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A novel automated bioreactor for scalable pro-cess optimisation of haematopoietic stem cell culture
19th Annual ISCT Meeting
E- depletion. However, CliniMACS plus IVIG reduces some immune modulating cell subsets (TCRgd, CD56/16, p<0.001) more efficiently and others (CD4, p<0.001) less efficiently than the Iso E- method. Our comparison of post-TCD HPC grafts demonstrate that similar TCD methods vary in their ability to remove different, and potentially important T-cell subsets including NK cells, and may provide an explantation for the varying clinical results observed in TCD-allograft 69 NEUTROPHIL CONTAMINATION IN AUTOLOGOUS PERIPH ERAL BLOOD STEM CELL PRODUCTS: IMPACT ON POSTTRANSPLANT OUTCOME W Xia Royal North Shore Hospital, Sydney, New South Walse, Australia Aim: To evaluate whether neutrophil contamination of HPC-A product could impact transplant related outcomes at a single centre. Methods: Between 2009 and 2012, 129 patients underwent HPC-A mobilization and collection. Apheresis was initiated once PB CD34 >15/uL and continued until > 2106/kg CD34+ cells were collected. The instrument modes of apheresis included Cobe Spectra manual, Cobe Spectra automated and Optia. Patient characteristics, collected HPC-A volume, neutrophil% in HPC-A, infused neutrophil dose, and transplant outcomes were evaluated. Results: 53%, 38% and 9% of patients received manual, auto and Optia mode collection. Manual mode, comparing auto mode, significantly caused larger collected HPC-A volume (mL) (308+122 vs 123+70, p<0.001); and more neutrophil% in HPC-A (40+19.3 vs 16+18.9, p<0.001). Increasing patient age significantly correlated to higher infused neutrophil dose (p¼0.005). Mean age of patients neutrophil dose of 5108/kg in HPC-A significantly delayed neutrophil engraftment vs HPC-A containing neutrophils <5108/kg, (14.24d+4.51 vs 12.5d+2.39, p¼0.019). Infused neutrophil number significantly correlated to neutrophil engraftment time (R2¼0.094, p¼0.001); but didn’t impact platelet engraftment, TRM and survivals. Conclusions: 1. Manual mode of apheresis was associated with greater volume and higher neutrophil contamination in HPC-A than auto mode. 2. Older age was associated with higher infused neutrophil dose in HPC-A 3. Infused neutrophils >5108/kg in HPC-A is associated with delayed neutrophil engraftment but did not impact on other post transplant outcomes. 70 QUANTIFICATION OF HAEMATOPOIETIC CHIMERISM BY REAL TIME PCR ASSAY VALIDATION K Kooloos The Royal Children’s Hospital, Parkville, Australia Following allogeneic stem cell transplantation, it is of great importance to determine whether the newly developed haematopoietic system is of donor or recipient origin. Quantitative Real Time PCR (Polymerase Chain Reaction) has recently been implemented at The Royal Children’s Hospital, Melbourne to replace the qualitative VNTR (Variable Number of Tandem Repeats) assay by gel electrophoresis to determine post transplant chimerism. Real Time PCR discriminates alleles using fluorescent dye-labelled probes for Single Nucleotide Polymorphisms (SNP). This test is used to monitor engraftment of the transplant and identify re-emergence of recipient cells, an indicator of engraftment failure or impending relapse. Three SNP probes from Applied BiosystemsÒ (rs1908777, rs3918344 & rs338773) were validated in the implementation of the Real Time PCR assay. Analysis was performed using Rotor-geneÔ software. The method validation was based on the following criteria and their respective results are as follows; reaction efficiencies using DDCT method 0.021, 0.029 & 0.064 (acceptable <0.1), primer accuracy showed correlation coefficient values of 0.99 for all primers, sensitivity of recipient DNA detection R2 values of 0.994, 0.996 & 0.9915 (acceptable 0.98), precision/reproducibility CV 6.4%, 6.2% & 6.1% over ten runs (acceptable 10%) and a comparison with existing cytogentics FISH XY testing showed pairings were significantly effective as indicated by P values of <0.0001 for all primers. This method provides rapid, simple and reliable results for the quantitation of DNA chimerism post transplant. Reference: Harries et al. 2005 Analysis of haematopoietic chimerism by quantitative realtime polymerase chain reaction.
