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Designing a CMC strategy for cell and gene therapy
S46
Poster Abstracts
Mesenchymal Stromal Cells (MSCs) have achieved a great potential in Regenerative Medicine, although limited information exists about bioprocess and production on Good Manufacture Practices (GMP)-grade, standardization and quality controls. The present work describes our experience on the validation and GMP production of 25 consecutive bone marrow (BM)-derived MSCs batches for clinical use. BM was obtained from posterior iliac crests of patients. Manufacturing included isolation by density-gradient centrifugation and plating, trypsinization and secondary expansion cultures and, harvesting and formulation of a suspension containing 4010E6 viable MSCs. Quality controls included cell counting and viability assessment; immunophenotyping; Sterility Test, Mycoplasma detection and Endotoxin Tests conducted according to European Pharmacopeia (Eur. Ph.) and, finally, Gram Staining. All products were shipped according to current EU Regulations. After validation, 25 MSCs batches for clinical application were manufactured. BM harvested volumes ranged from 98 to 163 mL (median 132,1mL) containing 8,38E8 to 4,29E9 viable Nucleated Cells (NCs) (median 2,53E9). Cell concentrations ranged from 5.74E6 to 3.56E7 NCs/mL (median 2.34E7). Final product volume was 10,0 mL (4-10,5 mL) containing a median of 4.02E7 (3.21E7-4.87E7) viable MSCs. Antigen expression was consistent with MSCs phenotype as described by ISCT. In a minority of the batches produced, we observed an increase of HLA-DR expression compatible with the activation on the MSCs. Sterility, Mycoplasma, Gram Staining and Endotoxin tests of the finished products were always negative. We have established a GMP protocol, using human sera as supplement, confirming its safety and feasibility. It is consistent and reproducible and the bioprocess developed demonstrated to be robust, safe and reliable. This work was cofounded by MEDCEL and FACTOCEL projects from the Spanish government (Ministerio de Ciencia e Innovación). 148 MULTICOLOUR FLOW CYTOMETRY QUANTIFICATION OF IMMUNOAFINITY SELECTED CMV ANTIGEN SPECIFIC T-CELL IN CLINICAL SCALE PREPARATIONS WITH TWO DIFFERENT DEVICES S Kloess1, R Esser1, M Marburger1, S Tischer2, C Priesner1, K Aleksandrova1, B Maecker-Kolhoff3, H Heuft2, L Goudeva2, R Blasczyk2, L Arseniev1, B Eiz-Vesper2, U Köhl1 1 Institute for Celltherapeutics, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany, 2Institute for Transfusion Medicine, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany, 3Clinic for Paediatric Haematology and Oncology, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany Virus-specific T-cells (CTL) can be selected via the CliniMACS CCS using either the CliniMACSPlus (Plus) or Prodigy (Miltenyi Biotec GmbH, Germany). Because of the low frequency of the target CTL a precise quality control (QC) of the final product is a challenge, even after re-stimulation for IFNg secretion. After proving donors’ CMV positivity and obtaining their written inform consent 3 lymphaphereses were conducted. Re-stimulation with MACS GMP PepTivator HCMVpp65 and selections of CMV-CTL were carried out with the Prodigy and Plus simultaneously starting with 0.5-1x109 leucocytes. Single platform flow cytometry (FCM) with a panel of up to 9 colours and a successive gating strategy was applied for QC: CD3, CD4, CD8, CD45, IFNg (positive) and CD14, CD19, CD56 (negative). 7AAD was used for viable staining and Fluorospheres for counting. The selections were less time consuming (<7 h hands-on time) with the Prodigy as compared to the Plus (>12 h). Both devices provided similar results for the target fractions: 0.29.4 vs. 2.4-10.7x106 CD45+/7AAD- cells in 7-9.6 ml and 40-43 ml, respectively. The overall 7AAD negativity (19-63%) and the cell concentration (0.160.65 x106 cells/ml) were relatively low. The corresponding proportions of CD3+IFNg+ cells ranged between 38.7-59.8% and 8.7-52.2%. Within 2 selection pairs predominantly CD8+/CD3+/IFNg+ cells were found (CD4:CD8 ratio 1:4.8). The last preparation contained more CD4+/CD3+/IFNg+ lymphocytes (CD4:CD8 ratio 4.8:1). The contaminating IFNg- T-cells were 2.9-11.3% and 5.1-36.7%, respectively. Although more than 1 x104 CTL were collected with both machines the cell concentration was <1x106 cell/ml. In combination with the relatively small volume extended analyses are not possible. Thus a multicolour single platform FCM is the method of choice for the accurate QC. Beside some initial technical difficulties with the CliniMACS Prodigy both machines seem to target cell fractions with similar characteristics.
