In this podcast, we spoke with Margherita Neri, Director of Vector Process Development, Milan Site at AGC Biologics, Andrew Laskowski, Global Product Manager Bioreactors at Cytiva and Andreia Pedregal, Upstream Applications Specialist Manager at Cytiva about large-scale viral vector manufacturing. Our conversation included discussions around scalability, AAV (adeno-associated virus) and lentivirus production platforms, adherent culture, and next generation bioreactor improvements.

I began the interview by asking Margherita about her work at AGC Biologics. She explained that as the Director of the Vector Process Development Unit, her team is responsible for process development of large scale viral vector production for gene therapy applications. Her team is also the first point of contact for new clients.

Next, we talked about the types of viral vector platforms that AGC Biologics operates. Margherita described that at their Milan site, they offer AAV (adeno associated virus) and lentiviral vector production platforms in adhesion and in suspension, at 50-to-200-liter scale with expansion planned for up to 1,000 liters.

I then asked her about some of the differences between adherent cell culture and suspension cell culture paths to commercial manufacturing. Margherita said that the first consideration is that most clinical trials in gene therapy have been sustained with vector produced from adherent cells, typically via processes performed using Cell Factory™ or Cell STACK®. Now that those gene therapy products are being commercialized, manufacturers need to increase scale and demonstrate comparability using a minimal comparability exercise. So, systems that allow adherent scale up are very useful in this process.

Suspension processes are appealing from a scalability point of view because historically they were used for traditional protein bioproductions which can be scaled up to 20,000 – 30,000 liters. Of course, this scale still needs to be demonstrated for vector production that is performed mainly using transient transfection at 200-500 liter scale for lentivirus and between 500-to-1,000-liter maximum scale for AAV. Margherita went on to say that another important aspect in comparability between adherent and suspension systems is quality of the vector in terms of impurity profiles. She said that with adherent processes, cells are attached to the growth support, and the levels of host cell protein and cell DNA are lower when compared to suspension processes. This is very important for lentiviral vector production that is used in vivo where the requirements for impurity levels are very challenging, especially considering that for lentiviral vectors there is currently no affinity step for purification.

I followed up by asking her how AGC Biologics can help customers that want to stay in adherent culture to scale up from current processes, for instance, from flatware to larger-scale production. She explained that when customers ask for a scale increase, they usually offer the iCELLis™ platform. First, they demonstrate at small scale the feasibility of the transition from flatware to the iCELLis bioreactor using the iCELLis Nano bioreactor. Using the iCELLis Nano bioreactor, AGC Biologics has developed a full upstream and downstream process that is highly representative of their process using the full-scale iCELLis bioreactor.

AGC Biologics can then propose that customers use the vector produced in the iCELLis scale-down model to perform a comparability study between a clinical vector and the future commercial or large-scale vector. This comparability should be based not only on the comparison of titers, residuals, and all the CQA, but also AGC Biologics suggests performing a test of cell transduction on the target cells (i.e. CD34 or T cells) and evaluation on these cells of transfection efficiency – vector copy number, residuals and functionality.

I followed up by asking Margherita about whether the iCELLis system was scalable. She said that yes, in their experience the system is scalable, with some work required to perform the scale up from the iCELLis Nano bioreactor to the iCELLis 500 + bioreactor. They did a lot of experiments to verify the scalability from the iCELLis Nano to the iCELLis 500 +bioreactor. After these preliminary studies the productivity data in the full-scale runs was very comparable to the runs that were obtained in the iCELLis Nano.

Next, I asked Andrew about any recent improvements that had been made to the iCELLis bioreactor. He explained that the iCELLis bioreactors have been commercially available for a little over ten years. The company has been launching new products and improvements to this family on an almost annual basis. For the iCELLis 500 bioreactor specifically, they launched a significant hardware upgrade in 2017 of the iCELLis 500 + and this upgrade allows the system to be implemented into GMP clean rooms much more easily. They have also continually upgraded the single-use system (the vessel) for the iCELLis 500. They began offering genderless sterile Presto™ connectors on the vessel also in 2017. In 2019, they integrated more stable and reliable single use sensors. “And most recently, in 2021, they launched an improved and more robust iCELLis 500+ bioreactor, called the Generation R vessel”.

