672 iCELLisTM Fixed Bed Technology for Adherent Cells Is an Efficient Scalable System for Viral Vector Production Applications Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The Ameri[.]
CLINICAL TRANSLATION OF VECTOR PRODUCTION AND PROTOCOL PREPARATION II 670 Probing the Stability of rAAV Capsids and Genomic Constructs Using Differential Scanning Calorimetry in Concert With Mobile Phase Modifiers James D Maratt,1 Samantha F Povlich,1 Christopher J Morrison,1 Gwendolyn M Wilmes,1 Samuel C Wadsworth,1 K Reed Clark.1 Pharmaceutical Development, Dimension Therapeutics, Cambridge, MA The recent resurgence of using rAAV vectors as commercially viable therapeutic agents has highlighted the need to identify stable capsid / genomic construct combinations which can both endure a typical cGMP manufacturing process and possess a long product shelf life In this work we investigate the inherent stability of various capsids and genomic constructs using differential scanning calorimetry (DSC) DSC measures the amount of energy required to increase the temperature of a sample and reference and can be used to determine the Tm of the molecule in question Numerous commercially relevant rAAV serotypes containing several genomic constructs (self-complimentary and oversized genomes) were subjected to melting curve analysis using DSC to compare stability Additionally, various mobile phase modifiers were added to solution in order to probe the stability of rAAV vector, using organics and kosmotropic / chaotropic salts These results demonstrate DSC as an effective tool to examine inherent rAAV vector capsid stability and support a role for the serotype and vector genome structure to affect the observed stability 671 Development of a Successful Lyophilization Process for Lentiviral Vector Clinical Batches Nicolas Delacroix,1 Gael Ouengue Mbele,1 Julien Deroyer,1 Guillaume Deplaine,1 Cecile Bauche.1 THERAVECTYS, Villejuif, France Since lentiviral vectors are incrinsingly used in various applications (vaccination, gene therapy, T-cell therapies…) and given that so far the best way to keep these vectors is a -80°C storage, more adapted ways of formulation and storage must be developped Previous attempts have been made to freeze-dry viral vectors : adenovectors and Moloney Monkey Leukemia Virus-based vectors (Cruz et al., 2006) and lentiviral vectors (Shin et al, 2010) None of these attempts have proved satisfactory as the final lyophilized products were not suitable for pharmaceutical applications (mainly due to unacceptable levels of contaminants observed in the final products) We developped an new method to freeze-dry our clinical grade batches of lentiviral vectors This method allows us to store the batches at -20°C and +4°C while maintaining the characteristics of the particules after reconstitution and the ability to use them as intended (direct injection, T-cell modification…) Our method allows the lyophilization of lentiviral vectors either produced in adherent cells cultures (with FBS) or in suspension cells cultures (without FBS and with synthetic culture medium) The vectors are protected during the freeze-drying process by the addition of a lyoprotectant (sucrose or threalose) and stability is evaluated over time Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Quality controls have been performed to evaluate the impurity profiles of the lentivector batches before the freeze-drying process and after the reconstitution of the particules (titer, total proteins, total DNA, HEK 293T proteins), and we demonstrated that the profiles were not impacted by the process and that the batches were still fitting the international pharmacopea recomandations We also made some immunogenicity evaluation of our vaccine candidates before and after the lyophilization process, and we were able to show that the process had no impact on the breadth and intensity of the cellular immune response we are able to elicit after a direct injection of the reconstituted particles in mice THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company THERAVECTYS develops lentiviral vectors used for human vaccination and immunotherapy applications (CAR-T cells and TCR) An in house cGMP facility is now up and running, allowing us to produce and charaterize our own lentiviral vector clinical batches A qualified freeze-dryer has been installed in the plant, to systematically encompass this formulation and repartition step in our bioproduction processes 672 iCELLisTM Fixed-Bed Technology for Adherent Cells Is an Efficient Scalable System for Viral Vector Production Applications Hanna P Lesch,1,2,3 Piia Valonen,2 Kati Heikkilä,2 Eevi Lipponen,1,2 Achim Müller,2 Eva Räsänen,2 Tarja Tuunanen,2 Joonas Malinen,3 Anne Martikainen,3 Tuula Salonen,3 Elisa M Nurminen,2 Minna Hassinen,1,2 Minna Karhinen,2 Nigel Parker,1,2 Robert Shaw,1,2 Seppo Ylä-Herttuala.3,4 FKD Therapies, Kuopio, Finland; 2FinVector Vision Therapies, Kuopio, Finland; 3Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; 4Science Service Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland 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 manufacturing using iCELLisTM Nano HEK293 cell cultivation, infection and harvest of the virus by lysing the cells inside the bioreactor were efficient, S267 Clinical Translation of Vector Production and Protocol Preparation II reaching total yield of 3.4 x 10^14 vp/batch When upscaling the process into 100 m2 cultivation area with iCELLis500, 1.2 x 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 673 Establishment of a National Reference Standard for Reverse Transcriptase Activity Assay and Its Applicability in Quality Control of Gene therapy Products Yonghong Li,1 Chen Wang,2 Lei Yu,3 Chunming Rao,1 Junzhi Wang.1 National Institutes for Food and Drug Control, Beijing, China; Beijing Tiantan Biological Products Corporation Limited, Beijing, China; 3Beijing Institute for Drug Control, Beijing, China The reverse transcriptase (RT) activity assay is widely used in the quality control of