Total Nucleated Cell Recovery Research Articles

Preparation of cryopreserved chimeric antigen receptor T cells for the locoreogional delivery to the neural axis

Background aimsIntracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure. MethodsWe developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20–25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity. ResultsThe developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%–88%; total nucleated cell recovery mean: 76.5%, range: 65.4%–82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours’ post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%–81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells. ConclusionsWe have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.

Single laboratory evaluation of umbilical cord blood units processing methodologies for banking.

To compare the efficiency of 3 different processing methods (Sepax, AutoXpress [AXP], and manual processing with hydroxyethyl starch [HES] sedimentation) used at Stemlab during a 10-year period. Historical data were compiled and the analytical results obtained for the 3 different methods were compared. The manual processing (HES) method yielded the highest level of total nucleated cell recovery after processing, and the AXP system yielded the highest CD34+ cell number. The red blood cell reduction was also significantly higher with the HES method. Also, HES showed comparable results to Toticyte technology for umbilical cord blood (UCB) processing. These results show that the HES method is as effective as automated technologies for UCB volume reduction; hence, it is a suitable methodology for private and public UCB banks. The HES method also proved to be superior to Toticyte technology for medical applications, with higher recovery yields of total nucleated cells after thawing and equivalent CD34+ cell recovery and functionality.

Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

Abstract 23 A First Insight into the Validity of Post-Thaw Cell Recovery, Viability, and Potency Over Time

IntroductionTesting of a thawed segment or representative sample of a cord blood unit (CBU) for cell recovery, viability, and potency forms part of pre-release testing. Not all CBUs that have had pre-release testing completed are shipped for transplant, due to postponed or cancelled shipment request, and are therefore kept in the inventory until they are searched again. In an effort to identify these CBUs for future expedited release, the World Marrow Donor Association (WMDA) initiated a project to flag them as “Ready to Ship” in the WMDA Search & Match Service. However, it is not known how long post-thaw results are valid before they must be repeated on another thaw. Such information can be beneficial not only for transplant centers to better assess CBU suitability but also for Cord Blood Banks (CBBs) to avoid unnecessarily exhausting their precious samples.ObjectiveThis study aimed to provide a first insight into the validity of post-thaw results over various lengths of time.MethodsNineteen CBUs, collected between 2003 and 2015 at the BMDI CBB and that had post-thaw testing performed twice, were analyzed for change in the total nucleated cell (TNC) recovery, total CD34 cell recovery, CD34 cell viability, and CFU-GM counts between two thaws.ResultsThe 19 CBUs were grouped into 4 groups based on their storage time between thaws. An average decrease in TNC recovery of 5.3% from first thaw to second thaw was observed for the entire dataset (P = .015). Four CBUs with a greater than 10% decrease had a bag TNC greater than 25 × 108. Individual CBU data is shown in Figure 1. No significant change between thaws was seen with total CD34 recovery (P = .738) or CD34 viability (n = 9 pairs, P = .354). A large variation in CFU-GM counts was observed between thaws. It is worth noting that all CBUs met CBB release criteria of >60% TNC recovery and that CD34 viabilities were above 70% (FACT requirement) in both thaws.Figure 1.Comparison of first and second thaw TNC recovery (A), total CD34 recovery (B), CD34 viability (C), and CFU-GM counts (D) of individual cord blood unit, grouped into 4 groups based on their storage time between thaws: 0-6 months (i), 2-3.5 years (ii), 6-8.5 years (iii), and 12 years (iv).DiscussionWhile results need to be confirmed on a larger dataset, our preliminary data shows very little change in post-thaw cell recovery and viability over various lengths of time (2 months-12 years), suggesting post-thaw results may be valid for longer than 10 years.

How do I warm HPC(A) products to maximize cell viability in the setting of cold agglutinin disease?

