- Email: [email protected]
Scale up of pluripotent stem cell cultivation and differentiation in a novel single-use bioreactor system
S78
Poster Abstracts
Table I. Patient N°
Platelet count before IA
IA sessions (N)
ACD-A (ratio)
Heparin
Relative treated plasma volume (-fold)
Blood Debit
Plasma debit
1 2
20 × 10E9/L 25 × 10E9/L
3 4
1:18 1:25
1300 UI/H Not used
2.0 1.5
82 50
37 26
Material, Patients and Methods: Two patients with acute myeloid leukemia in second complete remission were enrolled. The presence of anti-HLA antibodies was determined by lymphocytotoxicity and the titer by Luminex. Immunoadsorption was performed with polyclonal sheep anti-human IgG adsorbers (Miltenyi Biotec GmbH, Germany) on LIFE 18 apheresis system. In addition all patients received Rituximab 375 mg/kg bw in a single dose. Results: Data on patients and treatments are summarized in Table I. After immunoadsorption the two patients increased their post-platelet transfusion increments and could further-on be transfused with non-HLA compatible platelets. Conclusion: By a combination of Rituximab and repeated immunoadsorption sessions, the poly-HLA alloimmunization could be managed sufficiently to enable post-transfusion platelet increment. Immunoadsorption turned out to be a safe procedure without relevant clinical side effects even if the platelet count is below 25 × 10E9/L. 177 GMP VALIDATION OF LARGE-SCALE EXPANSION OF REGULATORY T CELLS FROM PATIENTS AFFECTED BY LIVER AND KIDNEY FAILURE C. Lavazza1, M. Vigano’1, T. Montemurro1, E. Montelatici1, S. Budelli1, M.G. Cannone1, S. Savelli1, F. Ulbar2, L. Catani2, V. Giudice3, M. Cescon4, G. La Manna5, R.M. Lemoli6, R. Giordano1 1 Cell Factory “F. Calori”, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy, 2 Department of Experimental, Diagnostic and Specialistic Medicine, Institute of Hematology “L. e A. Seràgnoli”, S. Orsola Hospital, Bologna, Italy, 3O.U. Immunohematology and Blood Bank, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 4Department of Organ Failure and Transplants, O.U. Transplant Surgery, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 5Department of Organ Failure and Transplants O.U. Nefrology, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 6 Clinic of Hematology, Department of Internal Medicine (DiMI), University of Genoa, AOU IRSSC S. Martino IST, Genova, Italy Regulatory CD4+CD25+ T lymphocytes (Tregs) can improve the outcome of solid organ transplantation through induction of immunologic tolerance. Due to the low frequency of Tregs in peripheral blood, their enrichment through isolation and ex vivo expansion is required to obtain a suitable quantity of cells for therapeutic use. In this study, we investigated, under good manufacturing practice (GMP) conditions, the performance of Tregs isolation, expansion and cryopreservation from leukapheresis of patients with hepatic and renal terminal failure. Steady state leukapheresis from 3 patients (a 47-year-old male with alcoholic cirrhosis and a male and a female, both 62-year-old, with end-stage kidney disease) were processed using GMP-compliant devices and reagents in our facility authorized for ATMPs production. The Tregs content was 1.9 ± 0.7% (171 ± 99 × 10^6 cells). After immunomagnetic CD8 depletion followed by CD25 enrichment, we recovered 52 ± 13% (82 ± 33 × 10^6 cells) of Treg, with a purity (expressed as percentage of CD4+CD25+ cells) of 71 ± 0.2%. FoxP3+ and CD127FoxP3+ Tregs were 62 ± 0.2% and 54 ± 0.1% of isolated cells, respectively. Forty millions of isolated cells (of which 29 ± 6 × 10^6 Tregs) were expanded in vitro in gas-permeable culture bags (Miltenyi Biotec) for 3 weeks in TexMACS GMP medium supplemented with rapamycin (both Miltenyi Biotec) and allogeneic, heat-inactivated plasma, in presence of IL-2 (Novartis) and antiCD3/CD28 beads (Miltenyi Biotec). At day 21, we obtained 4.5 ± 0.6 × 10^9 Tregs, with a 166 ± 38-fold expansion as compared to starting amount, resulting in a cell dose of 66 ± 8 × 10^6 Treg/Kg for a mean body weight of 70 Kg. Purity was 95 ± 0.1%; FoxP3+ and CD127-FoxP3+ were 91 ± 0.1% and 87 ± 0.1% of expanded cells, respectively; contaminating cells (CD8+, CD19+ or CD56+) did not exceed 0.9 ± 0.01%.
