Cord Blood Processing D-Efficiency! Next Generation Protocol Development For Celling Clinicians More Cells

Transcription

1 WHITE PAPER RESEARCH AND DEVELOPMENT UPDATES Cord Blood Processing D-Efficiency! Next Generation Protocol Development For Celling Clinicians More Cells Authors: Petra Cravens, Mindy Wilke-Douglas, Dalip Sethi, Ray DeGrella, Ella Severson, Mitchel Sivilotti CESCA THERAPEUTICS INC Citrus Road Rancho Cordova, CA USA

2 Introduction In the past five years we have seen a change in the transplant physician s expectation with respect to the minimum acceptable cell counts needed for cord blood transplantation (CBT). 1 In the early days of public cord blood banking, the cut-off for an acceptably high cell count, in terms of Total Nucleated Cells (TNCs), was approximately 900 Million cells per cord blood unit (CBU). It was in accordance with this understanding that public cord blood banks invested enormous resources in the accumulation of valuable biological stock and retain much of this inventory today. As would be expected, clinical results from the adoption of CBT by the medical field provided continuous feedback to cord blood banks (CBB). The net result is a trend leading CBBs to prioritize TNC counts: transplant physicians select CBUs based upon various factors, the most important of which is the quantity of TNCs due to a positive correlation with improved clinical outcomes (shorter engraftment times, reduced transplant mortality, etc.). 2,3,4 Manufacturers initially received an A for innovative device solutions, their grade may be quickly becoming a D-efficiency by not evolving alongside TNC count expectations. The CBB industry, by not leaving any stone left unturned, has pushed skyward the requirement of TNC counts in the absence of approved cell expansion techniques, as obtaining sufficient cell doses remains to be the main challenge CBBs face today. 1,5 In this effort, though medical device manufacturers initially received an A for innovative device solutions, their grade may be quickly becoming a D-efficiency by not evolving alongside TNC count expectations now as high as 1.6 Billion TNCs, an increase of 78%. This obvious lack of innovation begs the question of whether or not it is possible to improve rapid targeted cell isolation for high volume CBUs? In formulating a solution to this development challenge we were keenly aware that high TNC requirements (the new normal for public CBBs) result in the disproportionate selection of high volume CBUs versus low volume units (far higher than originally contemplated by device designers). Thus, in response to high volume CBU processing demand, we conducted this study to exploit our technological advances in CB processing to target higher volume CBUs. 6 Specifically, we developed and tested a new high volume CBU processing device which demonstrated an impressive 15.8 percentage point increase in TNC yields (an additional ~ Million TNCs) compared to the standard AXP processing method on high volume CBUs. Needless to say, this could make all the difference to a physician when faced with selecting their next CBU. PROPERTY OF CESCA THERAPEUTICS, INC PAGE 2 OF 8

3 Materials and Methods We conducted this study to exploit the flexibility of our technological advances in CB processing to target higher volume CBUs. Adult peripheral blood units (PB) and umbilical cord blood units (UCB) were collected in 70 or 35 ml of citrate phosphate dextrose (CPD), respectively. Blood units were maintained at ambient temperature (20 25 C) until processing. PB units were investigated 24 hours postcollection and UCBs were investigated at a mean age of 45.6 ± 6 hours using our new high volume processing device and compared to the standard AXP (as per the AXP System Operator and Maintenance Manual). 7 Processed blood samples were harvested from the freezing bag for hematological and flow cytometry analyses. Differences between pre- and post-processing samples were assessed using a Student s t-test. PROPERTY OF CESCA THERAPEUTICS, INC PAGE 3 OF 8

4 Results and Discussion Based on a proprietary method of sensory interrogation, our device distinguishes between density separated blood components and differentially selects target fractions for sequestration. The target cell population, for CBBs, is the fraction containing TNCs, which is found primarily between the least dense population of red blood cells (RBCs) and the lower interface of cell-free plasma. Though this target population of cells can be considered similar across human cord and peripheral blood samples, the concentration (of cells per unit volume) and distances traveled to achieve density equilibrium can vary extensively between samples within either blood type. In this case we were interested in evaluating the effects of extended time to density equilibrium as we see this as the pivotal consequence of higher volume CBUs. In the Figure 1, we conducted a comparison of various PB sample volume cohorts (N=30, 3 UCB per cohort) to isolate, in a controlled environment, the effect of sample volume on the TNC % recovery pre- versus post-processing with our device. The result of this investigation demonstrated that when processed using the standard AXP protocol, the TNC recovery in PB decreased with increasing volume. When repeated using our new high volume processing device, the volume effect was eliminated entirely, a demonstration of the powerful efficacy of our new technology. % TNC Recovery FIGURE 1. High volume processing device eliminates volume related reduction in TNC recovery in PB. *Indicates a statistically significant difference in the % of TNC recovered between our new high volume processing device versus the AXP Standard AXP New High Volume Device Starting PB Volume (ml) p=0.04 * p=0.00 * We developed and tested a new high volume processing 15.8 % POINT INCREASE IN TNC YIELDS device, which demonstrated a 15.8 % point increase in TNC yields (an additional ~ Million TNCs) compared to the standard AXP system on high volume CBUs. PROPERTY OF CESCA THERAPEUTICS, INC PAGE 4 OF 8

