Identification of relevant analytical methods for adeno-associated virus stability assessment during formulation...

Area of Science:

• Biotechnology
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Viral vectors are crucial for gene therapy, oncology, and vaccine development but exhibit inherent instability, necessitating cold storage.
• Optimizing viral vector formulations requires high-performance analytical methods that are sensitive, high-throughput, and consume minimal material.
• Adeno-associated viral vectors (AAVs) are prominent in gene therapy, posing unique analytical challenges for stability assessment.

Purpose of the Study:

• To evaluate analytical methods for their ability to indicate or predict the stability of adeno-associated viral vectors (AAVs).
• To identify suitable techniques for AAV formulation development that meet requirements for high sample throughput and low material consumption.
• To categorize analytical methods based on their predictive power for time-dependent long-term viral vector stability.

Main Methods:

• Extrinsic differential scanning fluorimetry (DSF) using SYBR Gold to detect AAV genome release.
• Nano differential scanning fluorimetry (nanoDSF) to analyze AAV capsid unfolding via intrinsic protein fluorescence.
• Size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) and UV detection for particle size distribution and genome load analysis.

Main Results:

• Extrinsic DSF and nanoDSF show significant potential for predicting AAV stability.
• SEC-MALS-UV is effective for stability indication of particle size distribution and genome load in AAVs.
• These methods fulfill critical requirements for AAV formulation development, including throughput and material usage.

Conclusions:

• Extrinsic DSF and nanoDSF are highly suitable techniques for predicting AAV stability during formulation development.
• SEC-MALS-UV provides stability-indicating measurements for AAV particle characteristics and genome content.
• Categorizing analytical methods by stability prediction power is essential for accelerating viral vector formulation and development.