Industrialization of adenoviral vector production in fixed bed bioreactor and amplification of primary liver cells in Xpansion® bioreactor: Autologous insulin producing cells for the treatment of diabetes, from bench to clinical scale

Diabetes is a major global health problem with over 370 million diabetics and an estimated 550 million by 2030. Current therapies rely on recombinant insulin injection to the patients several times a day to control glucose level but do not address the fundamental problem; the loss of insulin producing cells of the pancreas. Orgenesis developed a cell therapy to replace these cells by taking a small biopsy from a patient’s liver, growing the cells in flatware treating these cells with adenovirus vectors containing the genes required to transdifferentiate them to insulin producing cells. This approach allows the diabetic patient to be also the donor of his own therapeutic tissue, overcoming both the shortage in tissues availability from cadaver and also the immune suppression. To bring this cell therapeutic approach into the pre-clinical and clinical phases, Orgenesis and Pall combined their respective expertise to develop a strategy to manufacture both viral and cells products at large scale and with greater control by the usage of two single-use large scale bioreactors. For large scale viral production we used packed-bed iCellis® 500 disposable bioreactor that provides 3D controlled, perfusable system with low shear stress for adherent cells. The Xpansion 200 single-use bioreactor was used for growing the primary human liver cells under controlled culture conditions to generate cell mass required for curing a diabetic patient.In this study, we have optimized adenovirus serotype 5 manufacturing using the iCellis Nano bioreactor with different cultivation area up to 4 m2. HEK293 cell cultivation, infection and harvest of the virus in an adherent environment proved possible reaching total virus yield of 3.4e14 IU/batch. We have successfully scaled up the cell amplification process to the fully closed Xpansion platform technology. Results showed that 1-2 gr of patient’s liver biopsy was expanded to around 1.8 Billion cells in Xpansion 200 bioreactor representing more than the targeted dose requirement of 1 Billion cells per patient. Next step of the study is to focus on develop purified viral stocks, incorporating the viral trans-differentiation step into the developed cGMP cell expansion process

Case study: Single-use platform for complete process development and scale-up of an Adenovirus

Virus production for vaccines and gene therapy relies heavily on adherent cell culture based processes – typically trays, roller bottles or microcarriers. The introduction of the iCELLis® fixed-bed bioreactor and single-use purification operational units provides a full solution for rapid process development at small-scale in the iCELLis Nano bioreactor with direct, predictable process transfer to the large-scale iCELLis 500 bioreactors. The iCELLis 500 bioreactor provides a fixed-bed with 66 - 500 m² of surface area for cell growth. A 500 m² fixed-bed is equivalent to 5,882 roller bottles of 850 cm² or 278 HYPERStack 36. Coupled with other single-use upstream and downstream technologies, the iCELLis bioreactor and purification membranes provide a complete single-use platform for virus production. We have performed the complete process development and scale up of an adenovirus (Ad5) process using HEK-293 cells. The reference process using 2D flatware was transferred, optimized and scaled-up into the iCELLis Nano bioreactor (surface areas of 0.53 - 4.0 m²). Based on results in the iCELLis Nano bioreactor and virus requirements, the 66 m² large-scale iCELLis 500 fixed-bed bioreactor was selected. In order to simplify the seed train, the fully-closed, single-use Xpansion® multiplate bioreactor was used. The upstream yield from the iCELLis 500 66 m2 bioreactor was 1.04 x 1016 of Infectious Units (IFU). In this study we optimized and developed adenovirus purification manufacture processes using Mustang® Q with bind/elute strategy managed to reduce significantly the impurities such as HCP and HC-residual DNA and enriching full vs. empty capsid (\u3e90%). The eluted Ad5 from Mustang Q membrane is immediately processed through the UFDF for further concentration and buffer exchange to final virus formulation buffer. Final purified product was then sterile filtered and vialed for potency studies. Downstream processing utilized single-use systems with 62% recovery for a final purified yield of 6.42 x 1015 IFU. Analytical characterization of the virus met specifications and in vivo GLP toxicology testing results were comparable to material produced using the reference process. Scalable upstream and downstream strategies for production and purification of virus based product as such described here offers a fast-to-market, more cost effective alternative to traditional processes. We will review the iCELLis bioreactor platform and downstream purification platform and present as a case study the process development and scale up of the complete adenovirus (Ad5) process. Please click Additional Files below to see the full abstract