biological products including gene therapy products for it can detect all infectious retroviruses in biological starting materials, cell banks and final products But the lack of reference standard for RT activity results in significant systemic error and incomparability between laboratories To standardize RT activity assay, this study established a sensitive real time PCR and developed the first national reference standard, which was prepared from a raw material of recombinant moloney murine leukemia virus reverse transcriptase Our tests indicated that the candidate reference standard met relevant requirements of the national reference standard The activity value of candidate RT standard was determined as 26.4 U per ampoule by a collaborative calibration among four laboratories, with the within-laboratory geometric coefficients of variation (GCVs) ranging from 2.7% to 9.6% and the between-laboratory GCV values being 9.8% The thermal accelerated stability test showed that no loss of activity was observed when the candidate RT standard was stored at 4°C , 25°C and 37 °C compared with -20°C even after months The applicability study showed that the cell bank and viral bank of an adenovirus gene therapy product were positive and the final products were negative As a conclusion, the candidate preparation was suitable to serve as a Chinese national standard for RT activity assay and can be used in the quality control of gene therapy products 674 Development of the Retroviral Vector Manufacturing Process for MM-TK Therapy Simona La Seta Catamancio,1 Manuela Cota,1 Patrizia Mangia,1 Ralitsa Arnaudova,1 Paolo Morandi,1 Marisa Marzocca,1 Michele Manfredini,1 Francesca Rossetti,1 Elena Spoldi,1 Giuliana Vallanti,1 Gian-Paolo Rizzardi,1 Catia Traversari.1 MolMed S.p.a, Milan, Italy cell culture They allow to increase biological and process safety by reducing cleaning and sterilization procedures and the risk of cross contaminations In this context, we are currently evaluating different single-use, closed-system bioreactors for the manipulation of adherent packaging cell lines A feasibility study is on-going, using a single-use bioreactor based on the 2D multi-tray stacks technology Xpantion system (Pall LifeSciences) This study explores the role of gas control on cell growth and its impact on the RVV quality attributes evaluating key metabolites, as glucose and lactate, and viral titer during cell expansion In parallel a manufacturing process in 10-layers cell factories (CFs) has been developed and validated The results obtained with the Am12/ SFCMM-3 Mut2 #48 producer cell line grown up to 10 days in XP10 and in XP50 bioreactors showed an infectious viral titer on the reference cell line CEM A3.01, a transduction efficiency on T cells and an impurities level close to those obtained with the CFs Process development and comparability data will be presented in more detail 675 High Quality Plasmid DNA Manufacturing for Ex-Vivo Protein Synthesis and Viral Vector Production for Gene Therapy Neha Tiwari, Jill Beilowitz, Carlos Sampson, Dorothy Peterson Process Development, VGXI, Inc, The Woodlands, TX High quality plasmid DNA is required for a variety of therapeutic purposes Although use as a DNA vaccine is the most popular, a high quality plasmid DNA product is also required as the raw material for processes like viral vector production for gene therapy, ex-vivo protein synthesis, etc Contrary to a DNA vaccine, plasmid products used as raw materials have varied requirements in terms of purity and presence of contaminant levels in the final product It is essential to have a large scale plasmid production method which can be tailored for the specific plasmid product based on its potential use Our company has designed a plasmid production strategy, consisting of purification steps, for removal of specific contaminants It can be tailored to manufacture plasmid for specific uses at both research and large cGMP scale Plasmid pVGXIa, a raw material for Ex-vivo protein synthesis, was produced using a modified purification approach Since the plasmid was the raw material for protein synthesis and not a drug product, removal of only host cell protein (HCP) as a contaminant was critical during purification Plasmid paste from the fermentation process was purified using a modified step method designed for the removal of HCP The step method consisted of our company’s patented Airmix® technology, Ion Exchange separation and Buffer-exchange The final purified plasmid contained £0.2% of HCP Total circular form of plasmid DNA was 100% A 25% reduction in production cost and 10% increase in yield at the 10L scale were achieved by customizing the purification strategy This same approach can be utilized for cost effective production of high quality plasmid DNA for viral vector generation for gene therapy at large scale The MM-TK Drug Product (DP) is a medicinal product used as adjunctive therapy in leukaemia patients undergoing stem cell transplantation It is constituted of T lymphocytes genetically modified ex vivo with the γ retroviral vector SFCMM-3 Mut2 #48 encoding the HSV-TK Mut2 suicide gene and the cell surface marker ∆LNGFR During clinical development, different improvements were introduced in retroviral vector (RVV) manufacturing in order to obtain simple and robust processes producing RVV with low level of impurities and high performance in lymphocyte transduction Single-use bioreactors are currently replacing conventional bioreactors also for mammalian S268 Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... 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. .. Retroviral Vector Manufacturing Process for MM-TK Therapy Simona La Seta Catamancio,1 Manuela Cota,1 Patrizia Mangia,1 Ralitsa Arnaudova,1 Paolo Morandi,1 Marisa Marzocca,1 Michele Manfredini,1 Francesca... reference standard for RT activity results in significant systemic error and incomparability between laboratories To standardize RT activity assay, this study established a sensitive real time PCR and