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9th Annual Meeting of the American Society of Gene Therapy

Pre meeting session 002: Manufacturing Issues for Gene Based ProductsóHow to Get a Product Out the Door (Gwen Binder, PhD)

Boro Dropulic, MBA, PhD (Chair): Lentigen Corporation: Overview of product development.

Release testing for products includes testing for purity, potency, identity and safety. Each of these tests are unique to the individual biological product but adhere to common CFR guidelines. The rigor of testing is on a sliding scale of stepwise GMP compliance from preclinical to Phase III. The presentations in this session provide a broad overview of the status and issues in today's gene therapy-related manufacturing.

Anthony Green, PhD: Puresyn: Cost effective production of supercoiled plasmids.

Since plasmid production yields low product to contaminant ratios and are large they are difficult to purify. There is no current consensus release testing, and scalability remains a challenge. Increasing potency, delivery efficiency, stability, reducing costly manufacturing steps, and improving expression and fermentation conditions to increase yields, enable cost effective production. The method of purification can affect stability. These parameters should be determined early on to ensure translatable scale-up.

Andy Lin, PhD: Cell Genesys: Scalable manufacturing for oncolytic adenovirus.

Manufacturing changes typically are made in order to meet increased doses, and therefore it is important to think early about developing a scalable process. Comparability is better with processes that can be scaled in a linear fashion. Establishing potency specifications early on is important for the end of Phase II meeting. Reference material, expiration dates, and comparability studies for process changes are necessary. Production cell line specific antibodies are needed to determine host cell protein contamination. Efforts should be made early on for serum free production.

J. Fraser Wright, PhD: Children's Hospital of Philadelphia (CHOP): AAV production in a new GMP facility.

Vector is produced in transfected HEK 293 cells in roller bottles without serum. Tangential flow filtration reduces volume, and vectors are isolated by cell lysis and nuclease digestion. A major issue is empty capsid contamination presently separated by gradient centrifugation which will be replaced by scaleable column chromatography later. Purity is tested by protein gel. Activity is tested by cell transduction, and in vivo delivery in mice. Potency is the most challenging test, and a reference material is required.

Isabelle Riviere, PhD: Memorial Sloan Kettering (MSK): GMP retroviral vector production.

A master cell bank was established for production of a retroviral vector. Primary clone selection was performed on Hela cells then two primary cell selections as determined by titration, protein expression, and expression. Single insert clones were chosen for ease of characterization. Vector was produced in cell factories and collected in bags, and harvested at 32ºC. Vector stocks underwent step filtration and freeze thaw to ensure removal of cells. Current focus is on bioreactor scale-up, stable envelopes, serum free production, titration cell banks and lot to lot consistency.

Vladimir Slepushkin, MD, PhD: VIRxSYS: GMP lentiviral vector production.

There is a bottleneck to clinical application of lentiviral vector due to production issues. Virxsys currently produces lentiviral vector at the largest scale of 100L. Recent production adjustments are reduction in serum from 10% to 5%, increasing cell factories from 16-32, increasing collections to 3 from 2, and using 5L instead of 1L size exclusion columns. Current scale up for Phase III is ongoing and will utilize anion exchange chromatography.

Bruce Levine, PhD: Cell and Vaccine Production Facility, the University of Pennsylvania: Advances in GMP T cell processing.

The first lentiviral vector trial used a single dose of 10 billion T cells, grown under static conditions. For greater cell production enabling multiple doses, a new process utilizing a dynamic system (Wave) has been developed. This results in cell concentrations as high as 20 million/ml although 5-10 million/ml is optimal. A new focus in the CVPF is GMP development of artificial antigen presenting cells (K562 aAPCs) for expansion of various subsets of T cells.

Elizabeth Read, MD: National Institutes of Health: G-CSF mobilized hematopoietic stem cells (HSC) for chronic granulomatous disease (CGD).

An earlier trial for the most severe CGD used a retroviral vector and resulted in 48-89% transduction and poor gene expression. Therefore, an ongoing study is using busulfan preconditioning to enhance engraftment of cells transduced with a murine retroviral vector. Increasing the vector concentration and improving the cytokines used is expected to improve gene marking. The cellular product is subject to rapid release testing since the cells are not frozen.

Ray Harris, PhD: National Cancer Institute at the NIH: GMP HSV vector production.

HSV vector was produced in cell factories using vero cells in 10% FCS in a 3 day incubation. Harvesting occurs prior to cell lysis to reduce serum contamination, followed by mild salt lysis (~500 pfu/cell), chromatographic (sepharose) removal of DNA, concentration by Q sepharose, and final polishing by sepharose 4FF followed by a final benzonase step. Total recovery is 17%, and most is lost in the polishing step (56% to 17%). Final filtration is performed using a 0.45 µm filter to maintain titer (0.2 µm causes 90% loss). The current application is for intravascular delivery for liver cancer.