Our results provide a robust approach to obtain high numbers of GMPgrade autologous Tregs in patients who are candidate for liver and kidney transplantation, in order to induce immune tolerance. 178 SCALE UP OF PLURIPOTENT STEM CELL CULTIVATION AND DIFFERENTIATION IN A NOVEL SINGLE-USE BIOREACTOR SYSTEM B. Lee, Y. Hashimura, S. Karamil, R. Wesselschmidt PBS Biotech, Inc., Camarillo, California, United States Growing and differentiating pluripotent stem cells as aggregates suspended in bioreactors is considered a viable option for scaling up the manufacturing process to a commercial scale. The conventional cylindrical bioreactors with horizontal impellers require high agitation power in order to fully suspend the cell aggregates, which can cause high hydrodynamic shear stress to the cells. Anchorage dependent cells grown as aggregates can be more sensitive to hydrodynamic shear stress than suspended single cell cultures. In addition, the cells respond to the microenvironment in ways that may affect final product yield, quality, and potency. Identifing an acceptable range of agitation rates to achieve satisfactory cell growth and differentiation outcome of pluripotent stem cells in a bioreactor is a challenge, and this problem becomes worse as the size of bioreactor increases. Recently, a novel single-use bioreactor system using a Vertical-Wheel technology is introduced, which offers efficient fluid mixing, homogenous dissipation energy distribution, and uniform cell aggregate suspension with low power input. The vertical mixing mechanism not only requires very low power input to suspend cell aggregates uniformly, but also allows the low shear environment to remain constant across the full range of vessel sizes from 0.5 to 80 liters. The homogeneous distribution of turbulent energy dissipation rates inside Vertical-Wheel bioreactor generates uniform size of cell aggregates, and the desired size of cell aggregates can be achieved by controlling agitation rate. Since the size of cell aggregates is critical for differentiation kinetics as well as the yield of final target cell type, controlling the size of majority of cell aggregates can be beneficial for cell growth and differentiation process of pluripotent stem cells. This unique characteristic of the vertical mixing mechanism offers unparalleled scalability to achieve a consistent and robust manufacturing process for shear sensitive pluripotent stem cell products. The Vertical-Wheel single-use bioreactors have been evaluated with several different cell types including human bone marrow-derived mesenchymal stem cells (BM-MSC), embryonic and induced pluripotent stem cells (PSC’s). The results of physical measurements of Kolmogorov scales, the growth of various types of cells, and the differentiation kinetics of PSC’s in various sizes of VerticalWheel bioreactors will be discussed. 179 DEVELOPMENT AND OPTIMIZATION OF LARGE SCALE PRODUCTION OF ADENOVIRAL VECTOR AND AUTOLOGOUS INSULIN PRODUCING CELL USING ICELLIS® 500 AND XPANSION® 200 SINGLE-USE BIOREACTORS R. Legmann1, A. Reniers1, N. Kohlstrom1, B. Gardell1, T. Sanderson2, L. Bradbury2, H.A. Mallory1, F. Moncaubeig1, V. Aviv3, K. Ron3, I. Tzchori3, I. Meivar-Levy3, S. Ferber3 1 Process Development, Pall Life sciences, Westborough, Massachusetts, United States, 2ARD, Pall Life Sciences, Westborough, Massachusetts, United States, 3Orgenesis Ltd, Ramat Gan, Israel Diabetes is a major international health problem with over 370 million diabetics globally and an estimated 552 million by 2030. Orgenesis has developed an autologous cell therapy approach that allows the diabetic patient to be a donor of their own therapeutic tissue. Starting with a small biopsy from a patient’s liver, the biopsied cells are expanded in flatware, trans-differentiated into au-
Poster Abstracts
Table I. Patient N°
Platelet count before IA
IA sessions (N)
ACD-A (ratio)
Heparin
Relative treated plasma volume (-fold)