149 DESIGNING A CMC STRATEGY FOR CELL AND GENE THERAPY V Pimpaneau Voisin Consulting Life Sciences, Rennes, France Defining a CMC strategy is a key element to successful product development, regardless of product type. In recent years, the implementation of the ATMP regulation and guidelines together with the new quality paradigms developed in ICH guidelines (Q8-10), and the refinement of GMP requirements (Annex 2), all encourage science and risk based approach to become an integrated part of product development. Identifying the CMC regulatory requirements at any given development stage provides the opportunity to reflect on the product, its specific characteristics and the potential technical challenges or limitation that one may face during the course of development. This set of information will be critical in defining the needs in terms of product characterization tools, production scales, comparability plans, risk mitigation etc. and will help drive priorities and efforts throughout development. This presentation will also discuss how anticipating the CTD content of Module 3 early on can help identify potential gaps and define a regulatory frame as it relate to CMC requirements which, combined with a risk based approach, become the foundation of the CMC strategy that one can build upon as development evolves. The presentation will also highlight the importance of the characterization tools to help feed an efficient development cycle build on process and product monitoring and on comparability. Learning objective: Understand the key elements of CMC differences between ATMP and other medicinal products and how they influence product development. Discuss the possible approaches to overcome technical and regulatory challenges and how the knowledge of product specificities can allow the integration of a risk based approach. Learn how the development of the appropriate characterization tools and early planning of comparability strategies are essential to successful product development and to the preparation of the Module 3.
150 SPECTRA OPTIAÔ AND ELUTRAÔ FOR THE PRODUCTION OF MONOCYTE-DERIVED DENDRITIC CELL VACCINES GS Andreassen1, L Skoge1, M Lundby1, RJ Smith2, G Kvalheim1, D Josefsen1 1 Dep of Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2Biotech and Cell processing EMEA, Terumo BCT Inc, Zaventem, Belgium We investigated the Spectra Optia as a platform for collecting mononuclear cells (MNC) from unmobilised patients and the Elutra performance to further process these products to a monocyte enriched fraction for the generation of dendritic cells. A total of 14 patients were enrolled: Ovarian cancer (2), Prostate cancer (6), Glioblastoma (2), Lymphoma (2) and malignant melanoma (2). A single monocytapheresis procedure was completed for each patient, using standard settings. Samples were collected prior to apheresis, and from the harvested product. Cells were stored overnight before elutriation for further enrichment of monocytes and production of dendritic cell (DC) vaccines. Data are presented as Median (Range). During apheresis 2.10 (1.03 e 2.80) total blood volumes were processed in 209 (110 e 330) minutes. A monocyte yield of 2.53 x 109 (1.35 e 4.56) total monocytes was collected in a final volume of 60mL (40 e 100). Monocyte collection efficiency (CE1%) was also high at 62.9% (29.7% - 77.7%). We also observed a moderate correlation (r2 ¼ 0.61) between pre-apheresis monocyte count and monocyte yield per total blood volume processed. Nontarget cell residuals were extremely low, thus making ideal products for further monocyte enrichment by elutriation. A median of 2.10 x 109 (0.89 e 3.18) total monocytes was subsequently elutriated. After processing, monocyte purity increased from 30% (16% - 67%) before elutriation to 88.6% ( 71.0% - 97.0%) with an overall monocyte recovery of 64% (42% - 100%). We conclude that Spectra Optia is able to isolate and enrich a highly purified MNC fraction from patients in steady-state haematopoiesis. Moreover, MNC products collected on Optia are ideally suited for subsequent elutriation to obtain highly enriched monocytes. Importantly, dendritic cell vaccines made from the enriched monocytes show no change in phenotype and clinical efficacy when compared to DC vaccines made from COBE Spectra and elutriation on Elutra.