Tangential flow filtration and scalability in viral vector purification

Pall Biotech has linearly scalable tangential flow filtration technology that can cover processing volume ranges from less than a liter to over 2000 liters. Manual assemblies can be used with Pall TFF filters in scales under 20L and programmable skids are available for larger scales. In this document TFF linear scalability of up to 200 liters is covered starting from a 7L initial volume and maintaining similar pressures, processing times and yields at all scales. Using this scalable technology Pall biotech can help biologics companies go to clinical trials and to commercial scale manufacturing successfully. Please click Additional Files below to see the full abstract

Scaling up and industrialization the production and purification of viral vectors for therapeutic use: Challenges and progress

With several recent FDA approvals and a strong drug pipeline, gene therapy is coming of age. With this comes the requirement to ensure that there are robust manufacturing processes in place in order to scale with demand and to make these therapies readily accessible to those who need them. However, current manufacturing processes for gene therapies have often been developed with limited scalability in mind and large shifts in technology have to take place to enable industrialization. This also has to be done while keeping costs in mind. Here, we will present a case study which illustrates the challenges and solutions to scale both up and downstream process steps required to manufacture adenovirus. After implementation of a bioreactor, the bioreactor scale increased 125 fold, from 0.53m2 to 66 m2. With the implementation of several scalable unit operations on the downstream, this took 1 day as opposed to 3 – 4 days required for the entire optimized process generating purified viral vector for the successfully completion of a global in vivo toxicology study. Altogether, the practicalities around manufacturing virus to industrial scale. Please click Additional Files below to see the full abstract

Clinical Scale Manufacturing Of Autologous Insulin Producing Liver Cells For The Treatment Of Diabetes

Diabetes is a major global health problem with over 387 million diabetics causing 4.9 million deaths worldwide. Current therapies rely on recombinant insulin injection to the patients several times a day to control glucose levels but do not address the fundamental problem; the loss of insulin producing cells of the pancreas. Orgenesis developed a cell therapy to replace these cells by taking a small biopsy from a patient’s liver; growing the cells in flatware treating; and transdifferentiating these cells with adenovirus vectors containing genes for insulin production. This approach allows the diabetic patient to be the donor of their own therapeutic tissue, overcoming both the shortage in cadaver tissue availability and immune suppression. To bring this cell therapy approach into the pre-clinical and clinical phases, Orgenesis and Pall combined their respective expertise to develop a manufacturing strategy for both viral vector and cell products at large scale with the added benefit of greater process control incorporating two single-use large scale bioreactors. The Xpansion® 200 single-use bioreactor was successfully used to scale-up the human adult liver-derived cells proliferation process. By using the Xpansion platform, Orgenesis now has a reliable process to amplify their cells from 10-25 million up to 1.8-2 billion cells required for curing a diabetic patient. The cell mass generated by the bioreactor preserves their viability and potential for trans-differentiation. As a result of this successful co-development partnership, Orgenesis is moving forward with their process to large-scale clinical studies for GMP-compliant commercial manufacturing of AIP cell for transplantation. For large scale viral production we used the packed-bed iCellis® 500 disposable bioreactor that provides 3D matrix in a controlled system with low shear stress for adherent cells. In this study, we have optimized manufacturing for three adenovirus serotype 5 using the predictive small scale iCellis® nano and the manufacture scale iCellis® 500 bioreactor with cultivation area of 66 m2. By optimizing culture and infection parameters such as HEK293 cell seeding density, multiplicity of infection, time of infection, day of harvest, and media circulation parameters, yield was increased to 5.6x1015 infectious virus particle per batch (iCellis 50066). These cells and viral vector yields make the Xpansion® 200 and the iCellis® promising scalable technologies for the production of adenovirus products. The purified adenovirus hPDX-1, hNeuroD and hMafA were fully functional and successfully transduced the target liver derived cells. Next step of the study is to incorporate the viral trans-differentiation step into the developed cGMP cell expansion process. Please click Additional Files below to see the full abstract