Meet our team at the ESGCT: Oct. 24-27, 2023 in Brussels, Belgium
The ESGCT conference will take place October 24-27 2023 at the Tour & Taxis complex in Brussels, Belgium.
Meet our team during the event:
Stop by our booth #48 to meet our team during the ESGCT conference and learn more about our extended services to bring your cell and gene therapies to life… From process development up to commercial launch, Yposkesi is the full-service CDMO of choice, working by your side, to meet your timelines and budget.
Yposkesi will present several posters during the ESGCT conference:
Poster 1: Comparison of the transduction efficiency of T–cells using LVV produced from suspension- or adherent-based production systems.
A Parcelier, A Larbi, E Neveu, A Auffret Cariou, C Rousseaux, B Mullan, and S Charrier.
Gene transfer technology using lentiviral vectors (LVV) is one of the most promising therapeutic approaches for cancer immunotherapy (CAR-T or TCR cells). However, an important manufacturing challenge is the industrialization of lentiviral vector production, especially given the potentially variable quantity/volume requirements for these new applications. Therefore, we have recently launched a scalable lentiviral vector manufacturing process based on quadruple plasmid transient transfection of HEK293T cells grown in suspension in bioreactors in serum-free conditions (50L- 200L scale), called Lentisure™. This platform benefits from more than 10 years of cumulative experience in LVV manufacturing at Yposkesi. Given potential concerns between the product quality and product performance of LVV produced either by adherent- or suspension-based production systems, we have performed a comparison of LVV produced from both production systems.
The cell culture harvest from the suspension-based production system was purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The overall yield of manufactured lentiviral vector was higher than that obtained with an adherent process, for a comparable titre. The final LVV was characterized for infectious viral titre (IG/mL), particle content (ng p24), and process related impurities (protein and DNA).
We then evaluated the efficiency of this new lentiviral vector in parallel to a vector produced by an adherent manufacturing process. Results showed a comparable transduction efficiency of Jurkat cells and activated T-cells between lentiviral vectors produced by both adherent and suspension manufacturing processes, with a good correlation between multiplicity of infection (MOI), vector copy number and protein expression. We also demonstrated similar behaviour of T-cells transduced with a lentiviral vector produced either by an adherent or the new suspension processes.
In conclusion, lentiviral vectors manufactured by our Lentisure suspension platform resulted in a larger quantity of infectious and physical LVV particles, with comparable product quality, including transduction efficiency in T-cells, to lentiviral vector produced by more conventional methods (adherent-based LVV production). These studies may help clinical programmes who wish to transition between production systems (suspension, adherent) based on their clinical needs and future patient demand.
Poster 2: Scalability and bioreactor supplier change for GMP lentiviral vector manufacturing in a suspension cell culture system.
A Auffret–Cariou, N Avenier, S Charrier, S Findakly, B Mullan and C Rousseaux.
An important challenge in the manufacturing of lentiviral vectors (LVV) is to be able to provide different quantities of high-quality vectors for different needs, in a scalable manner that ensures comparable yield and product quality. To this end, we have recently launched a suspension lentiviral vector manufacturing process platform based on quadruple plasmid transient transfection of HEK293T cells grown in bioreactors in serum-free conditions at 50L and 200L scale, called Lentisure™. This platform benefits from more than 10years of cumulative experience in LVV manufacturing at Yposkesi.
However, the quantity of LVV obtained at 200L bioreactor scale with our process is often in excess of actual CMC needs for new therapeutic approaches, such as for cancer immunotherapies (CAR-T or TCR cells), especially at early stages of development. As such, we have scaled-down our 200L bioreactor scale lentivirus manufacturing process to a 50L bioreactor scale, while also switching to a different bioreactor equipment supplier, in order to adapt to these lower needs of high-quality vectors and at a more competitive cost. The bioreactor supplier change (new culture bioreactor technology) was performed for reasons of operational flexibility.
For the scale down approach, the cell culture conditions were optimized taking into account bioreactor geometries and the different gassing and mixing capacities of the new bioreactor platform. All other process parameters and steps were also reduced/scaled back (from 200L scale to 50L scale) to fit with the harvested volume to be purified (50L).
We demonstrate that the cell culture performance and product quality in both bioreactor systems were similar. Results will be presented for infectious viral titre (IG/mL), particle content (ng p24), protein expression (Flow cytometry) and process related impurities (protein and DNA). The work will demonstrate that the product quality profile of the lentiviral vectors produced by both suspension manufacturing processes at 50L and 200L are comparable, with good correlation for cell culture process performance, viral titre, and product quality. Finally, the results are also compared to similar data from the Lentisure LVV adherent manufacturing platform, which further show that the quality of the LVV produced is not impacted by the process (adherent or suspension) or scale (50L or 200L suspension) used for LVV manufacturing with the Lentisure platform.
In conclusion, we have scaled down an LVV suspension manufacturing process from 200L to 50L Bioreactor scale, while changing equipment supplier. Process performance and product quality was comparable between scales and equipment, and also between adherent and suspension manufacturing platforms, which highlights the adaptability of the LentiSure platform to adapt to changing needs (lower quantities, same quality) for LVV clinical programmes.
Poster 3: Evaluation of several process cell lysis reagents as replacements for Triton X-100 for rAAV production.
E Neveu, A Parcelier, N Avenier, D Desravines, C Rousseaux, B Mullan, and S Charrier.
