7 Essential Steps of the AAV Manufacturing Process

Efficient AAV manufacturing is essential to the success of gene therapy programs, yet scaling it reliably remains complex. From plasmid design prep to fill-finish, each step demands precision, innovation, and regulatory alignment. This article outlines the end-to-end AAV workflow, explores key optimization strategies, and highlights how Forge Biologics brings speed and flexibility through its FUEL™ platform.

What Is AAV and Why Is It Used in Gene Therapy?

Adeno-associated virus (AAV) is a small, non-pathogenic virus widely used in gene therapy as a delivery vector. It is widely considered to have a favorable safety profile, triggers only mild immune responses, and can be engineered to target specific tissues.

The 7 Essential Steps of the AAV Manufacturing Process

1. Plasmid Design and Preparation

The first step in the AAV manufacturing process is designing plasmids that carry the therapeutic gene of interest, the AAV replication and capsid (rep/cap) genes and helper functions. This is followed by manufacturing the high-quality research-grade or cGMP plasmids. Here are several key details:

AAV manufacturing involves a producer cell line or plasmids to begin the process: transfer plasmid (therapeutic gene + ITRs), rep/cap plasmid (serotype-specific proteins), and helper plasmid (adenoviral functions).
Plasmids must be highly pure, free of endotoxins, and under cGMP conditions if being used for clinical applications.
Scalability requires high-yield production systems and robust quality control for batch-to-batch consistency.
The plasmid design impacts transfection efficiency, AAV yield, and downstream AAV performance.

2. Upstream Process: Transfection and Cell Culture

Transient transfection of mammalian cells (often suspension-adapted HEK293 cells) in shake flasks or bioreactors initiates AAV production. These are the key features of upstream process development:

Suspension-adapted HEK293 cells are typically used for scalable AAV production in stirred-tank bioreactors.
Transfection is most commonly done using transfection reagents (such as polyethylenimine (PEI)), though electroporation may be used in certain systems.
Bioreactor parameters such as pH, dissolved oxygen, and agitation must be carefully optimized.
Upstream process optimization significantly affects overall AAV vector yield and quality.

3. Cell Lysis and AAV Harvesting

After production, cells are lysed, and the crude lysate is clarified to prepare for downstream purification. Considerations undertaken in this step include:

Cells are typically harvested 3-4 days post-transfection, once peak AAV expression is reached.
Lysis is performed using chemical or mechanical methods, followed by a nuclease treatment to degrade free DNA.
The clarified lysate is filtered to remove debris, preparing it for downstream purification.
Timely and effective harvesting is essential to maximize recovery and minimize degradation.

4. Downstream Purification

Chromatography and filtration steps remove impurities and isolate AAV particles, often followed by full/empty capsid separation. Downstream purification includes:

Affinity chromatography techniques are performed utilizing different resins such as POROS AAVX/AAV9, etc.
Tangential flow filtration (TFF) concentrates the in-process material and exchanges buffers for subsequent steps.
Empty vs. full capsid separation is performed to enrich the therapeutic vector population, and can be achieved using either Cesium Chloride Ultracentrifugation or Ion-Exchange Chromatography.
Downstream steps aim to produce a more pure and potent vector, and ensure regulatory compliance for clinical use.

5. Process Development, Optimization, and Scale-Up Strategies

While not always considered a "step" in the manufacturing run itself, process development is a parallel activity and is utilized to optimize each stage, ensure scalability, and reduce risk during clinical and commercial manufacturing. Some of these parallel activities are:

Optimizing processes from bench-scale to cGMP production while maintaining yield and product quality.
Translating early research methods into scalable, cGMP-ready processes by optimizing upstream and downstream parameters.
Applying Design of Experiments (DOE) to identify ideal transfection conditions, plasmid ratios, and purification settings.

6. Formulation and Fill-Finish

The AAV product is buffer-formulated and aseptically filled into final containers under cGMP conditions. Here are some of the key details or considerations:

AAV is formulated into stabilizing buffers that preserve potency and prevent aggregation.
Cryoprotectants and surfactants may be added to enhance stability during storage.
Aseptic, single-use systems are used in cGMP environments for sterile fill-finish operations.
Final drug product is filled into vials or syringes under strict regulatory controls and labeled accordingly.

7. Analytical Development, Quality Control, and Release Testing

Rigorous testing ensures the final product meets safety, potency, and purity specifications before release. For example:

Developing and qualifying product-specific analytical methods to assess potency, purity, genome titer, and critical quality attributes (CQAs) early in the process.
Ensuring process robustness and product consistency by linking production parameters to analytical readouts for smoother scale-up and regulatory readiness.

