Supporting Rapid Development with a Standardized Single-Use Manifold and AAV Platform Process
February 3, 2021
Despite all the challenges it has brought, COVID-19 is shaping an evolution in the drug and vaccine development industry. Suppliers like Pall are working in more collaborative ways than ever before to bring vaccine candidates to market, leveraging single-use platform technologies to speed process development and ultimately, commercialization efforts.
In the first blog of our rapid vaccine development series, Clive Glover, Director of Cell and Gene Therapy, discussed how new integrated platform technologies are enabling process development times as short as eight weeks. In this next blog in the series, we take a deeper dive into the types of technologies that are making this progress possible.
Standardizing Single-Use (SU) Manifoldsfor AAV Platform Processes
It is no secret that single-use technologies (SUT) are becoming more ingrained in bioprocesses as time goes on. Before the pandemic was even a consideration, the teams at Pall and Cytiva were doing work to establish standard platforms for monoclonal antibody (mAb) and viral vector manufacturing. This strategic approach was in response to the ever-growing need for flexible common designs to reduce development times across the biopharma industry. Self-contained manifolds can be adjusted according to the specific requirements of unit operations, providing great flexibility to optimize efficiency, such as through maximization of filter usage and minimization of holdup volume.
Leveraging standard designs reduces the need for new validations and aligns with a set list of parts and components that can be preordered, leading to dramatically reduced lead times. Scaling is further simplified when working with Pall’s Process Development Services (PDS) Group because of extensive experience working with this equipment.
In addition, one universal bio-container bag can be used across the platform (storage, mixing bioreactor, etc.), further reducing the amount of validation required.
Using SUT to Attack COVID-19
For the COVID-19 project with AstraZeneca, the teams worked to standardize not only each unit operation, but the SU manifolds that connect all the operations together. This includes technologies from both Pall and recently acquired Danaher sister company, Cytiva.
The need to standardize was a necessity for this project because there are more than20 different sites manufacturing CHAdOx1nCov-19/AZD1222, each using the 50 or so consumables required for the manufacturing process. If each site had its own customized version, there would need to be more than1,000 parts!
By standardizing the SU manifolds as much as possible, the supply chain is simplified; it is possible to work with preferred components and ensure that these parts get to where they are needed on time rather than becoming a bottleneck for vaccine manufacturing.
And while the specific details of the process established for the Oxford/AZ COVID-19 vaccine cannot be disclosed, information on Pall’s platform for scale-up of a typical Ad5 viral vector intended for use in a gene therapy product can be presented.
In the upstream, Pall’s Xpansion® multiplate bioreactor is used for the seed train and the iCELLis® 500 bioreactor for cell culture. Downstream, three low-shear and scalable technologies are applied for:
- Cell clarification via depth filtration
- Membrane chromatography for purification and polishing and tangential flow filtration (TFF) for concentration after clarification
- Diafiltration after polishing
Starting with an initial volume of 5 L in the Xpansion bioreactor, 3 L of final product can be produced containing >10^12 viral particles using adherent cell culture. The iCELLis bioreactor system is the most mature single-use, pre-sterilized, controlled, and automated adherent cell culture solution available in the market for use in cGMP manufacturing environments. It has already been used for cGMP production of viral vectors, which helps to streamline the approach.
However, because no two processes are alike, the downstream portion of the platform was built with flexibility in mind to accommodate specific requirements, such as additional chromatography and hold steps. With this approach, the platform serves as a starting base and then the equipment is matched to the process, thus ensuring faster implementation of robust solutions. Using this platform, it is therefore possible to achieve operational efficiencies in the order of 30%. The platform can also be adapted easily to suspension culture if desired.
Digging Deeper into an Ad5Process
Process development projects typically began with reproduction of the research-scale flatware process, followed by transfer to the iCELLis Nano bioreactor for process development, which included optimization of the cell-culture and virus harvest conditions. Three runs are then performed and found to give reproducible results, demonstrating the robustness of the process. The process is then scaled to the iCELLis 500 bioreactor using the Xpansion 200 bioreactor in a simplified seed train.
Crude virus is harvested directly onto media for clarification using Seitz® V100P depth filters, which are designed specifically for clarification of viruses and virus-like products. The virus recovery was >90% for this step. Mustang® Q membrane chromatography for capture and semi-polishing provided highly robust and effective removal for host-cell contaminants and empty and partial capsids, with recovery of 96% with >90% full capsids and a total process yield at this point of >65%. SDS-Page analysis throughout the process allowed for monitoring of the product concentration and purity over time.
Importantly, the virus titer was shown to be scalable from benchtop to manufacturing scale. In addition, scaling 125-fold from the initial flatware (0.33 m2) to the iCELLis 500 bioreactor (66 m2) generated a 158-fold increase in virus titer, which translates to an increase of nearly 1 log. Furthermore, the properties of the virus obtained in the iCELLis 500bioreactors were comparable to those of the virus produced using the initial flatware process.
Notably, the downstream process was highly streamlined and scalable, reducing the typical time required from 3-4 days to just one.
Enabling Rapid Large-Scale Production of AAV Vectors
By fully integrating the adenovirus manufacturing platform using adherent cell culture, the team can support reproducible large-scale production of many different adenoviral vectors. Large quantities of high-quality viral vector can be manufactured in compliance with GMP requirements suitable for clinical manufacturing with a total process time of just over 5 weeks, which is important when working on time-sensitive projects.
As importantly, the ability to use this platform process for multiple adenoviral vectors without the need for further optimization can dramatically accelerate development times and reduce overall costs.
Join us next time as we conclude our rapid development series with a look at the six lean product and process development principles (LPPD principles) and the role they play in projects like this.
In the meantime, discover more vaccine blogs by Pall experts here.
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Clive Glover – Director, Cell & Gene Therapy
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