Introduction: Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A165) and a FLAG-tagged pre-processed short form D (DΔNΔC, Ad.VEGF-DΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. Objective: To compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A165, Ad.VEGF-DΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-DΔNΔC) where the FLAG tag is removed. Methods: We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. Results: VEGF expression was higher after Ad.VEGF-DΔNΔC-FLAG and Ad.VEGF-DΔNΔC transduction compared to Ad.VEGF-A165 in all EC types (*p<0.001). Tube formation was higher in ECs transduced with Ad.VEGF-DΔNΔC in all species compared to other constructs (***p<0.001, *p<0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p<0.001). Conclusion: We observed high transduction efficiency and functional effects of Ad.VEGF-DΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial.

rAd-IFN is a recombinant adenoviral gene therapy vector encoding IFNα2b gene for the treatment of refractory non-muscle invasive bladder cancer. The vector transduces bladder wall cells where IFNα2b gene is expressed leading to death of cancer cells. The advanced testing strategy to determine the pharmacological activity of rAd-IFN drug product involves three key assays: 1. Infectious titer of the virus, quantitative assay 2. Expression of the transgene (IFNα2b), semiquantitative assay 3. Potency (IFNα2b mediated cell killing), quantitative assay The infectivity and transgene expression assays have been performed for batch release and stability monitoring of activity during Phase 2 and will remain unchanged in principle for Phase 3 and commercial use. In the infectivity assay the cells supporting adenovirus replication are infected with three concentrations of adenovirus and left to produce the virus for two days. The percentage of infected cells is then determined with a flow cytometer utilizing a fluorescently conjugated antibody against an adenoviral structural protein. Samples are analysed in parallel with a reference standard and infectivity is given as relative Infectious Units / ml. In expression assay, the IFNα expression capability of the virus preparation is determined by infecting IFN insensitive cells with the rAd-IFN virus and the concentration of produced IFNα is measured with a commercial IFNα ELISA (enzyme-linked immunosorbent assay) from cell culture supernatants For Phase 3 a new potency assay is developed and added to release and stability testing in order to provide evidence that batches of rAd-IFN are able to produce active IFNα2b which has a relevant pharmacological effect. In this assay cells are transduced using multiple dilutions of reference standard and test samples leading to expression of IFNα2b and subsequent cell death. Cell killing efficiency is determined using colorimetric method measuring dehydrogenase activity of the living cells. Relative potency of test sample is determined against reference standard response curve after testing parallelism by equivalence test. All activity assays will be fully validated according to ICH Q2 (R1) prior to release testing of Phase 3 clinical study material (Accuracy, Precision, Specificity, Linearity and Range, System Suitability and Robustness). The three validated assays will provide enhanced quantitative measure of biologic function of the rAd-IFN vector and thus demonstrate the quality and efficacy of drug product batches.

Single use disposable technology faces further challenges in producing viral vectors in sufficient large quantities especially where adherent cells are needed. Scaling up the adherent system has proven to be troublesome. The PALL iCELLis® disposable fixed-bed bioreactors offer a novel option for viral vector manufacturing in large quantities in an adherent environment. We have made process development in iCELLis Nano, where the cultivation area varies between 0.53 - 4 m2, after which we went forward to iCELLis 500, where the cultivation volume can be upgraded to 500 m2 (66 - 500 m2). iCELLis 500 has proven to be ideal to satisfy upstream demand and large-scale downstream purification process was developed to supply high quality recombinant adenovirus based gene products in our fully-licensed GMP manufacturing facility for pre-clinical and clinical trials The process is initiated by HEK293 cell mass expansion in suspension mode using Biostat® CultiBag RM (Sartorius Stedim Biotech S.A.) bioreactor. The expanded suspension cell mass is inoculated into iCELLis 500 for further expansion in adherent mode. This is infected by Working Viral Seed Stock and subsequent virus is released from the infected cells by chemical lysis. Downstream process contains Benzonase digestion, clarification, concentration and conditioning by crossflow ultrafiltration, capture and polishing by anion exchange chromatography, and final concentration and formulation is achieved using crossflow filtration. All product contact parts are fully disposable. Several batches have been produced with consistent results. Further validation of the process for commercial manufacturing is currently ongoing. We established a scalable, large-scale manufacturing process to supply high quality recombinant adenovirus based gene products in our fully-licensed GMP manufacturing facility for clinical trials. Alongside this we have validated assays which are providing a relevant quantitative measure of biologic function of the vector and demonstrating the quality and comparability of drug product batches in Phase 3 and commercial use.

Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.

To date many early phase gene therapy trials have been successful. However, phase III and commercial phase have brought further challenges in producing viral vectors in sufficient large quantities. Upscaling the process on suspension cells is feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome and costly. The PALL iCELLisTM disposable fixed-bed bioreactors offer an efficient option for viral vector manufacturing in large quantities in an adherent environment. In iCELLisTM Nano, the cultivation area 0.53-4 m2 for smaller batches, is ideal for process development purposes. In iCELLIs500 the cultivation area varies between 66 and 500 m2 and is ideal to satisfy demand for phase3/commercial requirements. We have optimized adenovirus type 5 manufacturing using iCELLisTM Nano. HEK293 cell cultivation, infection and harvest of the virus by lysing the cells inside the bioreactor were efficient, reaching total yield of 3.4 × 10^14 vp/batch. When upscaling the process into 100 m2 cultivation area with iCELLis500, 1.2 × 10^16 vp/batch were produced. iCELLisTM technology is applicable also to other vector types which require for example plasmid transfection. Virus can also be harvested by perfusion from the medium. Lentiviral vector production in 293T cells was tested in iCELLisTM Nano and we achieved high plasmid transfection efficiency, leading to the comparable titers and productivity as in flasks. To conclude, iCELLisTM equipment has provided us an efficient way to manufacture large batches of different kinds of gene therapy products suitable for large preclinical animal models and up to phase III trial and beyond.