Real-Time, Ultrasensitive, and High-Throughput Monitoring of Adeno-Associated Virus Production via an...

Area of Science:

• Biotechnology
• Biosensing
• Gene Therapy Manufacturing

Background:

• Conventional adeno-associated virus (AAV) quality control methods are time-consuming, hindering gene therapy process optimization.
• There is a critical need for rapid, label-free, and high-throughput quantification techniques for AAV vectors used in gene therapy.

Purpose of the Study:

• To develop and validate an aldehyde-functionalized metasurface plasmon resonance (ALMetaSPR) biosensor for rapid, label-free, and high-throughput AAV quantification.
• To create two complementary platforms: a 96-well high-throughput system and a portable process analytical technology (PAT) device for real-time AAV production monitoring.

Main Methods:

• Development of an ALMetaSPR biosensor utilizing antibody-based capture for AAV quantification.
• Implementation of a 96-well high-throughput system for 15-minute AAV analysis.
• Integration of microfluidics and smartphone interfaces into a portable MetaSPR-based PAT (MSPR-PAT) device for real-time monitoring.

Main Results:

• The ALMetaSPR biosensor accurately quantifies AAV-2 and AAV-5 titers label-free.
• The 96-well system is 8-fold faster than ELISA; the MSPR-PAT enables real-time online monitoring.
• Achieved a broad dynamic range (6.25 × 10^09–4.00 × 10^11 GC/mL) with a 250-fold sensitivity improvement over existing label-free methods (LOD: 1.24 × 10^09 GC/mL).
• Biosensor surface regeneration over 42 cycles without performance loss significantly reduces costs.

Conclusions:

• The ALMetaSPR biosensor addresses a critical bottleneck in gene therapy manufacturing.
• This innovation integrates high-throughput screening, real-time process analytics, and one-step operation for efficient AAV production.
• The developed platforms offer a significant advancement in AAV quality control, accelerating gene therapy development and production.