Cornerstone Biomanufacturing Development Services for Adenoviruses (AdV) provide comprehensive solutions, including optional upstream AdV production parameter assessment and scale-up capabilities to 10 liters. Emphasis is placed on DSP-focused optimization to ensure a holistic approach, enhancing overall process efficiency and effectiveness.
In downstream processing, feasibility studies are available, with options for single-step or full processes. These methods ensure superb purity, achieving over 99% total DNA and protein reduction, while maintaining the robustness of the downstream method. Detailed reports with results and SOPs are provided, along with support after completion, to ensure seamless integration and success in production endeavours.
Cornerstone® Adenovirus Process Development solves these crucial challenges:
Adenovirus Upstream Process Development
Adenovirus Downstream Process Development
Adenovirus Upstream Process Development
Adenovirus Downstream Process Development
This phase often demands significant time and resources, involving products from various producers with different licensing agreements and royalties, driving up costs.
The experienced team optimizes product combinations for peak performance, reducing development time. A streamlined process is also available using Sartorius raw materials.
Frequently the viral seed used in early phases is not highly concentrated nor pure, creating challenges in reproducibility and scale-up. As in other viral vector processes, upstream process development is usually not immediately connected to downstream, leaving a possibility for some upstream choices to significantly impact recovery and/or purity of the final product.
Before starting, the viral seed is thoroughly characterized using the adeno analytical toolkit, ensuring a smooth transition between scales and viral seed batches, and enhancing upstream process robustness. A combination of classical and DoE experimental approaches is employed to investigate parameters that impact both USP and DSP success.
The smallest scale in adenovirus 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.
The Ambr® 250 modular system, featuring 6 single-use vessels operating in parallel, is utilized to reduce variability and accelerate optimization. Processes developed at this scale are scalable across the entire Sartorius portfolio, offering savings of up to 4-5 times 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, USP is optimized to enhance DSP and reduce costs. Scaling up to 5L or 10L allows for the production of sufficient material to test the entire process using scalable products. The entire bioprocess can be performed without freezing material between USP and DSP for shipment, closely mimicking GMP conditions.
In adenovirus upstream processing, analytics play a crucial role in ensuring efficiency and quality. Key parameters such as viable cell density and viability are monitored. Spent media analytics, including glucose, glutamine, pyruvate, lactate, ammonia, and LDH, provide valuable metabolic insights. d/ddPCR is used for viral genome (vg) titers, alongside PATfix Adeno. Total protein and DNA measurements assess target molecules and impurities, integrating USP and DSP. Tools like BioPAT ViaMass, BioPAT Trace, and PATfix Adeno enable real-time monitoring. Additionally, a comprehensive toolbox for downstream sample evaluation ensures thorough adenovirus process control.
The lysis step depends on the chromatography purification that follows. Consequently, the ratio of the components in the lysis buffer has to be adapted.
To define optimal lysis conditions, a holistic approach based on accumulated experience is employed. Different combinations of components in the lysis buffer are tested with subsequent steps in mind. This approach optimizes lysis to align with the following steps while focusing on achieving the highest yield.
To clarify the lysate several steps of filtration are used, firstly coarse and then fine filtration. Sartorius filters with cut off 3, 0.8 and 0.45 μm are used. Expected issues are related to shear stress to which the virus is exposed and filtration scale-up.
Each filtration step is optimized, in regards to expected issues, with an aim to achieve a step recovery close to 100%. If the starting material does not behave as expected, additional steps are incorporated into the clarification process. The filtration scale-up is meticulously planned and tested, provided the material quantity permits.
By performing ultrafiltration and diafiltration we partially purify the product and minimize the volume of material. The possible issues in this step can occur if the material is not clarified enough, if the membrane area is not sufficient, the procedure is time-consuming and product is exposed to shear stress.
The ultrafiltration and diafiltration steps are ensured to be carried out correctly by carefully analyzing various parameters, conducting small-scale tests, and drawing on previous experiences.
The decision on the cleaning steps is influenced by the application, the initial material, customer requirements and other factors. The two most common options are noted in the scheme. Challenges are not expected in this phase.
The final formulation step can be part of the chromatographic step otherwise it is a separate step. If the purification process does not allow combining chromatography and formulation in a single step, an additional chromatographic step or DF is required.
