Risk Assessment and Comparability Analysis of Adherent Versus Suspension Cultures for Gene Therapy Manufacturing

Introduction

Adeno-associated viruses (AAVs) have emerged as a leading platform for gene therapy, offering a safe and effective method for in vivo gene delivery. 

​However, scaling up AAV production to meet clinical demand remains a significant challenge. Early-phase AAV production often relies on adherent cell cultures, but as patient populations grow, transitioning to suspension cultures becomes necessary for improved scalability and yield. 

This blog explores TKD Solutions’ approach to de-risking the transition from adherent to suspension cultures for AAV gene therapy manufacturing. By combining risk assessment and comparability analysis, TKD Solutions ensures a smooth and validated process change, enabling large-scale production without compromising product quality or safety.

AAVs provide a safe and efficacious platform for in vivo gene delivery as AAVs are non-pathogenic and do not integrate into host genomes yet enable sustained gene delivery to target tissue.

Why Transition to Suspension Cultures?

The growing number of AAV gene therapies in clinical trials—over 250—highlights the need for robust and scalable production methods. Adherent cell cultures, while effective for small-scale production, become logistically infeasible as patient populations increase. For example, a Phase 1/2 trial required over 400 ten-layer adherent cell stacks to produce doses for just six patients. ​

Suspension cultures, utilizing bioreactors, offer a more efficient solution for late-phase production.

TKD Solutions’ Approach to De-Risking the Transition

Transitioning from adherent to suspension cultures involves significant process changes, which can introduce risks to product quality and safety. TKD Solutions developed a comprehensive method to evaluate and mitigate these risks:

• Risk Assessment: A side-by-side comparison of adherent and suspension processes was conducted, scoring process attributes based on severity, occurrence, and detection of failure. 
• ​Comparability Analysis: Analytical tools were used to compare critical quality attributes (CQAs) of AAV products from both production methods. 
• Validation: The results ensured that the suspension process met clinical demand without introducing new risks.

Key Findings from the Risk Assessment

The risk assessment identified critical process attributes that could impact product quality and safety. High-risk attributes included cell lysis, empty/full capsid separation, and dilution to target titer. However, these risks were consistent across both adherent and suspension processes, indicating no additional risks were introduced by the transition.

Results of the comparability report showed no deviations between culture conditions.

Comparability Analysis Results

TKD Solutions conducted a detailed comparability study, evaluating CQAs such as strength, purity, identity, safety, and general quality across one adherent GMP lot and three suspension lots. 

The results showed:

• All assays passed acceptance criteria. 
• Suspension cultures demonstrated moderate improvements in certain attributes, such as a higher percentage of full capsids. 
• No deviations were observed between adherent and suspension culture conditions.

Analytical Methods Used

The comparability study included various analytical methods to ensure product consistency:

• Strength: Vector genome titer and infectious titer assays confirmed comparable dosing titers and functionality. 
• Identity: Genomic and protein identity assays verified the consistency of the AAV genome and capsid proteins. 
• Purity: Full-to-empty particle ratios and residual chemical assays demonstrated high purity levels across all lots.
• Safety: Endotoxin, sterility, and replication-competent AAV assays ensured product safety.

Conclusion

TKD Solutions’ combined approach of risk assessment and comparability analysis enabled a seamless transition from adherent to suspension cultures for AAV gene therapy manufacturing. The validated suspension process met clinical demand while maintaining product quality and safety. As more lots are produced, further process improvements and controls will be implemented to enhance scalability and reproducibility.

Read the full white paper to learn more about our science-driven consulting for advanced therapies.

Resources:

^[1] Vigene Biosciences. Wang, JH., Gessler, D.J., Zhan, W. et al. Adeno-associated virus as a delivery vector for  gene therapy of human diseases. Sig Transduct Target Ther 9, 78 (2024).  https://doi.org/10.1038/s41392-024-01780-w 

^[2] https://www.insights.bio/cell-and-gene-therapy-insights/journal/article/3198/are-we-there-yet-after-250aavbased-clinical-trials-do-we-have-a-wellpaved-road-toward-firstinhuman-entry#:~:text=After%20250+%20AAV%2Dbased%20clinical%20trials%2C%20do%20we%20 have%20a%20well%2Dpaved%20road%20toward%20 first%2Din%2Dhuman%20entry?&text=Here%20we%20analyze%20these%20streamlining%20efforts%20 in,regulatory%20filing%20strategy%2C%20for%20AAV%2Dbased%20gene%20therapy. 

^[3] Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum. Gene Ther. 2011; 22:595-604 

^[4] TKD Solutions AAV9 Process Risk Assessment Report (platform based)

^[5] FDA. VISIPAQUE (iodixanol) injection, for intra-arterial or intra-venous use. https://www.accessdata.fda.gov/ drugsatfda_docs/label/2017/020808s026lbl.pdf. FDA Highlights of Prescribing Information.