Each of these steps must be tightly controlled to ensure safety and efficacy, especially as programs move toward clinical or commercial use.

Recent Technological Innovations in AAV Manufacturing

Recent advances in AAV manufacturing are transforming scalability and efficiency for gene therapy programs. Higher-yielding platforms—like Forge’s FUEL™ platform—offer increased productivity, scalability, and consistent quality attributes. High-throughput screening tools, including mini bioreactor systems, allow for rapid evaluation of constructs and conditions to inform early development decisions.

At the same time, innovations like automation are enhancing process understanding and control. Real-time analytics and automated systems help optimize conditions and improve reproducibility, accelerating the path to cGMP. Together, these tools are setting a new standard for reliable, high-quality AAV production.

What to Know Before Partnering with AAV Manufacturing Experts

Before selecting a CDMO for AAV manufacturing, it’s essential to evaluate their technical capabilities, timeline from process development to clinic and commitment to quality. Look for partners with proven expertise in plasmid manufacturing, scalable upstream and downstream processes, and cGMP-compliant manufacturing. The ability to support programs with regulatory strategy, analytical development, and release testing is equally important—especially as your program advances toward clinical milestones.

Integrated end-to-end AAV manufacturing can significantly streamline development by reducing handoffs, aligning workflows, and ensuring consistent quality from research to production. For gene therapy developers, a fully integrated CDMO often translates to fewer delays, more predictable outcomes, and a smoother path from concept to clinic.

Scaling AAV production often reveals challenges that aren’t visible during early development. Common issues include low vector yields, inconsistent full-to-empty capsid ratios, and limitations in process scalability or reproducibility. Manufacturing platforms that work at small scale may behave differently in large bioreactors, and optimizing each step requires careful coordination. A strong CDMO partner can help anticipate and overcome these hurdles through a platform experience, analytical insight, and robust process development.

Why Forge Biologics Is a Leader in AAV Manufacturing

Forge Biologics has established itself as a leader in AAV manufacturing by combining deep gene therapy expertise with a purpose-built facility and infrastructure designed to accelerate programs from preclinical stages through commercialization. At the center of this operation is the Hearth, Forge’s 200,000 sq. ft. cGMP facility that houses end-to-end capabilities under one roof—including plasmid production, process and analytical development, toxicology-grade and clinical-grade manufacturing, and quality control labs. This integration enables a seamless process, reducing program risk, and shortening timelines for those developing complex AAV-based gene therapies.

What sets Forge apart is its focus on manufacturing innovation, exemplified by a suite of proprietary technologies designed to improve scalability and yield. Forge’s FUEL™ platform delivers an efficient manufacturing foundation with advanced technologies, proven processes, and product-specific optimizations. Powered by Ignition Cells™, Forge’s proprietary suspension HEK293 cell line, and paired with modified rep/caps and pEMBR 2.0™, Forge’s platform has been shown to improve titers by 6- to 22-fold in client programs.

Beyond platforms and facilities, Forge brings a true partnership model to each engagement. From the earliest stages of molecular development through IND-enabling studies, cGMP manufacturing, and commercial readiness, Forge’s cross-functional teams provide strategic guidance every step of the way, in addition to a client dedicated team overseeing each program. The company’s in-house regulatory experts support IND submissions and agency interactions, while its Analytical Development and Quality teams help ensure products meet evolving regulatory standards. Whether working with emerging biotech startups or established gene therapy developers, Forge delivers customized, scalable solutions that help clients navigate complexity and bring transformative therapies to patients faster.

Frequently Asked Questions

How is AAV produced in large quantities?
AAV is produced at scale using a suspension-based cell culture system in stirred-tank bioreactors. Transient transfection or stable producer cell lines introduce the necessary genetic components, enabling efficient vector production suitable for clinical and commercial applications.

What cell lines are used for AAV production?
Commonly used cell lines for AAV manufacturing include HEK293 cells and their derivatives, such as Forge’s proprietary HEK293 Ignition Cells™.

How do you separate full and empty AAV capsids?
Full and empty AAV capsids can be separated using density-based ultracentrifugation or advanced chromatography methods. Forge employs scalable purification strategies that enrich full capsids to support efficacy and regulatory expectations.

Is AAV manufacturing cGMP-compliant at Forge?
Yes. Forge operates a purpose-built cGMP facility for end-to-end AAV manufacturing, with robust quality systems, validated processes, and regulatory oversight to ensure compliance for clinical and commercial production.

How long does it take to produce clinical-grade AAV?
Timelines for cGMP batch delivery are highly dependent on the specifics of a program, including vector design, process readiness, and scope of work. Connect with our Client Development team to discuss your program and receive a more detailed scope of work based on your program needs.