S50 Poster abstracts

Materials and Methods: PRP bags were treated in an ultrasonic bath for 30minutes at 20 KHz. Platelet lysis was evaluated by PDGF-AB release. Effi-ciency was tested by measuring BM-MSC cumulative population doubling(cPD), differentiation capacity and immunogenic properties. Safety was eval-uated by karyotyping.

Results: 74% of PDGF-AB was released. PL significantly (p¼0.01) enhancedBM-MSC proliferation rate compared to FBS. cPD in 10% PL obtained bysonication was better compared to 10% PL obtained by freezing/thawing (18.9vs. 17.4 p<0.01). BM-MNC expressed MSC markers and were able to differ-entiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSC and T-cells were co-cultured in close contact, immunosuppressive activityof BM-MSC was maintained. Cell karyotype evidenced no genetic alterations.

Conclusions:We have developed a fast method for the production of PL bysonication that could be a safe, efficient and fast substitute for FBS.

176LARGE VOLUME WHOLE BLOOD MONONUCLEAR CELLENRICHMENT USING A COBE SPECTRAN Almezel, S Pinkard, T LeemhuisHoxworth Blood Center; University of Cincinnati Academic Health Center,Cincinnati, OH

Several adoptive immunotherapy approaches for treatment of infectiouscomplications, relapsed malignancy, and/or graft rejection begin witha mononuclear cell enrichment procedure, either as part of the leukapheresisprocedure, or as the first step in the target cell isolation procedure. Non-mobilized PB apheresis products often contain many more cells than needed,are somewhat risky to the donor, and can be logistically complicated to obtain.Perhaps more donors would be willing and able to give a unit of blood for suchinvestigational studies than would be willing to travel to a donor center for anapheresis procedure. We have utilized volunteer donor whole blood units todemonstrate that it is feasible to achieve more than adequate MNC purity andrecovery from whole blood using the COBE Spectra, provided there is suffi-cient WBC and RBC content to be able to discern an interface. The SpectraWBC and BMP tubing sets and BMP program were utilized. A minimumblood volume of 400 mL was transferred to the BMP tubing set and connectedto the instrument. The collect pump was manually opened once the interfacewas established using a Colorgram setting of 1-2% hematocrit. Total bloodvolume processed after interface was established was 4-6 times the productvolume. Starting with 450 mL of peripheral blood, we were able to isolate up to13�108 TNCs with a mean MNC purity of 79%, 74% MNC recovery, and98% RBC depletion. The ability to enrich for immune cell populations ina closed system, without exposing the cells to Ficoll make this procedure usefulfor a variety of immunotherapeutic applications that utilize lymphocytes (DLI,Treg, or NK) or monocytes (APC, Dendritic cells) as final products for infu-sion, or as intermediate products requiring further processing. MNC puritycould likely be improved with procedural modifications if recovery is not theprimary objective.

177IMPLEMENTING PROCESS DEVELOPMENT IN THE ACA-DEMIC SETTINGJL Macpherson, CY Kan, MW Keir, B Shen, A Jaramillo, JE RaskoRoyal Prince Alfred Hospital, Sydney, Australia

Background: Delivery of compliant clinical cell therapy products requirescell processing or manufacture within the strict confines of a QualityManagement System. Located in a large teaching hospital adjacent to one ofAustralia’s largest universities we are regularly approached by academicinvestigators who seek to develop their latest basic research discovery for usein patients. Often, the effort and skills required to translate basic researchdiscoveries to the clinic is under-appreciated during early discussions.Contract Manufacturing Organisations offer some services, but their avail-ability in our region is limited and they are often beyond the budget ofacademics.

Methods: We received funding to establish a laboratory where scale up,evaluation of clinically relevant reagents, assay development, product charac-terisation and writing of procedures can occur. We identified suitable nearbylaboratory space, and conducted a review of equipment and infrastructurerequirements. A list of potential cell therapy products for manufacture was

compiled and the processing requirements referenced to the equipmentavailable in our GMP laboratory. The need to develop product characterisationassays was considered in the selection of equipment. Models of staffing andtraining were reviewed and compared.

Results: Infrastructure funding was obtained from the Australian Govern-ment, and matched by philanthropy for operational costs for the first 2 years.Equipment requirements were defined and quotes sought. Following refur-bishments, the laboratory has been fitted out with equipment compatible withqualification and training objectives. To make the most of limited resources,specialist staff skilled in cell therapy production work alongside the researchteam during development. A specific process development example will bediscussed.

Conclusion: Implementation of any major clinical development projecttakes time and allocation of resources. The long-term benefits of investing ina process development laboratory is that the GMP laboratory can remainfocussed on the manufacture of compliant products.

178CYTOCOMPATIBILITY TESTING OF CELL DELIVERY DEVICESMAY BE CRUCIAL FOR CELL THERAPIESN Ramachandran, M HilesCook Biotech, West Lafayette, IN

Cellular therapies are gaining evidence for efficacy in medicine, and most ofthem are being delivered to the patient through tubular instruments such asneedles and catheters. Not withstanding sheer and fluidic forces that mayinfluence these extremely sensitive cells, it is important to consider the cellinteractions directly with the materials that compose the delivery devices. Infact, there is a whole other level of device compatibility, deemed cyto-compatibility, that is often overlooked and in need of better definition andstandardization. Within the delivery devices, cells may adhere to the surfacesand be lost, may get stimulated to die by a specific processing aid used inmanufacturing, or may simply get activated or pushed down an undesireddifferentiation pathway. To detect these failure modes we have adopteda multipronged approach wherein a combination of direct contact and indirectcontact assays and tests are used to assess the cytocompatability of deliverydevices. Some these tests will be used for design verification and some for lotrelease testing. Consideration is given to which cell types or populations ofcells to use for testing, what exposure times are relevant, and what specificcellular functions to measure. Testing was carried out under conditions atleast as extreme as can be expected in clinical use. We have chosen to examineseveral aspects of cytocompatibility, including cell counts, viability, metabolicrate of cells, and phenotype changes, by comparing common catheter mate-rials to various controls. We have established a strict set of acceptance criteriaand would like to stimulate the dialogue of how standardization of these testscan be achieved and what is necessary and reasonable. Copyright (c) 2006Microsoft Corporation. All rights reserved.

179GRANULOCYTES - AN EXPLORATION OF SOMETHING NEW -SPECTRA OPTIA�

MJ DarbyPeter Mac, Melbourne, Australia

History: Patient with hairy cell leukaemia (HCL) received cladribine inductionchemotherapy. Complications were GI bleeding requiring a right hemi-colectomy and ileostomy, and uncontrolled neutropaenic sepsis (MRSA, VREand Staph Epidermidis and Candida Globrata) requiring ICU admission.Granulocyte infusionswere given; 4BuffyCoatsTransfusions (ARCBS) followedby 10 from volunteer donors. There was prompt and progressive improvementallowing discharge home on day 63. Neutrophil count was 6.2�109/L on day 76.

Aim: To assess the use of the TerumoBCTSpectra Optia� apheresis device andtheLDL/leukoreductionmethod for thecollectionof volunteer donorgranulocytes.

Background and Method: Until recently, the preferred method of Gran-ulocyte Collection was using the Cobe Spectra� device and Dextran 70% asa sedimentating agentwith sodiumcitrate added as an anticoagulant. In the absenceof a registered sedimentating agent, we evaluated theTerumoBCTSpectraOptia�

apheresis device using the IDL filler, IDL kit, sodium citrate as an anticoagulantand the Granulocyte program minus HES option. All donors received GCSF(5mcg/kg) andDexamethasone (8mg) starting the day prior to thefirst collection.