Engineering Better AAV Manufacturing: From Vector Design to Scalable Purification

Adeno-associated virus (AAV) has become an important delivery platform for gene therapies, but manufacturing remains complex. Yield, full capsid content, process reproducibility, scalability and purification performance cannot be solved independently.

Research from APC and our academic collaborators is exploring AAV manufacturability across the development pathway, from the design of plasmids and production cell lines through to transfection optimization and downstream purification.

Together, these studies point toward a broader principle: better AAV manufacturing starts by designing manufacturability into the process, rather than trying to solve it at the final unit operation.

1. Design the vector and its production components

Modified Plasmids and Inverted Terminal Repeats Enhance Adeno-Associated Virus Production and Performance

Plasmid DNA is a significant contributor to the cost and complexity of transient AAV production. This study examined whether redesigning plasmid backbones and targeted modification of inverted terminal repeats (ITRs) could improve plasmid production without compromising AAV yields.

Modification of the pHelper plasmid backbone increased plasmid yield by 140%. A targeted ITR modification increased pTransgene plasmid yield by 57% and transgene expression by 28%, while maintaining AAV yield.

The findings demonstrate that manufacturability can be influenced before transfection begins. Rational design of the genetic starting materials may reduce upstream material requirements while maintaining or increasing the performance of the resulting vector.

Full publication

• Donohue, N., Bogdanovic, A., Conheady, J., Davin, S., Barron, N. and Glennon, B. (2026). Modified Plasmids and Inverted Terminal Repeats Enhance Adeno-Associated Virus Production and Performance. International Journal of Molecular Sciences, 27, 7603. DOI: *Editor’s choice 10.3390/ijms27177603
  https://doi.org/10.3390/ijms27177603

2. Design the production host

What Makes an “Ideal” Cell Line for Recombinant Adeno-Associated Virus Production?

The host cell is not simply the vessel in which AAV is produced. Growth characteristics, transfection efficiency, cellular response to Rep and Cap expression, secretion, apoptosis, immune signalling and metabolism can all influence vector yield and quality.

This review evaluates eight cell systems used for AAV production and considers the characteristics that could define a purpose-built production host. Desirable traits include compatibility with suspension culture and chemically defined media, appropriate helper-gene activity and the potential to engineer characteristics such as improved secretion, apoptosis resistance and reduced antiviral responses.

Rather than accepting the limitations of today's production hosts, the paper asks a more fundamental question: what would a cell line look like if it were designed specifically for AAV manufacturing?

Full publication:

• Conheady, J., Donohue, N., Bogdanovic, A., Davin, S., Glennon, B., Barron, N. and Smales, C.M. (2026). What Makes an “Ideal” Cell Line for Recombinant Adeno-Associated Virus Production? Biotechnology and Bioengineering. DOI: *Editor’s choice 10.1002/bit.70260
  https://doi.org/10.1002/bit.70260

3. Design the process around product quality

Enhanced Recombinant Adeno-Associated Virus (AAV) Biomanufacturing: DOE-Enabled Transfection Optimization for Maximum Full Capsid Yield and Robust Scale-Up

Triple transfection is governed by multiple interacting variables, making one-factor-at-a-time optimization poorly suited to building a robust and scalable AAV process.

In this study, a two-stage Design of Experiments approach was used to identify and model the transfection parameters affecting transfection efficiency, genome titre, capsid titre and full capsid percentage. DNA amount, transfection-reagent volume, complexation time and pTransgene ratio emerged as important determinants of process performance.

The optimized process delivered an approximately four-fold improvement in full capsid percentage while maintaining genome titres, and the conditions were translated through stepwise scale-up from small-scale experiments to a 50 L WAVE bioreactor.

The work demonstrates the value of moving from parameter optimization to multivariate process understanding, particularly where the objective is not simply greater titre, but a more manufacturable product profile.

Full publication

• Bogdanovic, A., Donohue, N., Glennon, B., McDonnell, S. and Whelan, J. (2026). Enhanced Recombinant Adeno-Associated Virus (rAAV) Biomanufacturing: Design of Experiment (DOE) Enabled Transfection Optimization for Maximum Full Capsid Yield and Robust Scale-Up. Biotechnology Journal, 21, e70249. DOI: *Editor’s choice 10.1002/biot.70249
  https://doi.org/10.1002/biot.70249

4. Design downstream processing as a platform

Towards a Platform Chromatography Purification Process for Adeno-Associated Virus (AAV)

AAV downstream processing remains substantially less standardized than established biologics manufacturing. Processes are frequently developed around individual serotypes, creating repeated development effort and making predictable scale-up more difficult.

This review examines the product characteristics that could be exploited to develop a more broadly applicable chromatography platform, including capsid surface charge, structural properties, size and interactions with receptors and metal ions. It also considers chromatography modes, formats and commercial factors required to translate scientific understanding into a GMP-ready purification strategy.

The goal is not necessarily a single fixed purification recipe for every AAV product. It is a platform built around common scientific principles that allows development teams to begin from a better understood and more transferable starting point.

Full publication

• Bogdanovic, A., Donohue, N., Glennon, B., McDonnell, S. and Whelan, J. (2025). Towards a Platform Chromatography Purification Process for Adeno-Associated Virus (AAV). Biotechnology Journal, 20, e202400526. DOI: *Editor’s choice* 10.1002/biot.202400526
  https://doi.org/10.1002/biot.202400526

A connected approach to AAV manufacturability

Taken together, this body of research reflects APC’s broader approach to AAV development:

• Starting-material design influences the efficiency and economics of production.

• Host-cell characteristics shape productivity, product quality and the biological response to vector production.

• Process conditions determine how effectively those biological and genetic systems translate into consistent, scalable production.

• Downstream strategy must separate the desired product from an impurity profile already influenced by the decisions made upstream.

• AAV-specific analytics use fit-for-purpose and novel approaches developed around the unique characteristics of AAV to better understand product quality and inform process development decisions.

• Connected CMC data preserves the evidence, context and decision history generated across development, helping teams carry process understanding forward into scale-up, transfer and manufacturing.

APC scientists work across these areas, connecting vector and plasmid design, host-cell biology, process development, analytical characterization, scale-up and downstream purification. iAchieve provides the digital layer that helps keep this knowledge connected and usable as the program progresses.

The opportunity is therefore bigger than optimizing individual unit operations. It is to build manufacturability into AAV development from the beginning, using deeper process understanding to improve robustness, scalability and product quality across the development lifecycle.