Our Cornerstone Process Development Services for AAV offer comprehensive upstream solutions, including transfection-based methods, scale-up to 10 liters, and optimization to maximize titer and full capsids. We also integrate DSP-focused optimization for a holistic approach.
In downstream processing, we specialize in serotype-agnostic AAV capture, advanced polishing methods, and analytical development. Our services cover all serotypes, with extensive experience in AAV8 and AAV9. We offer feasibility studies, single-step or full processes, ensuring exceptional purity with over 85% full AAV and 80-95% recoveries in the polishing step. Detailed reports with results and SOPs are included.
Cornerstone® AAV Process Development solves these crucial challenges:
AAV Upstream Process Development
- Raw Material Benchmarking
- Small Scale Optimization of Transfection Parameters
- Optimization of Scale-Independent Bioreactor Parameters
- Scale-up to 10L and Verification, Robustness of USP and DSP
AAV Downstream Process Development
- Clarification
- Ultrafiltration | Diafiltration (UF | DF)
- Capture
- Polishing
- Sterile Filtration
Cornerstone for AAV Upstream Process Development
- Transfection-based: We can collaborate using packaging or producer cell lines if you already have them or can secure the necessary license.
- Scale up to 10 liters
- Maximization of titer and % full capsids – after first round we are at 5E+10 vg/mL and >20%F on a small scale, after optimization reaching over 50% AAV8 on a 10L scale, over 40% AAV9 on a 5L scale
- DSP-focused optimization for holistic approach
This phase often demands significant time and resources, involving products from various producers with different licensing agreements and royalties, driving up costs.
Our experienced team optimizes product combinations for peak performance, saving you development time. We also offer a streamlined process using Sartorius raw materials.
At this scale, most process developments aim to maximize AAV titer. Optimizing the percentage of full capsids requires purified AAV, and analytical throughput often limits this phase.
We use a blend of classical and DoE experimental approaches to investigate parameters affecting both USP and DSP success. Various analytical tools help us evaluate target particles and critical impurities early in AAV process development.
The smallest scale in AAV production is typically 2L. Conducting parallel experiments at this scale requires a significant investment in multiple 2L vessels or more time, increasing variability between conditions. Total volumes escalate quickly, driving up costs.
We use the Ambr® 250 modular system with 6 single-use vessels operating in parallel, reducing variability and speeding up optimization. Processes developed at this scale are scalable across the entire Sartorius portfolio, saving up to 4-5x in raw materials compared to the 2L scale.
Frequently, DSP optimization begins only after the upstream process USP is established, limiting the ability to adjust both bioprocess parts in tandem.
From the smallest scales, we optimize our USP to enhance DSP and reduce costs. By scaling up to 5L or 10L, we produce sufficient material to test the entire process using scalable products. Additionally, we can perform the entire bioprocess without freezing material between USP and DSP for shipment, closely mimicking GMP conditions.
The Role of Analytics in AAV Upstream Processing
In AAV upstream processing, analytics are vital for efficiency and quality. We monitor key parameters like viable cell density and viability. Our spent media analytics (glucose, glutamine, pyruvate, lactate, ammonia, LDH) provide metabolic insights. We use d/ddPCR for viral genome (vg) titers, and PATfix AAV Switcher and monolith multiwell plates for empty vs. full capsid evaluation. Total protein and DNA measurements assess target molecules and impurities, integrating USP and DSP. Tools like BioPAT ViaMass, BioPAT Trace, and PATfix AAV Switcher enable real-time monitoring. Additionally, our toolbox for downstream sample evaluation ensures comprehensive AAV process control.
Bridging the Gap Between AAV Upstream and Downstream
When designing the bioprocess, we develop the upstream process (USP) for AAV with the downstream process (DSP) in mind. The typical DSP includes standard protocols such as TFF with nuclease treatment, capture on CIMmultus SO3, and polishing on CIMmultus QA. To maximize efficiency, downstream can be co- optimized with upstream.
How do we achieve this?
We use a blend of classical and DoE (collaboration with MODDE) experimental approaches to thoroughly investigate parameters affecting both USP and DSP success. Our analytical tools, such as PATfix AAV Switcher, evaluate target particles and critical impurities early in process development, seamlessly integrating USP and DSP.
This ensures the downstream team starts with materials that have:
- A high percentage of full AAV capsids
- A low percentage of empty capsids
- Minimal protein and DNA impurities
- This comprehensive approach guarantees optimal AAV production and robust process control.
AAV Downstream Process Development Services
- Serotype agnostic platform consisting of 5-step DSP process, with overall process recovery up to 50%
- Covering DSP services up to Clinical Phase I
- The serotype-agnostic AAV capture step achieves up to 30% higher recovery compared to the process using the affinity capture step
- Efficient and reproducible AAV E/F separations across scales (laboratory-process development-manufacturing) achieving superb AAV purity with high recoveries employing CIMmultus QA HR
- Reduction of up to 5 log of process related impurities (hcDNA and HCP).