High titers of cold agglutinins jeopardize the quality of an apheresis product meant for autologous or allogeneic transplant. Management of transplant patients with cold agglutinin disease (CAD) is often experience-based and under reported, yet decisions must be made quickly to optimize product management and patient outcomes. There remains a lack of data quantifying cell recovery and viability when using various warming methodologies. To expand the published experimental data on this subject, our human cellular therapy lab compared cellular recoveries and viabilities after manipulation of cryopreserved apheresis products through various warming methodologies: (1) extended warming in a water bath, (2) warming via blood warmer and infusion pump, and (3) warming in a water bath followed by infusion pump as a control to assess potential shear stress effects. The presented studies demonstrate that all methods of product warming produce the same rates of recovery of total and viable cells across vital cell types prior to patient administration. Statistically, use of an extended water bath protocol provided a marginal benefit in recovery of total nucleated cells, though this effect is diminished when products are held for an extended period to simulate a delay in administration. These results can inform decisions to improve patient care and minimize product manipulation and loss. Centers are encouraged to use this information to guide proactive measures to establish a standard operating procedure to manage CAD cases.

Red blood cell depletion in small-volume bone marrow processing using manipulation with third-party red blood cells: A comparison of the performance of the COBE spectra and the spectra Optia systems.

For pediatric recipients, red blood cells (RBCs) are added to bone marrow (BM) collections before low RBC volume BM processing using COBE Spectra (COBE) or Spectra Optia (Optia). However, the processing efficiency of this approach has not been evaluated. This study aimed to evaluate RBC depletion and nucleated cell subpopulation recovery rates in third-party RBC-manipulated BM products processed with the COBE or Optia. We retrospectively collected data on RBC depletion from low RBC volume BM with third-party RBCs (manipulated group) and on conventional large-volume, BM (unmanipulated group) processing performed between September 2010 and December 2021. All procedures were performed using COBE or Optia. The median residual RBC volume in the manipulated group was 9.5 ml in COBE and 2.5 ml in Optia (p=.01). The median total nucleated cell (TNC) and mononuclear cell (MNC) were comparable between the manipulated groups using each cell separator (TNC, 40.8 vs. 47.1%; MNC, 78.3 vs. 79.4%). The manipulation did not adversely affect TNC and MNC recoveries in either device. In addition, Optia achieved similar CD34+ cell recovery to that in large-BM-volume processing using the same device (147.5 vs. 184.5%, p=.112). During a follow-up period, neutrophil engraftment was achieved in all patients who received third-party RBC-manipulated grafts, and platelet engraftment was achieved in all cases, except one. The addition of third-party RBC to low RBC volume BM collections from or for pediatric patients does not have any negative impact on either RBC depletion or hematopoietic cell recovery during processing with the widely used cell separator.

Washing transplants with Sepax 2 reduces the incidence of side effects associated with autologous transplantation and increases patients' comfort.

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) is routinely used in various hematologic malignancies. However, dimethylsulfoxide contained in cryopreserved grafts can cause adverse events (AEs). Forty-three ASCTs were performed with Sepax 2 washed grafts between 7/2016 and 10/2019. The aim of this study was to determine whether washing out dimethyl sulfoxide (DMSO) from transplants using the Sepax 2 (S-100) device is safe and reduces the incidence of DMSO-associated AEs. The washing procedure was automated and that resulted in the satisfactory recovery of total nucleated cells, CD34+ cells, and colony forming units of granulocyte and macrophages (85%, 80%, and 84%, medians). Time to engraftment of leukocytes, granulocytes, and platelets as well as the number of neutropenic days did not differ when compared to 20 consecutive ASCTs without washing. The AE occurrence was lower compared to unwashed grafts: 81% versus 78% during and shortly after grafts administration, 76% versus 69% in the following day. We conclude that the washing of cryopreserved transplants using Sepax 2 was feasible with a high recovery of hematopoietic cells, did not influence time to engraftment, and resulted in the satisfactory reduction of AEs and improved tolerance of the procedure.

The Post-Thaw Recovery of Cryopreserved Chimeric Antigen Receptor (CAR) T-Cells during Manufacture Is Better Than That of Cryopreserved Peripheral Blood CD3+ Cells