Blood Debit
Plasma debit
1 2
20 × 10E9/L 25 × 10E9/L
3 4
1:18 1:25
1300 UI/H Not used
2.0 1.5
82 50
37 26
Material, Patients and Methods: Two patients with acute myeloid leukemia in second complete remission were enrolled. The presence of anti-HLA antibodies was determined by lymphocytotoxicity and the titer by Luminex. Immunoadsorption was performed with polyclonal sheep anti-human IgG adsorbers (Miltenyi Biotec GmbH, Germany) on LIFE 18 apheresis system. In addition all patients received Rituximab 375 mg/kg bw in a single dose. Results: Data on patients and treatments are summarized in Table I. After immunoadsorption the two patients increased their post-platelet transfusion increments and could further-on be transfused with non-HLA compatible platelets. Conclusion: By a combination of Rituximab and repeated immunoadsorption sessions, the poly-HLA alloimmunization could be managed sufficiently to enable post-transfusion platelet increment. Immunoadsorption turned out to be a safe procedure without relevant clinical side effects even if the platelet count is below 25 × 10E9/L. 177 GMP VALIDATION OF LARGE-SCALE EXPANSION OF REGULATORY T CELLS FROM PATIENTS AFFECTED BY LIVER AND KIDNEY FAILURE C. Lavazza1, M. Vigano’1, T. Montemurro1, E. Montelatici1, S. Budelli1, M.G. Cannone1, S. Savelli1, F. Ulbar2, L. Catani2, V. Giudice3, M. Cescon4, G. La Manna5, R.M. Lemoli6, R. Giordano1 1 Cell Factory “F. Calori”, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy, 2 Department of Experimental, Diagnostic and Specialistic Medicine, Institute of Hematology “L. e A. Seràgnoli”, S. Orsola Hospital, Bologna, Italy, 3O.U. Immunohematology and Blood Bank, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 4Department of Organ Failure and Transplants, O.U. Transplant Surgery, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 5Department of Organ Failure and Transplants O.U. Nefrology, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, 6 Clinic of Hematology, Department of Internal Medicine (DiMI), University of Genoa, AOU IRSSC S. Martino IST, Genova, Italy Regulatory CD4+CD25+ T lymphocytes (Tregs) can improve the outcome of solid organ transplantation through induction of immunologic tolerance. Due to the low frequency of Tregs in peripheral blood, their enrichment through isolation and ex vivo expansion is required to obtain a suitable quantity of cells for therapeutic use. In this study, we investigated, under good manufacturing practice (GMP) conditions, the performance of Tregs isolation, expansion and cryopreservation from leukapheresis of patients with hepatic and renal terminal failure. Steady state leukapheresis from 3 patients (a 47-year-old male with alcoholic cirrhosis and a male and a female, both 62-year-old, with end-stage kidney disease) were processed using GMP-compliant devices and reagents in our facility authorized for ATMPs production. The Tregs content was 1.9 ± 0.7% (171 ± 99 × 10^6 cells). After immunomagnetic CD8 depletion followed by CD25 enrichment, we recovered 52 ± 13% (82 ± 33 × 10^6 cells) of Treg, with a purity (expressed as percentage of CD4+CD25+ cells) of 71 ± 0.2%. FoxP3+ and CD127FoxP3+ Tregs were 62 ± 0.2% and 54 ± 0.1% of isolated cells, respectively. Forty millions of isolated cells (of which 29 ± 6 × 10^6 Tregs) were expanded in vitro in gas-permeable culture bags (Miltenyi Biotec) for 3 weeks in TexMACS GMP medium supplemented with rapamycin (both Miltenyi Biotec) and allogeneic, heat-inactivated plasma, in presence of IL-2 (Novartis) and antiCD3/CD28 beads (Miltenyi Biotec). At day 21, we obtained 4.5 ± 0.6 × 10^9 Tregs, with a 166 ± 38-fold expansion as compared to starting amount, resulting in a cell dose of 66 ± 8 × 10^6 Treg/Kg for a mean body weight of 70 Kg. Purity was 95 ± 0.1%; FoxP3+ and CD127-FoxP3+ were 91 ± 0.1% and 87 ± 0.1% of expanded cells, respectively; contaminating cells (CD8+, CD19+ or CD56+) did not exceed 0.9 ± 0.01%.