The goal of the harvest clarification process is to produce a clarified lysate that is free of cellular debris and other contaminants, thereby improving the efficiency and yield of subsequent purification steps. In AAV harvest clarification, several filtration steps and types of filters are typically used to ensure the removal of cell debris and other impurities, commonly primary filtration with larger pore sizes (e.g. 0.5-1.0 µm), followed by secondary filtration step with smaller pore sizes (e.g., 0.2-0.45 µm) to further remove fine particles and reduce bioburden.
Concentrating samples (up to 50x) can be a time-consuming process with low capacities, leading to precipitation and aggregation of AAV due to complexation.
An additional step with DNase treatment reduces the required amount of nuclease enzyme typically added to the lysate (up to 10x higher volumes). By operating in high salt conditions and using a salt-tolerant DNase enzyme, the virus is less prone to aggregation and complexation. This approach enhances the efficiency and reliability of AAV processing.
Different types of AAV and their chimeras require a versatile platform solution.
Our platform is designed to accommodate all AAV serotypes, with a capture process that is serotype-agnostic and binds to all. Only the initial salt concentration and pH need to be defined. Operating at low pH (3.5-5.0) is advantageous for viral inactivation, which is a crucial aspect of clinical studies. This approach ensures broad applicability and effectiveness in AAV processing.
The separation of empty and full capsids (E/F) is challenging and serotype-dependent, requiring high dilutions (20-50x) between the capture and polishing steps. Additionally, AAV aggregation is common during dilution in low salt conditions. Our solution involves using Design of Experiments (DoE) to optimize the process and implement new methods. Transitioning between SO3 and QA can be achieved through pH elution, eliminating the need for high dilution. We also use multimodal columns to load at higher salt concentrations, enhancing process efficiency and stability.
Due to aggregation, viral loss occurs. By using an appropriate formulation buffer, we prevent aggregation.
The Role of Analytics in AAV Downstream Processing
In AAV downstream processing, we employ a range of analytical methods to ensure quality and efficiency. Throughout the process, we use dPCR, PATfix, and DNA/protein assays to monitor impurity reduction, aggregation, and the empty/full (E/F) capsid ratio. These methods are applied consistently throughout the entire downstream process, ensuring optimal AAV production and robust process control.
Why Choose Cornerstone® AAV Process Development Services?
By partnering with us, you’ll benefit from:
- Higher resolution between peaks for more accurate subpopulation detection and separation.
- Significantly improved purity of full AAV capsids.
- Serotype versatility across AAV2, AAV5, AAV8, AAV9, and others.
- Cutting-edge analytical tools for handling complex AAV samples.
Additional Resources
Read the article AAV Capsids Separation is Critical for Advancing Gene Therapies for more detailed insights
Hear From Our Customers
Do You Have a Question?
FAQ About AAV Process Development
We can develop and optimise an entire upstream process, from a small scale to 10L, beginning with the GOI and RepCap of your choice. We will procure all Sartorius products for the process development phase, and we can also offer assistance with the licensing of all those that will be implemented in your final process. As long as you can obtain a suitable licence, products from other vendors are welcome as well. We consistently develop upstream processes with ongoing input from our downstream team. Consequently, unless otherwise agreed upon, we will develop a process that aligns with our typical DSP workflow using standard protocols (TFF with nuclease treatment, capture on CIMmultus SO3, and polishing on CIMmultus QA). In order to maximise the bioprocess efficiency, downstream can be additionally co-optimised with upstream. If you require assistance with improving your plasmid sequences or media formulation, we can team up with our colleagues at Sartorius Polyplus and Sartorius Xell to provide the most effective support for your requirements.
Our facilities are located in Ajdovscina, Slovenia. We can support you with process development in our modern, fully-equipped BSL 1 and 2 facilities. Take a virtual tour of our laboratories.
For best results, we suggest thorough optimization of the transfection conditions, that involves cells, DNA and transfection reagent. Combining a DoE approach with spent media analysis can help in understanding why titers might be lower in certain conditions
The easiest strategy to prevent downstream losses is to find a proper balance between your desired product and critical impurities that can make your downstream inefficient and expensive. Frequently, the upstream conditions that maximise AAV titer may not necessarily maximise full capsid production. We encourage starting with a small-scale DoE screening using both titer and %F readouts, then scaling up to the required scale.
By working with us, you can take the advantage of our chromatography-based analytical PATfix AAV platform, which is designed to facilitate process monitoring and quality control. Furthermore, we have a team of expert analytics who developed a set of fully optimised in-house q, d, and ddPCR analytics designed to target a variety of components of GOI constructs, including ITRs, promoters, polyA sequences, plasmid DNA backbone, and more. In addition to mentioned analytical portfolio, Cornerstone process development service now also includes Oxford Nanopore sequencing.
Consult Our Experts
Our experts would be happy to discuss your project. Fill the contact form below or send us an email to cornerstone@biaseparations.com