The process is developed with a minimum number of steps to prevent product loss. The final process considers clients’ needs, available equipment, timelines, and other relevant factors.
In adenovirus downstream processing, a variety of analytical methods are employed to ensure quality and efficiency. The PATfix Adeno analytical platform is used for detecting and semi-quantifying the product, as well as monitoring host cell-related impurities released during cell lysis. Additionally, d/ddPCR is utilized for quantitative analysis of viral genome (vg) titers. A cell-based infectivity assay is conducted to assess infectious titer, serving as a crucial analytic to select conditions that do not affect product infectivity. Total protein and total DNA are also monitored.
In summary, each analytical method mentioned is used to measure target molecules and critical impurities, integrating the steps of downstream processing into a coherent and successful process.
The lysis step depends on the chromatography purification that follows. Consequently, the ratio of the components in the lysis buffer has to be adapted.
To define optimal lysis conditions, a holistic approach based on accumulated experience is employed. Different combinations of components in the lysis buffer are tested with subsequent steps in mind. This approach optimizes lysis to align with the following steps while focusing on achieving the highest yield.
By partnering with us, you’ll benefit from:
Read the article Bridging Upstream and Downstream for Improved Adenovirus 5 Bioprocess for more detailed insights
Read the article Bridging Upstream and Downstream for Improved Adenovirus 5 Bioprocess for more detailed insights
To achieve optimal results, we recommend the DoE approach in the optimization of the infection step, including the use of a viral seed that has been extensively characterized. In these experiments, production phase duration and its influence on impurity accumulation and infectious titer are frequently overlooked. Similar to other vectors, the combined use of a DoE approach and spent media analysis can be helpful with identifying the reasons for lower titers in specific conditions.
A well-defined upstream process, with determined robustness at each step, is critical to batch-to-batch consistency. This is best accomplished with an analytical toolset capable of monitoring both product and impurities. By collecting data and establishing critical process parameters (CPPs) during robustness studies, you can more effectively manage your process. Aside from the standard cell culture parameters, nutrients and metabolites in the spent medium, and the physical titer of the product itself, it is important to track impurities critical to your chosen downstream method. Functional titer monitoring typically takes longer to provide data, and while it is not appropriate for at-line process monitoring, it is crucial to do analytics on samples taken during each manufacturing process as a part of quality control strategy.
We can develop and optimize an entire upstream process, from a small scale to 10L, beginning with the construct of your choice. We will procure all Sartorius products utilized during 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 + CIMmultus QA). In order to maximize the bioprocess efficiency, downstream can be additionally co-optimized with upstream.
By working with us, you will have the opportunity to take advantage of our chromatography-based PATfix Adeno analytical tool that can be further optimized to your needs. Furthermore, we have a team of expert analytics who developed a set of fully optimized in-house qPCR and dPCR analytics designed to target a variety of components of GOI constructs, including promoters, polyA sequences, and more. Adenovirus in-house assays have been developed by our cell culture experts on a variety of cell lines. These assays can be customized to meet your specific needs.
There are multiple options for how to approach the DSP development, with the most effective being the Cornerstone process development. We approach every material with a careful analysis and a specific plan, offering the final process with high yield, outstanding purity and results supported by orthogonal analytics.
We are using several orthogonal analytical methods to determine the product concentration and yield as well as to determine impurities and their removal. One of the most important analytical methods we employ are: PATfix Adeno (HPLC analytics enables detection and semi-quantification of the product and monitors host cell related impurities released from the cells during the cell lysis), cell-based infectivity assay (method enables assessment of infectious titer and helps us choose the conditions not affecting the infectivity of the product), d/ddPCR for vg titers (quantitative analysis of product titer), total protein and total DNA concentration methods.
It depends of the application. In general CIMmultus QA and OH can be used for capture step and CIMmultus QA for polishing step.
Binding capacity largely depends on the material used to determine it. We were using nuclease treated diafiltered feed stream and determine the capacity to be 4 – 7 E+12 vg/mL.
The purification method is very robust and can be used with materials coming from adherent as well as suspension cell lines. Method can be further optimized to meet the certain criteria according to client wishes and adapted to clients needs.
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