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71 SUCESSFUL COLLECTION OF PERIPHERAL HEMATOPOIETIC PROGENITOR CELLS (HPC-A) FROM LOW MEAN CORPUSCULAR VOLUME (MCV) DONORS BY MODIFYING PROGRAMMED SEPARATION FACTOR OF COBE SPECTRA APHERESIS DEVICE S Sharma, LF Sarhan, FA Rahman, A Tbakhi King Hussein Cancer Center, Amman, Jordan Potential blood and marrow transplantation donors must meet established donor evaluation criteria and be free of malignancies, major diseases, transmittable infectious diseases, and inherent metabolic disorders. At the time of collection, complete blood count (CBC) of the donor should be normal. Donors with microcytosis due to iron deficiency can be treated with iron supplementation before donation. However, donor deferral may be warranted if significant hemoglobinopathy is present. HLA matched, related transplants are preferable over unrelated transplants, considering post-transplant complications and morbidities. The chance for finding related donor in the Middle East is significantly higher as compared to many western countries. However, the incidence of thalassemia and other hemoglobinopathies is also higher in this region. Therefore, if a related donor, who is a full HLA match, but has a trait such as thalassemia minor, is the only related donor, can still be considered as a potential donor, instead of an unrelated transplant. Low MCV donors mobilize well with granulocyte colony stimulating factor (G-CSF), however, the collection efficiency of peripheral hematopoietic progenitor cells (HPC-A) from such donors by apheresis is poor. We have found that by manually modifying programmed separation factor of COBE Spectra apheresis device, it is possible to obtain comparable number of HPC-A from low MCV donors, equal to the control group with normal MCV (Table below). The only drawback was increased platelet loss from the donor due to higher sedimentation rate. All recipients engrafted well, related both, to the white blood cells and platelets.
72 A NOVEL AUTOMATED BIOREACTOR FOR SCALABLE PROCESS OPTIMISATION OF HAEMATOPOIETIC STEM CELL CULTURE E Ratcliffe Loughborough University, Loughborough, United Kingdom Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. Here we report the proliferation and erythroid commitment (CD71+, CD235a+) of HSCs in a new automated process development platform (ambrTM; advanced microscale bioreactor) designed for scaled-down optimisation of stirred tank production systems. The workstation comprises 2415 mL replicate independent suspension culture bioreactors and offers precise control of the culture environment through online monitoring and automated control of temperature, pH, gassing, stirring, cell density and liquid handling. Cell proliferation was relatively robust to cell density and oxygen levels and reached 6 population doublings over 10 days. Maximum culture densities for a simple fed batch system were established to be in the order of 107 cells/mL. This system will be valuable for the further HSC suspension culture optimisation necessary to apply conventional stirred tank technology to scaled manufacture of HSC derived products. HSCs are a good candidate for suspension culture production systems as many of the relevant cell types are naturally non-adherent. This should facilitate their clinical production due to the easy availability of large biopharmaceutical suspension bioreactors. The data reported here allows evaluation of HSC fed batch culture limits and the economics of a suspension reactor production system. It is likely that achieving densities in excess of those reported will require a non-batch approach to cell produced factor removal and nutrient supply. This scaled down system of stirred tank operation can successfully support HSC proliferation and differentiation and therefore provide a valuable tool for further exploring the limits of HSC bioprocessing.
E- depletion. However, CliniMACS plus IVIG reduces some immune modulating cell subsets (TCRgd, CD56/16, p<0.001) more efficiently and others (CD4, p<0.001) less efficiently than the Iso E- method. Our comparison of post-TCD HPC grafts demonstrate that similar TCD methods vary in their ability to remove different, and potentially important T-cell subsets including NK cells, and may provide an explantation for the varying clinical results observed in TCD-allograft 69 NEUTROPHIL CONTAMINATION IN AUTOLOGOUS PERIPH ERAL BLOOD STEM CELL PRODUCTS: IMPACT ON POSTTRANSPLANT OUTCOME W Xia Royal North Shore Hospital, Sydney, New South Walse, Australia Aim: To evaluate whether neutrophil contamination of HPC-A product could impact transplant related outcomes at a single centre. Methods: Between 2009 and 2012, 129 patients underwent HPC-A mobilization and collection. Apheresis was initiated once PB CD34 >15/uL and continued until > 2106/kg CD34+ cells were collected. The instrument modes of apheresis included Cobe Spectra manual, Cobe Spectra automated and Optia. Patient characteristics, collected HPC-A volume, neutrophil% in HPC-A, infused neutrophil dose, and transplant outcomes were evaluated. Results: 53%, 38% and 9% of patients received manual, auto and Optia mode collection. Manual mode, comparing auto mode, significantly caused larger collected HPC-A volume (mL) (308+122 vs 123+70, p<0.001); and more neutrophil% in HPC-A (40+19.3 vs 16+18.9, p<0.001). Increasing patient age significantly correlated to higher infused neutrophil dose (p¼0.005). Mean age of patients neutrophil dose of 5108/kg in HPC-A significantly delayed neutrophil engraftment vs HPC-A containing neutrophils <5108/kg, (14.24d+4.51 vs 12.5d+2.39, p¼0.019). Infused neutrophil number significantly correlated to neutrophil engraftment time (R2¼0.094, p¼0.001); but didn’t impact platelet engraftment, TRM and survivals. Conclusions: 1. Manual mode of apheresis was associated with greater volume and higher neutrophil contamination in HPC-A than auto mode. 2. Older age was associated with higher infused neutrophil dose in HPC-A 3. Infused neutrophils >5108/kg in HPC-A is associated with delayed neutrophil engraftment but did not impact on other post transplant outcomes. 