and you can view and print sections as desired. Like all our products, we continuously update and improve the AAV SOP Guidance Manual, and you can rest assured that you’ll always have the most up-to-date documentation when you apply a software update to ASTRA.
Dedicated AAV analysis methods
ASTRA’s System Database contains a dedicated folder with methods for additional functionality. The optimized AAV Method you created in ASTRA’s Method Builder, customized for your specific instrument parameters, is still saved in your User folder. The System folder makes it easy to navigate directly to the right location for additional processing using Apply Method or One-to-Many; plus you can further improve accuracy of the results by optimizing capsid and DNA extinction coefficients.
Capsid extinction coefficient
Whenever both UV absorbance and differential refractive index (dRI) data are collected for a single component, like a protein or plasmid, ASTRA can measure the UV extinction coefficient using the UV absorbance, dRI signal, and known dn/dc for the sample. Simply measure SEC-MALS data for a control sample of 100% empty AAV using your usual AAV method. Then apply the new ASTRA method for measuring the capsid extinction coefficient, and ASTRA will automatically calculate the protein extinction coefficient for your peak. You can save the value to a custom method for that serotype, ready for transfer to process development or quality control.
DNA extinction coefficient
Although the extinction coefficient for free ssDNA is well known, the tight packing inside the AAV capsid can cause the absorbance for encapsidated DNA to differ relative to free DNA, similar to the hypochromic effect in dsDNA. Accurate capsid (protein) and DNA extinction coefficients are important for optimal accuracy and linearity, and we recommend determining the DNA extinction coefficient for each combination of serotype and genome.
This measurement can be done directly in ASTRA from SEC-MALS data. You don’t need to know the exact composition of your AAV sample or have perfectly purified full AAV. Just apply the new method for measuring the DNA extinction coefficient (DNA ext coef – nonempty AAV) to any data collected for a sample with >3x1011 total AAV particles (e.g., 30 µL injection at 1013 AAV/mL) and Vg/Cp > 0.5. ASTRA will use the UV and dRI signals together with information you provide about your capsid protein to determine the refined DNA extinction coefficient. Then, save the new values of capsid and DNA extinction coefficients to a custom method for fast, super accurate analysis of future samples with the same serotype-gene pair.
Conclusion
Wyatt provides a full suite of instruments and methods for complete quantitation and characterization of your AAV products, including SEC-MALS, FFF-MALS, and DLS solutions. For a comprehensive overview of all of the system options Wyatt offers, visit our AAV Services page.
Do you have a question? Contact our experts here in Customer Support. We’re happy to help! Call +1 (805) 681-9009 option 4 or email support@wyatt.com.