Poster Abstracts
Mesenchymal Stromal Cells (MSCs) have achieved a great potential in Regenerative Medicine, although limited information exists about bioprocess and production on Good Manufacture Practices (GMP)-grade, standardization and quality controls. The present work describes our experience on the validation and GMP production of 25 consecutive bone marrow (BM)-derived MSCs batches for clinical use. BM was obtained from posterior iliac crests of patients. Manufacturing included isolation by density-gradient centrifugation and plating, trypsinization and secondary expansion cultures and, harvesting and formulation of a suspension containing 4010E6 viable MSCs. Quality controls included cell counting and viability assessment; immunophenotyping; Sterility Test, Mycoplasma detection and Endotoxin Tests conducted according to European Pharmacopeia (Eur. Ph.) and, finally, Gram Staining. All products were shipped according to current EU Regulations. After validation, 25 MSCs batches for clinical application were manufactured. BM harvested volumes ranged from 98 to 163 mL (median 132,1mL) containing 8,38E8 to 4,29E9 viable Nucleated Cells (NCs) (median 2,53E9). Cell concentrations ranged from 5.74E6 to 3.56E7 NCs/mL (median 2.34E7). Final product volume was 10,0 mL (4-10,5 mL) containing a median of 4.02E7 (3.21E7-4.87E7) viable MSCs. Antigen expression was consistent with MSCs phenotype as described by ISCT. In a minority of the batches produced, we observed an increase of HLA-DR expression compatible with the activation on the MSCs. Sterility, Mycoplasma, Gram Staining and Endotoxin tests of the finished products were always negative. We have established a GMP protocol, using human sera as supplement, confirming its safety and feasibility. It is consistent and reproducible and the bioprocess developed demonstrated to be robust, safe and reliable. This work was cofounded by MEDCEL and FACTOCEL projects from the Spanish government (Ministerio de Ciencia e Innovación). 148 MULTICOLOUR FLOW CYTOMETRY QUANTIFICATION OF IMMUNOAFINITY SELECTED CMV ANTIGEN SPECIFIC T-CELL IN CLINICAL SCALE PREPARATIONS WITH TWO DIFFERENT DEVICES S Kloess1, R Esser1, M Marburger1, S Tischer2, C Priesner1, K Aleksandrova1, B Maecker-Kolhoff3, H Heuft2, L Goudeva2, R Blasczyk2, L Arseniev1, B Eiz-Vesper2, U Köhl1 1 Institute for Celltherapeutics, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany, 2Institute for Transfusion Medicine, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany, 3Clinic for Paediatric Haematology and Oncology, Medizinische Hochschule Hannover, Hannover, Lowee Saxony, Germany Virus-specific T-cells (CTL) can be selected via the CliniMACS CCS using either the CliniMACSPlus (Plus) or Prodigy (Miltenyi Biotec GmbH, Germany). Because of the low frequency of the target CTL a precise quality control (QC) of the final product is a challenge, even after re-stimulation for IFNg secretion. After proving donors’ CMV positivity and obtaining their written inform consent 3 lymphaphereses were conducted. Re-stimulation with MACS GMP PepTivator HCMVpp65 and selections of CMV-CTL were carried out with the Prodigy and Plus simultaneously starting with 0.5-1x109 leucocytes. Single platform flow cytometry (FCM) with a panel of up to 9 colours and a successive gating strategy was applied for QC: CD3, CD4, CD8, CD45, IFNg (positive) and CD14, CD19, CD56 (negative). 7AAD was used for viable staining and Fluorospheres for counting. The selections were less time consuming (<7 h hands-on time) with the Prodigy as compared to the Plus (>12 h). Both devices provided similar results for the target fractions: 0.29.4 vs. 2.4-10.7x106 CD45+/7AAD- cells in 7-9.6 ml and 40-43 ml, respectively. The overall 7AAD negativity (19-63%) and the cell concentration (0.160.65 x106 cells/ml) were relatively low. The corresponding proportions of CD3+IFNg+ cells ranged between 38.7-59.8% and 8.7-52.2%. Within 2 selection pairs predominantly CD8+/CD3+/IFNg+ cells were found (CD4:CD8 ratio 1:4.8). The last preparation contained more CD4+/CD3+/IFNg+ lymphocytes (CD4:CD8 ratio 4.8:1). The contaminating IFNg- T-cells were 2.9-11.3% and 5.1-36.7%, respectively. Although more than 1 x104 CTL were collected with both machines the cell concentration was <1x106 cell/ml. In combination with the relatively small volume extended analyses are not possible. Thus a multicolour single platform FCM is the method of choice for the accurate QC. Beside some initial technical difficulties with the CliniMACS Prodigy both machines seem to target cell fractions with similar characteristics.