70 QUANTIFICATION OF HAEMATOPOIETIC CHIMERISM BY REAL TIME PCR ASSAY VALIDATION K Kooloos The Royal Children’s Hospital, Parkville, Australia Following allogeneic stem cell transplantation, it is of great importance to determine whether the newly developed haematopoietic system is of donor or recipient origin. Quantitative Real Time PCR (Polymerase Chain Reaction) has recently been implemented at The Royal Children’s Hospital, Melbourne to replace the qualitative VNTR (Variable Number of Tandem Repeats) assay by gel electrophoresis to determine post transplant chimerism. Real Time PCR discriminates alleles using fluorescent dye-labelled probes for Single Nucleotide Polymorphisms (SNP). This test is used to monitor engraftment of the transplant and identify re-emergence of recipient cells, an indicator of engraftment failure or impending relapse. Three SNP probes from Applied BiosystemsÒ (rs1908777, rs3918344 & rs338773) were validated in the implementation of the Real Time PCR assay. Analysis was performed using Rotor-geneÔ software. The method validation was based on the following criteria and their respective results are as follows; reaction efficiencies using DDCT method 0.021, 0.029 & 0.064 (acceptable <0.1), primer accuracy showed correlation coefficient values of 0.99 for all primers, sensitivity of recipient DNA detection R2 values of 0.994, 0.996 & 0.9915 (acceptable 0.98), precision/reproducibility CV 6.4%, 6.2% & 6.1% over ten runs (acceptable 10%) and a comparison with existing cytogentics FISH XY testing showed pairings were significantly effective as indicated by P values of <0.0001 for all primers. This method provides rapid, simple and reliable results for the quantitation of DNA chimerism post transplant. Reference: Harries et al. 2005 Analysis of haematopoietic chimerism by quantitative realtime polymerase chain reaction.
S23
71 SUCESSFUL COLLECTION OF PERIPHERAL HEMATOPOIETIC PROGENITOR CELLS (HPC-A) FROM LOW MEAN CORPUSCULAR VOLUME (MCV) DONORS BY MODIFYING PROGRAMMED SEPARATION FACTOR OF COBE SPECTRA APHERESIS DEVICE S Sharma, LF Sarhan, FA Rahman, A Tbakhi King Hussein Cancer Center, Amman, Jordan Potential blood and marrow transplantation donors must meet established donor evaluation criteria and be free of malignancies, major diseases, transmittable infectious diseases, and inherent metabolic disorders. At the time of collection, complete blood count (CBC) of the donor should be normal. Donors with microcytosis due to iron deficiency can be treated with iron supplementation before donation. However, donor deferral may be warranted if significant hemoglobinopathy is present. HLA matched, related transplants are preferable over unrelated transplants, considering post-transplant complications and morbidities. The chance for finding related donor in the Middle East is significantly higher as compared to many western countries. However, the incidence of thalassemia and other hemoglobinopathies is also higher in this region. Therefore, if a related donor, who is a full HLA match, but has a trait such as thalassemia minor, is the only related donor, can still be considered as a potential donor, instead of an unrelated transplant. Low MCV donors mobilize well with granulocyte colony stimulating factor (G-CSF), however, the collection efficiency of peripheral hematopoietic progenitor cells (HPC-A) from such donors by apheresis is poor. We have found that by manually modifying programmed separation factor of COBE Spectra apheresis device, it is possible to obtain comparable number of HPC-A from low MCV donors, equal to the control group with normal MCV (Table below). The only drawback was increased platelet loss from the donor due to higher sedimentation rate. All recipients engrafted well, related both, to the white blood cells and platelets.
72 A NOVEL AUTOMATED BIOREACTOR FOR SCALABLE PROCESS OPTIMISATION OF HAEMATOPOIETIC STEM CELL CULTURE E Ratcliffe Loughborough University, Loughborough, United Kingdom Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. Here we report the proliferation and erythroid commitment (CD71+, CD235a+) of HSCs in a new automated process development platform (ambrTM; advanced microscale bioreactor) designed for scaled-down optimisation of stirred tank production systems. The workstation comprises 2415 mL replicate independent suspension culture bioreactors and offers precise control of the culture environment through online monitoring and automated control of temperature, pH, gassing, stirring, cell density and liquid handling. Cell proliferation was relatively robust to cell density and oxygen levels and reached 6 population doublings over 10 days. Maximum culture densities for a simple fed batch system were established to be in the order of 107 cells/mL. This system will be valuable for the further HSC suspension culture optimisation necessary to apply conventional stirred tank technology to scaled manufacture of HSC derived products. HSCs are a good candidate for suspension culture production systems as many of the relevant cell types are naturally non-adherent. This should facilitate their clinical production due to the easy availability of large biopharmaceutical suspension bioreactors. The data reported here allows evaluation of HSC fed batch culture limits and the economics of a suspension reactor production system. It is likely that achieving densities in excess of those reported will require a non-batch approach to cell produced factor removal and nutrient supply. This scaled down system of stirred tank operation can successfully support HSC proliferation and differentiation and therefore provide a valuable tool for further exploring the limits of HSC bioprocessing.