BTEC at NC State University Enhances AAV Purification with PATfix Platform
Integration of PATfix improves viral vector research efficiency and effectiveness, says Shriarjun Shastry
The Golden Leaf Biomanufacturing Training and Education Center (BTEC) at NC State University is advancing viral vector research, particularly adeno-associated viruses (AAVs). Shriarjun Shastry (Arjun) and his team are developing serotype-agnostic peptide ligands for universal AAV purification. They integrated the PATfix platform for simultaneous analyses, improving purification methods like size exclusion chromatography (SEC) and steric exclusion chromatography. PATfix’s inline pH and conductivity sensors optimized gradient monitoring, enhancing AAV purification efficiency and effectiveness.
Meet the Expert: Shriarjun Shastry
Shriarjun Shastry (Arjun) joined BTEC in 2018, bringing expertise from his role in the Process Sciences Investigations Team at Regeneron Pharmaceuticals, Inc. He specializes in downstream processes and is actively involved in BTEC’s AAV and mRNA process development projects, focusing on purification. Arjun holds a bachelor’s degree in biotechnology engineering from BVB College of Engineering and Technology, India, and a Master of Science in biopharmaceutical engineering from New Jersey Institute of Technology, Newark. He is currently pursuing a part-time PhD in Chemical Engineering at North Carolina State University.
Arjun has been conducting research over the past few years, recently utilizing the PATfix system to streamline his research process. He collaborates closely with the NC Viral initiative at NC State, working with various viral vectors, including lentivirus, adenovirus, and AAV.
Research Focus on AAV Purification
The AAV production process involves multiple upstream and downstream steps, including chromatography and TFF. Key challenges include loss of transduction activity at low pH and the high cost of affinity resins, which can compromise capsid integrity. Through this research, a sequence of peptides was identified for purification studies.
These peptides were designed to bind at neutral pH, facilitating elution under similar conditions. Previous data were primarily derived from plate-based assays that determined the concentration of specific molecules. However, visual confirmation of the entire sample composition is more effective. This realization prompted the integration of advanced analytical methods, including the use of the PATfix platform, to enhance the research process.
Enhancing AAV Purification with PATfix
The development of serotype-agnostic peptide ligands for the universal purification of AAVs has introduced unique challenges. Traditional assays offered limited insights into sample composition, prompting researchers to adopt advanced analytical techniques. The PATfix platform emerged as a key solution to improve visualization and analysis in the purification process.
- Comprehensive Sample Analysis
The PATfix system offers multiple detectors, allowing for comprehensive sample analysis from a single injection. With capabilities such as UV, MALS, fluorescence, and refractive index detection, it provides valuable insights into sample composition.
“One very cool feature of the system is its inline pH and conductivity sensors, which help when running gradients. You don’t just rely on percentage A and percentage B to make assumptions about the gradient; you can actually see it forming through changes in conductivity and pH,” explains Arjun.
- Effective Purification Validation
Utilizing PATfix for size exclusion chromatography (SEC) demonstrated clear separation between AAV peaks and impurities, confirming the effectiveness of the peptide ligands. The system facilitated the observation of a distinct AAV peak alongside residual impurities, visually validating the purification process.
- Universal Binding Across Serotypes
Research also revealed that one peptide acted as a universal binder across various AAV serotypes, achieving consistent results. The ability to quantify capsid amounts using standard curves further improved the reliability of the findings.
Future Directions in Chromatography
Looking ahead, BTEC is committed to advancing AAV purification processes through ongoing research and innovation. Arjun’s work, particularly with the PATfix platform, positions the center to make significant contributions to gene therapy and vaccine development. Current efforts are focused on exploring steric exclusion chromatography and ion exchange chromatography methods to separate empty and full capsids, with promising initial results indicating potential for quantifying full capsid percentages in feed samples.
Through the integration of the PATfix platform and by leveraging these innovative approaches, BTEC aims to enhance its capabilities in AAV purification, ultimately supporting the broader goals of advancing therapeutic solutions in the field.