Triton X-100 is a detergent used to lyse production cells and release AAVs during rAAV manufacturing processes, which also has a complementary activity of detergent viral inactivation on enveloped adventitious viral contaminants. Due to its degradation to 4-tert-octylphenols, which have harmful endocrine effects, the use of Triton X-100 is no longer permitted by the European Union (REACH regulations).
In this work, we have evaluated four different cell lysis reagents for rAAV production and also for viral detergent inactivation properties. For cell lysis, results show that the required duration of cell lysis (10, 30, 60, 90 and 150 min) is different for each lysis reagent, but after 150min of incubation, a comparable rAAV production is reached for all agents. Secondly, the lysis reagents do not have similar efficiency as regards adventitious viral inactivation compared to Triton X-100. As previously shown by others, we have confirmed that Polysorbate-type reagents are sufficient to lyse cells, but not to inactivate enveloped viral particles and it is necessary to add a solvent such as TnBP, which is already used for viral inactivation in plasma products, to ensure viral inactivation.
Regarding our results, a new detergent, Simulsol (SL-11W) seems to be promising for both cell lysis and adventitious viral inactivation, and has the advantage that an additional solvent is not required to be added for adventitious viral inactivation effects. Whereas Triton X-100 and SL-11W have distinct membrane-disruptive effects in terms of their mechanisms of action, we have confirmed SL11-W effects cells lysis during the production of rAAV2/8 and rAAV2/9 vectors (bulk harvest) and have also demonstrated its efficacy for viral inactivation.
In conclusion, different polysorbate detergents demonstrate different properties (required incubation times) for AAV process cell lysis and require additional solvents if adventitious viral agent inactivation is also desired, and SL-11W is a potential candidate to replace Triton X-100 for rAAV production.
Poster 4: Leveraging platform and process characterization data to accelerate cell and gene therapy process validation and commercialization.
B Mullan on behalf of BioPhorum Cell and Gene Therapy consortium member companies.
There is clearly a strong desire within the cell and gene therapies (CGT) field to create common platforms and approaches to develop, manufacture, and validate these novel therapies, which for some gene therapies only differ by their genetic payload. As of the beginning of 2023, almost 30 different CGTs have been approved by regulatory authorities, with diverse delivery vehicles and routes of administration to address a variety of diseases. Many more products are in development and the number is expected to grow exponentially in the coming years.
Even with this progress, the CGT industry is still facing many challenges including limited material availability for development and clinical use, weak product understanding and analytics, limited prior knowledge, and high cost of goods, among others. To address some of these challenges, there is a strong desire to create common approaches to manufacture these novel therapies. Such “platforming” approaches, including leveraging prior knowledge from decades of manufacturing of traditional biologics, require a strong understanding of the regulatory requirements, manufacturing facility and equipment design, and production process design and control across therapeutic technology platforms. Strategically planning what is needed at what phase of development can be crucial in accelerating commercialization of these products, significantly cutting down the time needed to reach patients.
Despite interest in such approaches, and some initiatives in this area, there is currently no available guidance or best practice to aid CGT developers in this regard. To fill this gap, CGT companies from BioPhorum have evaluated opportunities to leverage process development and characterization knowledge across both CGT and other therapeutic technology platforms to accelerate subsequent process validation activities using experience gained with similar product types.
An overview of the regulatory environment and manufacturing approaches will be presented, followed by identification of specific opportunities for leveraging prior knowledge and experience in a phase appropriate manner. Examples of approaches that various companies in the BioPhorum Cell and Gene Therapy (CGT) consortium are taking will be presented. These examples and approaches are informed by the results of a survey on this topic from 20 member companies within BioPhorum CGT. The evaluation focuses particularly on manufacturing aspects and covers both cell and gene therapies. This work will also be published by BioPhorum as a white paper later in 2023.
We believe this initiative will be a valuable resource to CGT developers as the industry strives to move towards with such platforming and prior knowledge-based approaches, which are already well established for other therapeutic modalities.
Poster 5: Current approaches and considerations for viral clearance in cell and gene therapy (CGT) manufacturing processes.
B Mullan on behalf of BioPhorum Cell and Gene Therapy consortium member companies.
The assurance of viral safety in cell and gene therapy (CGT) products poses a unique challenge as the viral vector is a key component of both in vivo and ex vivo gene therapies, creating unique challenges to clear or inactivate potentially present adventitious viral agents from the manufacturing process, when the therapeutic product itself is a virus. Although viral clearance strategies for general biological drug manufacturing and vaccine production will be applicable to these products, there will be unique challenges and considerations for CGT modalities.
BioPhorum Cell and Gene Therapy (CGT) is an industry-wide consortium that supports the quest for better and faster development of cell and gene therapies. The BioPhorum CGT validation workstream published a white paper in June 2023 that details in a single location the major relevant existing guidance and advice on viral clearance for viral vectors with an emphasis on adeno-associated viral (AAV) vectors. This works highlights the unique considerations for CGT, provides potential options for resolving these challenges, and proposes a ‘gold standard’ approach for viral clearance for the manufacture of cell and gene therapies.
Key thematics and messages from the paper will be presented here, including the regulatory landscape, phase appropriate considerations, viral clearance and inactivation mechanisms, suggested strategies for viral clearance validation, and modes of viral clearance.
We believe that these approaches will be a valuable resource for the CGT sector in order to continue to ensure that CGT remain safe for patients, through sharing the experience and expertise of the BioPhorum CGT member companies, and leveraging well-established approaches for viral clearance from other applicable therapeutic modalities.