Automated plasmid purification system enables increased AAV production.

Area of Science:

• Biotechnology
• Gene Therapy Manufacturing
• Molecular Biology

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapies, but high manufacturing costs hinder development and access.
• Plasmid DNA (pDNA) purification is a significant bottleneck in AAV production, especially at larger scales.
• Current manual plasmid purification methods are time-consuming and limit scalability.

Purpose of the Study:

• To compare the efficiency and outcomes of manual versus automated plasmid DNA purification systems for AAV production.
• To evaluate the impact of scale (Maxi, Mega, Giga) on purification time, yield, and endotoxin levels.
• To investigate the underlying mechanisms for differences in AAV production titres observed between purification methods.

Main Methods:

• Plasmid DNA purification using conventional manual kits and a scalable automated system at Maxi, Mega, and Giga scales.
• AAV vector production via PEI-based transfection of HEK293F cells using purified pDNA.
• Analysis of AAV9 titres, endotoxin levels, and pDNA complex formation using dynamic light scattering.

Main Results:

• Automated purification significantly reduced processing time, with savings increasing at larger scales (e.g., 8 hours saved at Giga scale).
• The automated system generally resulted in lower yields (3-67% reduction) and higher endotoxin levels compared to manual methods.
• Surprisingly, plasmids purified by the automated system led to a 14-fold increase in AAV-MAX production titres, attributed to larger pDNA transfection complexes formed by the automated system's elution buffer.

Conclusions:

• Automated plasmid purification offers substantial time savings for AAV manufacturing, particularly at scale.
• The choice of purification method and its associated elution buffer can critically impact transfection efficiency and subsequent biologics production.
• Optimization of plasmid preparation methods, including elution buffer composition, holds potential for improving transient production of AAV and other biologics.