Qualification of the COSTIM assay to determine potency and use in clinical trials

19th Annual ISCT Meeting S51

Result: We collected 10 granulocyte products from 4 donors, 3 males,median age 34 years (range 31-35). All 4 donors tolerated the collection well,two donors experienced minor hypotensive episodes. The characteristics[median (range)] of the 10 collection episodes were; donor pre white cell count29.3�109/L (8.8-37.2); total blood volume processed 7972 mL (6000-9884 mL); procedural time 153.5 minutes (104-200); product volume 450 mL(242-670; neutrophil count 3.31�1010/L (0.87-92.22�1010/L); neutrophilincrement 0.12�109/L(0-0.32�109/L).

Conclusion: Granulocytes collected using TerumoBCT Optia� weresuccessful in treating our patient with HCL and severe poly microbial sepsis.A transfusible dose was achieved in all donations. Retrospective comparisonwith historical collections (Cobe Spectra/Dextran 70%) and maximisation ofcollection efficiency are pending.

180QUALIFICATION OF THE COSTIM ASSAY TO DETERMINEPOTENCY AND USE IN CLINICAL TRIALSM Buchholz1, J Knauer2, J Lehmann2, M Hass2, S Gargosky31Prima BioMed GmbH, Leipzig, Germany, 2Fraunhofer-Institute for CellTherapy and Immunology IZI, Leipzig, Germany, 3Prima BioMed Ltd,Redwood City, CA

Potency is one of the key quality control criteria for batch release of a drug forpharmaceutical use. We have undertaken the qualification of a potency bioassayfor our product Cvac, an autologous dendritic cell pulsed with recombinanthuman mucin 1 fusion protein. The challenge with bioassays for cellulartherapies is the inherent individual variability as well as the complexity ofassessing a biological system. Functionally, dendritic cells (DC) activate T cellsin vivo by presenting a T cell with a specific antigen which results in T cellproliferation and release of cytokines. T cell stimulation and proliferation(DNA synthesis) can be assessed. The COSTIM-assay published by Shankaret al. (2004) was foundational in the assay development. Additional workundertaken to optimise the assay included assessment of washing techniques,DC:T cell ratios, BrdU-incubation time and stopping, as well as themeasurement systems.

Product was co-cultured with suboptimal stimulated (aCD3) T cells(COSTIM). Product co-cultured with T cells in the absence of aCD3 (MLR)and T cells alone suboptimal stimulated with aCD3 served as negativecontrols.

Five different Cvac batches were used in the testing on three consecutive days.Every sample was measured in five replicates per day. The intra assay precision(well to well variation within plate) of raw data showed an average coefficient ofvariation (CV) of 9.02% (Range of 1.4 to 21.9%) while mean CV for stimulationindex (COSTIM/MLR) was 13.24% (Range 4.3 to 23.8%). The inter dayprecision (day-to-day variation within analyst) showed an average CV of 12.82%(Range 2.4 to 25.9%) and 19.82% (Range 6.4 to 30.6%) for raw data and stim-ulation index respectively. This assay is being applied to patient samples of theCANVAS clinical trial with the goal of evaluating potency in vitro with long termclinical outcomes.

181STEMVISION�: A BENCH-TOP INSTRUMENT FOR AUTO-MATED AND STANDARDIZED COUNTING OF ALLHEMATOPOIETIC COLONY TYPES IN CFU ASSAYS OFHUMAN BONE MARROW, CORD BLOOD AND MOBILIZEDPERIPHERAL BLOOD CELLSO Egeler, C Grande, N Yuan, B Wognum, S Woodside, A Booth,SJ Szilvassy, T Thomas, AC EavesSTEMCELL Technologies Inc., Vancouver, BC, Canada

