Advances in Downstream PAT for Biologics, Vaccines and Gene Vectors

Significance of the Topic

Real-time monitoring of critical quality attributes in downstream processing of biologics, vaccines and gene vectors is essential to ensure product safety, consistency and efficiency. Downstream process analytical technology based on multi-angle light scattering provides direct measurements of molecular weight, particle size, concentration and payload, enabling rapid decision making and improved process control.

Study Objectives and Overview

This work reviews advances in real-time multi-angle light scattering (RT-MALS) applied to preparative and process development laboratories. Key goals include integrating RT-MALS with common downstream unit operations, demonstrating its capability to monitor aggregation, discriminate product from impurities, optimize pool collection and ensure viral vector content and integrity.

Methodology and Instrumentation

Multi-angle light scattering measures scattered light intensity at multiple angles to determine weight-average molar mass, root-mean-square radius, particle concentration and viral payload by correlating scattering data with UV absorbance at 260 and 280 nm. RT-MALS brings these measurements in-line or on-line during processes such as chromatography and tangential flow filtration. The technology uses an ultraDAWN MALS detector coupled with OBSERVER real-time software, interfaced via OPC-UA or analog signals to process controllers. Dilution capabilities enable accurate measurements without disrupting flow.

Used Instrumentation

• ultraDAWN multi-angle light scattering detector
• OBSERVER real-time data analysis software
• Fast protein liquid chromatography systems
• Tangential flow filtration units
• Auxiliary pumps for on-line dilution
• Process controllers with OPC-UA connectivity

Main Results and Discussion

Case Study 1: Flow-through Protein Purification

• Hydrophobic interaction chromatography removes monoclonal antibody aggregates in real time
• % high molecular weight species calculated from weight-average molar mass triggers fraction collection
• Monitoring ensured optimal yield and defined quality regardless of feed variations

Case Study 2: Adenovirus Purification

• UV alone could not distinguish virus from impurities, while RT-MALS confirmed constant viral radius and integrated titer
• Validated data agreed with offline reference, reducing analytical turnaround by two weeks

Case Study 3: Viral Vector Ultrafiltration

• RT-MALS set a titer threshold to signal concentration end point and tracked average virion size to detect degradation
• Permeate monitoring identified membrane fouling in real time without processing delays

Case Study 4: AAV Enrichment

• Ion-exchange chromatography monitored full and empty capsids via vector genome to capsid ratio
• OBSERVER calculated titers and VgCp inline, matching offline mass photometry and enabling targeted collection
• Identified aggregate peaks by size to avoid co-purification

Benefits and Practical Applications

• Direct measurement of critical quality attributes in real time enhances process understanding
• Reduction of offline analytical delays accelerates process development and scale-up
• Improved yield, quality and productivity in production runs
• Greater flexibility when transferring processes across scales and manufacturing sites

Future Trends and Applications

Integration of RT-MALS with advanced process automation and digital twins will enable predictive control of downstream operations. Expansion to novel modalities, including cell and gene therapies, and coupling with machine learning for adaptive process adjustments will further enhance product consistency and reduce time to market.

Conclusion

RT-MALS represents a powerful downstream PAT tool, offering continuous insight into molecular and particulate attributes during purification and concentration steps. Its implementation streamlines workflows, elevates control over critical quality parameters and supports robust manufacturing of biologics, vaccines and gene vectors.

Reference

Patel N, et al. Monitoring monoclonal antibody aggregate removal using RT-MALS in flow-through HIC. mAbs. 2018;10(7):945-950.