For the iCELLis Nano, they launched the mPath™ control tower with a new docking station and Pall Link software back in 2018. Then they launched the second-generation vessel and weldable manifolds in 2020. They recently launched mPath Link software, which is their second-generation control software. It includes new features such as enhanced multiphase recipes, a mobile phone application that allows monitoring and control of the bioreactor from the convenience of your phone, as well as the ability to receive e-mail or text message alarm notifications remotely. Finally, they have validated the bioreactor to a higher standard of electromagnetic compatibility. The system has immunity against more severe electromagnetic interference events, which makes it a more stable and reliable system.

AGC Biologics was one of the first companies to receive electromagnetic compatibility upgrade; I asked Margherita about the improvements. She said that with the previous version of the iCELLis Nano that they experienced some electrical issues that affected some runs, but after the upgrade, they didn’t experience any more events and the upgrade improved their successful batch completion rate by over ten percent.

Next, I asked Margherita if she had any data that she could share on how the scaled-down iCELLis Nano is representative of the results in the iCELLis 500. She shared that they have completed more than 100 runs in the iCELLis Nano and about 10 runs in the iCELLis 500, both with LV and AAV vectors. The process development work was performed with GFP transgene as the gene of interest. They obtained very comparable titers in the iCELLis Nano and the iCELLis 500 in the bulk vector. For example, for AAV they used the 333 square meter packed-bed and they obtained 7×10 vector genomes per ml (AAV6) and 1×10 VG/mL (AAV8). For lentiviral vector production, they used the 133 square meter packed-bed and they produced an average of 4×10 per ml. This data was confirmed with different genes of interest from different clients.

Next, I asked Margherita what she found to be the biggest benefits of working with the iCELLis portfolio. She said that it was the first instrument that allowed them to scale up their gold standard process previously in Cell Factory vessel. They were able to scale up to 133 square meters very consistently, exploiting efficient control of gases and temperature that of course was absent in the traditional Cell Factory asset. The iCELLis system also allowed them to move to a more compact and closed system suitable for GMP requirements, particularly important with a commercial product. She also emphasized that they have had a very good experience with Pall Life Sciences (now Cytiva) technical support that helped them transition from the Cell Factory to the iCELLis bioreactor.

I followed up by asking Margherita about specific examples of the technical support she received. She said that she can give very good feedback for the technical support. From a scientific and engineering point of view, they were supported in the development of their platforms, in particular by Andreia who helped them understand the instrument and provided local technical support in Italy. This was a key factor in ensuring the speed and readiness of intervention in case there were any issues with the instrument.

Since Andreia was part of our panel, it was the perfect opportunity to ask her about the kind of support that she provides for iCELLis system customers overall. Andreia started by saying that she was representing the worldwide upstream field application team at Pall Life Sciences (now Cytiva), a team of scientists with bioreactor and bioprocess expertise. Their mission is to help customers successfully run their processes with iCELLis technology. The team listens to their customers to understand the process that has been developed. They then translate that process or help design a process that will be transferred to the iCELLis system. Then they come to the client site and conduct operator training to execute that experimental plan. She added that when they finish that training, the collaboration is only beginning as they plan follow up meetings to discuss the data generated and discuss process optimization and process scale-up.

I then asked Margherita about how they have integrated the iCELLis Nano and iCELLis 500 + bioreactors into their manufacturing platform. She explained that they have the AAV platform with the 333 square meter packed-bed that allowed them to obtain 480 liters of viral vector. They also developed a lentivirus platform with the 133 square meter packed-bed to obtain 200 liters of viral vector, and now they are able to offer that platform to their clients. They are also working to increase the packed-bed to 500 square meters as the maximum size for both vectors in order to reach 800 liters of bulk vector. Margherita continued that she thinks it is important to be able to offer this kind of technology to clients and it has been very useful for them to invest in this area.

I closed the interview by asking Andreia about delivery of the 200 iCELLis 500+ bioreactor systems to customers and what she thought customers saw as the biggest benefit of the system. She said that iCELLis technology enables manufacturing of biologics by reducing the process footprint. The iCELLis bioreactor can replace up to 6,000, 850 cm roller bottles or 800 Cell Factory vessels. This ultimately leads to a reduction of the operation costs that are associated with manual operation of all that flatware. Another result is the reduction of process risks linked to the demanding number of manual operations. Lastly, she mentioned the scalability from bench to manufacturing scale and the ease of use and implementation including the features that were designed to operate the iCELLis 500+ in a GMP clean room.

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