5 Results and Discussion (continued) To further explore and define reasoning for the superior performance of our new high volume technology with high volume blood units, a small group of PB samples (n=3) was processed and evaluated for TNC composition in sequential 5 ml aliquots starting from the bottom of the processing bag (RBC layer) through the plasma layer. The cumulative percentage of TNC recovered was determined for each aliquot and the results shown in Figure 2. The result of this investigation was encapsulated by the steepness of the TNC percentage recovery curve in our high volume device versus the AXP. In effect the high volume device appears to have created an environment where cell density equilibrium can be reached in a compressed volume while the device simultaneously targets and extracts cells without disturbing the hard-won density interface. We believe we have, as a result, demonstrated a connection between the amelioration of undisturbed cell focusing and target cell recovery efficiency through advancements in our proprietary technology. Of particular interest is the volume in which the high volume device focuses the TNC fraction 15mLs (>5mLs lower than typical industry protocols permit). With such a reduction in volume, one could expect to see much lower red blood cell concentrations and the potential flexibility to use smaller storage bags (freezer bags) or more interestingly to contemplate innovative cryopreservation cocktails with the intent of improving cell viability post-thaw; an area of advancement that CBBs and clinicians would find very attractive. FIGURE 2. High volume processing device results in a compression of TNC location within density fractionated PB compared to the AXP. Cummulative TNC % Recovery Cummulative PB Volume (ml) Standard AXP New High Volume Device PROPERTY OF CESCA THERAPEUTICS, INC PAGE 5 OF 8

6 Results and Discussion (continued) The final step of this investigation was to apply the advancements of the high volume device to UCB. To this end (Figure 3), we selected UCBs ranging in volume from 80mL to 170mL (including anticoagulant) and separated them into seven groups of defined volume range (15 ml maximum variance range). Each volume cohort (N=34, distributed across UCB cohorts per market prevalence) was processed using the high volume device or the AXP. Akin to our PB studies, the high volume device produced a consistently higher TNC percentage recovery, one that was a statistically significant improvement in comparison to the AXP in UCB volumes of 110mL and greater with a P value < On a percentage basis, the high volume technology improved recoveries by 15.8 percentage points for large volume UCBs (>110 ml). Further analysis (mass balance) of the post-processing bag set compartments revealed that the percentage (%) of TNC remaining in the plasma of large volume UCBs processed with the high volume device or the AXP was negligible and similarly we observed a remarkable decrease of the percentage (%) TNC present in the RBC bag. As a result, we can hypothesize that the increase in TNC recoveries observed with the high volume device was due to a more complete isolation and transfer of TNC into the freezing bag. To further confirm the safety and efficiency of the high volume device, we investigated both the CD34+ cell recovery and viability using flow cytometry (Table 1). Our results demonstrate robust percentage (%) recoveries in CD34+ populations and CD34+ cell viabilities, with no significant difference between pre- and post-processing UCB samples nor between processing devices. In general, CD34+ cell viability pre- and post-processing is %; however, our study demonstrates a declining percentage viability likely due to the age of our CBU ( hours). 2 Industry averages for CBU age are usually between 12 and 36 hours. Average TNC % Recovery FIGURE 3. In UCB greater than 110mL, the high volume processing device significantly increased TNC recovery versus the AXP. *indicates a statistically significant difference between the high volume device and the AXP p = 0.01 p = 0.00 p = 0.00 p = 0.01 * * * * Standard AXP New High Volume Device UCB Volume Range (ml) TABLE 1. CD34+ cell viability is sustained after processing with the high volume processing device. Standard AXP High Volume Device CD34+% Recovery 91.5% ± 12.1% 97.3% ± 10.5% Pre-processing Viability 87.4% ± 6.1% 85.1% ± 7.9% *Note: Cord blood median age hours. Post-processing Viability 88.5% ± 5.5% 83.8% ± 7.9% PROPERTY OF CESCA THERAPEUTICS, INC PAGE 6 OF 8

7 Summary In summary, the implementation of a new software (firmware) controlled technology designed to improve cellular focusing and disturbance-free isolation has spawned a customized device for improved TNC recovery from high volume cord blood units. Enhanced TNC recovery, however, does not appear to be the only benefit we can draw from this new automated device. As a consequence (and welcome side-effect) we hypothesize the advantage of reduced cell volume and concomitantly lower red blood cell content, both important breakthroughs, can translate into hypothetical gains in cell viability, cell potency, dose volume flexibility and perhaps even open the door to considerations for customized cellular products, products whose contents can be reproduced in a defined, controlled process environment. Additionally, these new devices could facilitate ingenuity in our users cryopreservation protocols. In addition to scientific gains, we envision that our new high volume processing technology may potentially create new financial economies as a result of i) elimination of UCB splitting (usage of fewer processing consumables) and ii) potentially maximizing/optimizing cryopreservation Dewar footprints in the event freezer bag technologies begin to accommodate these advancements. REFERENCES 1. Ballen K, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood : Rubenstein P. Cord blood banking for clinical transplantation. Bone Marrow Transplantation , Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med. 1998; 339 (22): Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilicalcord blood from unrelated donors. N Engl J Med. 2001; 344 (24): Barker JN, Byam C, Scaradavou A. How I treat: the selection and acquistion of unrelated cord blood grafts. Blood : Querol S. Towards more rational allogenic CB inventories. Abstract presented at the World Cord Blood Congress III, Rome 2011; Published in: Cord blood banking: current status. Hematology. 7. AXP System Operator and Maintenance Manual. ACKNOWLEDGMENTS The authors would like to thank: Blood Source in Rancho Cordova, CA for providing peripheral blood samples, New York Blood Center in New York, NY for providing cord blood samples, and Dr. Elisabeth Semple, Scientific Director at Cells for Life in Ontario, Canada, for contributing to preliminary studies and offering valuable support and guidance. PROPERTY OF CESCA THERAPEUTICS, INC PAGE 7 OF 8

8 CESCA THERAPEUTICS INC Citrus Road Rancho Cordova, CA USA PROPERTY OF CESCA THERAPEUTICS, INC PAGE 8 OF 8