The colony-forming unit (CFU) assay is the most reliable in vitro method toenumerate hematopoietic progenitor cells in human blood or bone marrow(BM). In this assay, progenitors cultured in cytokine-supplementedMethoCult�medium produce colonies of mature blood cells that are counted after 1-2 weeks.Typically, colonies generated by lineage-restricted ormulti-potential progenitorcells are scored manually using an inverted microscope according to morpho-logical criteria that can be difficult to apply consistently. This subjectivity cancause high intra/inter-individual and -laboratory variation in assay results. Toimprove the accuracy and reproducibility of the human CFU assay, we havedeveloped an instrument, STEMvision�, that uses image analysis software toidentify, classify and count colonies produced by erythroid, myeloid and

multi-potential progenitors in BM, cord blood (CB), and mobilized peripheralblood (MPB). Automated colony counts in 14-day CFU assays were highlycorrelated with manual counts. For BM assays (n¼163) the correlation coeffi-cients (r2) for total CFU, BFU-E and CFU-G/M/GM were 0.88, 0.63 and 0.83,respectively.The r2 values for automated andmanual counts of totalCFUs, BFU-E and CFU-G/M/GM in CB andMPB cell assays were 0.94, 0.91 and 0.81 (CB,n¼52) and 0.98, 0.96 and 0.93 (MPB, n¼129), respectively. For CFU-E andCFU-GEMM that are too rare to facilitate such statistical analysis,STEMvision� counts were comparable to manual counts produced by 2-7expert technicians who scored w50 BM, CB and MPB assays. Importantly,STEMvision� colony counts were more reproducible than manual counts:coefficient of variation (CV)¼ 5% for total colonies in a 14-dayCFU assay of CBvs. z 25% as reported for NMDP laboratories and 11% among STEMCELLTechnologies staff. The improved speed, accuracy and reproducibility of humanCFU assays scored using STEMvision� facilitates standardization of the assayfor research and clinical laboratories, and cord blood banks.

182CELL THERAPY DISTRIBUTED MANUFACTURING, MAN-AGING COSTS AND RISKSMJ McCall, DJ WilliamsLoughborough University, Loughborough, United Kingdom

Comparability (demonstration of product equivalence) is required aftera process change, when a second facility or location or new raw material isbrought on stream or when multiple sites of manufacture are created. Theability to demonstrate comparability across multiple sites is critical to allowprocess improvement, secure economies of scale and scope and enable supplyof equivalent cell therapies at acceptable costs in order to reach patients inmultiple markets and locations.

Solution of the problem hinges on satisfying the regulator of the compa-rability between manufacturing sites. This work presents a cost model thatquantifies the financial impact of defining and meeting an acceptable regulatoryburden at an early stage in order to inform decisions in the product develop-ment process.

This work quantifies the cost, time and risk associated with establishingmultiple sites of manufacturing for cell therapy products. A scenario and rulesbased mixed-integer linear programming (MILP) formulation is presented forcapacity planning in multi-markets that takes account of adoption and clinicaltrials outcome uncertainty. Results show the financial and temporal cost ofbuild, validating and operating multiple sites of manufacture. The model takesaccount of initial validation costs for each facility and the ingoing burden ofcomparability demonstration. High impact areas are the interdependence ofproduct shelf life (and distribution potential) and the number of sites needed tosatisfy a global market.

An application comparing alternatives to satisfying the regulatory burden ofdemonstrating comparability across multiple sites indicates that the currentlyanticipated requirements to measure the manufacturing and characterisationoutputs of each site against each other site including the sourcing of additionalclinical data is highly resource intensive and strategies for managing this arehighlighted.

This work arose from the VALUE TSB, project and was co-funded by theEPSRC Doctoral Training Centre in Regenerative Medicine.

183THE REGULATION OF ADVANCED THERAPY MEDICINALPRODUCTS IN EUROPE AND THE ROLE OF ACADEMIAKF Pearce1, MO Hildebrandt2, S Scheding3, U Köhl4,E Mischak-Weissinger4, A Hauser5, M Edinger5, H Greinix6, N Worel6,J Apperley7, MW Lowdell8, AM Dickinson11Institute of Cellular Medicine, Medical School, University of Newcastle uponTyne, Newcastle upon Tyne, United Kingdom, 2TUMCells, TechnicalUniverity Munich, Munich, Germany, 3Lund University, Stem Cell Center,Sweden, Lund, Sweden, 4Hannover Medical School (MHH), Hannover,Germany, 5University Hospital, Jose Carreras Center, Regensburg,Germany, 6MedizinischeUniversitaetWien,Vienna, Austria, 7Imperial College,London, United Kingdom, 8University College, London, United Kingdom

AdvancedTherapyMedicinal Products (ATMPs) aremedicinal products based ongene therapy, somatic cell therapy or tissue engineering. Regulation (EC) No1394/2007 has been designed to ensure their free movement within the European