Our results provide a robust approach to obtain high numbers of GMPgrade autologous Tregs in patients who are candidate for liver and kidney transplantation, in order to induce immune tolerance. 178 SCALE UP OF PLURIPOTENT STEM CELL CULTIVATION AND DIFFERENTIATION IN A NOVEL SINGLE-USE BIOREACTOR SYSTEM B. Lee, Y. Hashimura, S. Karamil, R. Wesselschmidt PBS Biotech, Inc., Camarillo, California, United States Growing and differentiating pluripotent stem cells as aggregates suspended in bioreactors is considered a viable option for scaling up the manufacturing process to a commercial scale. The conventional cylindrical bioreactors with horizontal impellers require high agitation power in order to fully suspend the cell aggregates, which can cause high hydrodynamic shear stress to the cells. Anchorage dependent cells grown as aggregates can be more sensitive to hydrodynamic shear stress than suspended single cell cultures. In addition, the cells respond to the microenvironment in ways that may affect final product yield, quality, and potency. Identifing an acceptable range of agitation rates to achieve satisfactory cell growth and differentiation outcome of pluripotent stem cells in a bioreactor is a challenge, and this problem becomes worse as the size of bioreactor increases. Recently, a novel single-use bioreactor system using a Vertical-Wheel technology is introduced, which offers efficient fluid mixing, homogenous dissipation energy distribution, and uniform cell aggregate suspension with low power input. The vertical mixing mechanism not only requires very low power input to suspend cell aggregates uniformly, but also allows the low shear environment to remain constant across the full range of vessel sizes from 0.5 to 80 liters. The homogeneous distribution of turbulent energy dissipation rates inside Vertical-Wheel bioreactor generates uniform size of cell aggregates, and the desired size of cell aggregates can be achieved by controlling agitation rate. Since the size of cell aggregates is critical for differentiation kinetics as well as the yield of final target cell type, controlling the size of majority of cell aggregates can be beneficial for cell growth and differentiation process of pluripotent stem cells. This unique characteristic of the vertical mixing mechanism offers unparalleled scalability to achieve a consistent and robust manufacturing process for shear sensitive pluripotent stem cell products. The Vertical-Wheel single-use bioreactors have been evaluated with several different cell types including human bone marrow-derived mesenchymal stem cells (BM-MSC), embryonic and induced pluripotent stem cells (PSC’s). The results of physical measurements of Kolmogorov scales, the growth of various types of cells, and the differentiation kinetics of PSC’s in various sizes of VerticalWheel bioreactors will be discussed. 179 DEVELOPMENT AND OPTIMIZATION OF LARGE SCALE PRODUCTION OF ADENOVIRAL VECTOR AND AUTOLOGOUS INSULIN PRODUCING CELL USING ICELLIS® 500 AND XPANSION® 200 SINGLE-USE BIOREACTORS R. Legmann1, A. Reniers1, N. Kohlstrom1, B. Gardell1, T. Sanderson2, L. Bradbury2, H.A. Mallory1, F. Moncaubeig1, V. Aviv3, K. Ron3, I. Tzchori3, I. Meivar-Levy3, S. Ferber3 1 Process Development, Pall Life sciences, Westborough, Massachusetts, United States, 2ARD, Pall Life Sciences, Westborough, Massachusetts, United States, 3Orgenesis Ltd, Ramat Gan, Israel Diabetes is a major international health problem with over 370 million diabetics globally and an estimated 552 million by 2030. Orgenesis has developed an autologous cell therapy approach that allows the diabetic patient to be a donor of their own therapeutic tissue. Starting with a small biopsy from a patient’s liver, the biopsied cells are expanded in flatware, trans-differentiated into au-