149 DESIGNING A CMC STRATEGY FOR CELL AND GENE THERAPY V Pimpaneau Voisin Consulting Life Sciences, Rennes, France Defining a CMC strategy is a key element to successful product development, regardless of product type. In recent years, the implementation of the ATMP regulation and guidelines together with the new quality paradigms developed in ICH guidelines (Q8-10), and the refinement of GMP requirements (Annex 2), all encourage science and risk based approach to become an integrated part of product development. Identifying the CMC regulatory requirements at any given development stage provides the opportunity to reflect on the product, its specific characteristics and the potential technical challenges or limitation that one may face during the course of development. This set of information will be critical in defining the needs in terms of product characterization tools, production scales, comparability plans, risk mitigation etc. and will help drive priorities and efforts throughout development. This presentation will also discuss how anticipating the CTD content of Module 3 early on can help identify potential gaps and define a regulatory frame as it relate to CMC requirements which, combined with a risk based approach, become the foundation of the CMC strategy that one can build upon as development evolves. The presentation will also highlight the importance of the characterization tools to help feed an efficient development cycle build on process and product monitoring and on comparability. Learning objective: Understand the key elements of CMC differences between ATMP and other medicinal products and how they influence product development. Discuss the possible approaches to overcome technical and regulatory challenges and how the knowledge of product specificities can allow the integration of a risk based approach. Learn how the development of the appropriate characterization tools and early planning of comparability strategies are essential to successful product development and to the preparation of the Module 3.
150 SPECTRA OPTIAÔ AND ELUTRAÔ FOR THE PRODUCTION OF MONOCYTE-DERIVED DENDRITIC CELL VACCINES GS Andreassen1, L Skoge1, M Lundby1, RJ Smith2, G Kvalheim1, D Josefsen1 1 Dep of Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2Biotech and Cell processing EMEA, Terumo BCT Inc, Zaventem, Belgium We investigated the Spectra Optia as a platform for collecting mononuclear cells (MNC) from unmobilised patients and the Elutra performance to further process these products to a monocyte enriched fraction for the generation of dendritic cells. A total of 14 patients were enrolled: Ovarian cancer (2), Prostate cancer (6), Glioblastoma (2), Lymphoma (2) and malignant melanoma (2). A single monocytapheresis procedure was completed for each patient, using standard settings. Samples were collected prior to apheresis, and from the harvested product. Cells were stored overnight before elutriation for further enrichment of monocytes and production of dendritic cell (DC) vaccines. Data are presented as Median (Range). During apheresis 2.10 (1.03 e 2.80) total blood volumes were processed in 209 (110 e 330) minutes. A monocyte yield of 2.53 x 109 (1.35 e 4.56) total monocytes was collected in a final volume of 60mL (40 e 100). Monocyte collection efficiency (CE1%) was also high at 62.9% (29.7% - 77.7%). We also observed a moderate correlation (r2 ¼ 0.61) between pre-apheresis monocyte count and monocyte yield per total blood volume processed. Nontarget cell residuals were extremely low, thus making ideal products for further monocyte enrichment by elutriation. A median of 2.10 x 109 (0.89 e 3.18) total monocytes was subsequently elutriated. After processing, monocyte purity increased from 30% (16% - 67%) before elutriation to 88.6% ( 71.0% - 97.0%) with an overall monocyte recovery of 64% (42% - 100%). We conclude that Spectra Optia is able to isolate and enrich a highly purified MNC fraction from patients in steady-state haematopoiesis. Moreover, MNC products collected on Optia are ideally suited for subsequent elutriation to obtain highly enriched monocytes. Importantly, dendritic cell vaccines made from the enriched monocytes show no change in phenotype and clinical efficacy when compared to DC vaccines made from COBE Spectra and elutriation on Elutra.