As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy extend beyond early phase trials, larger scale cell manufacturing processes incorporating cryopreservation steps become a logistical necessity. Mononuclear cells (MNC) collected by leukapheresis, which serve as the pre-culture “starting” fraction (PeC) for CAR T-cell production, may need to be cryopreserved prior to cell transportation to a centralized manufacturing facility. PeC aliquots may be cryopreserved for repeat cultures in cases of primary culture failure or for patient re-dosing. Further, cryopreservation of the post-culture (PoC) CAR T-cell product is essential in order to ease post-manufacture transportation, ensure completion of regulatory testing prior to product release and avoid product wastage due to unanticipated patient-related delays. The effect of cryopreservation on CAR T-cell viability, expansion, phenotype and transduction efficiency merit evaluation prior to routine use.From 2012 to 2017, data was obtained on 160 consecutive autologous CAR T-cell cultures initiated for patient infusion on 6 clinical trials of different CAR T-cell types (1. Anti-CD19-pediatric trial (n=58), 2.Anti-GD2 (n=14), 3.Anti-BCMA (n=21), 4.Anti-CD22 (n=43), 5. Anti-CD19-adult trial (n=22), 6. Anti-CD30 (n=2)).Following autologous leukapheresis collection, PeC was either cryopreserved-thawed (PeC-C) or used fresh (PeC-F) for CAR T culture initiation, depending on patient needs. Thereafter, cell selection, transduction-expansion and harvest were performed according to trial protocol specific instructions. The harvested PoC was again cryopreserved (PoC-C) or infused fresh (PoC-F). Data on patient demographics, number, timing and duration of cryopreservation, Total Nucleated Cell (TNC) count, viability, fold expansion (FE), transduction efficiency (TE), CD3% and CD4/CD8 ratios was collected. Post thaw TNC recovery (PTR) % was calculated as TNC*viability at thaw/TNC* viability at cryopreservation.Afterdiscarding 13 products due to manufacturing failure (MF) during culture, 147CAR T infusates were prepared for pediatric or adult patients with hematologic malignancies (135, 91.8%) or solid tumors (12, 8.2%). Mean age was 27.7 years (±19.9), weight 60.7 kg (±26.3), median CAR T-cell dose of 1x106 CAR-transduced viable CD3+ cells/kg (Range: 1x105 - 1x107). Gamma-retroviral or lentiviral vectors were used for CAR delivery in 88 (59.9%) and 59(40.1%) infusates, respectively. Pre-culture lymphocyte concentration included ficoll alone (42, 28.6%), flask adherence and/or elutriation (14, 9.5%), CD3.CD28 and/or CD4/CD8 selection (68, 46.3%). Of the 147 infusates, PeC-C and PoC-C were 69 and 78, respectively. Median duration of PeC-C and PoC-C was 6 days (Range: 3-868) and 9 days (Range: 1-408), respectively. TNC and CD3 PTR were lower in PeC-C than in PoC-C (Figure). At harvest (PoC), Mean FE, TE, CD3% and CD4/CD8 ratio were 12.8 (±12.5), 54.3 (±21.9), 97.6 (±4.4) and 1.9 (±2.0) respectively. In multivariate analyses, FE, TE, CD3% and CD4/CD8 ratios at harvest were unaffected by PeC-C status or duration. TE differed by trial. Twelve out of 13 (92%) MFs were due to failure of PeC transduction and/or expansion. Of the 12 MFs, 6 products were PeC-C (50%); 6 were PeC-F (50%). Among the 6 PeC-C MFs, none of the failures could be attributed to cryopreservation. In fact, 4 of the 6 PeC-C cultures were re-initiated with an additional PeC-C and all 4 yielded a successful product.Pre-culturecryopreservation impacts viable TNC and CD3% recovery during CAR T-cell manufacture compared to post-culture cryopreservation. Within this PeC fraction, CD3+ cells appear to have better viable recovery than the whole TNC fraction. This is likely due to a higher (differential) loss of monocytes and NK cells within TNCs. Despite these findings, most products manufactured after initial cryopreservation met clinical dose requirements. FE, TE, CD3% and CD4/CD8 ratios were unaffected by initial cryopreservation. The post thaw viability of CAR T-cells cryopreserved post culture was greater than cryopreserved peripheral blood CD3+ cells. Cryopreservation at either end of CAR T-cell manufacture is a viable strategy and may ease logistical constraints associated with large scale cell production. If optional, CAR T-cells cryopreserved post manufacture may be preferable to cryopreserving the starting fraction. [Display omitted] DisclosuresKochenderfer:Bluebird bio: Research Funding; Kite Pharma: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.

Analysis of outcomes of single-unit cord blood transplantation with umbilical cord blood units processed with two different red blood cell sedimentation reagents.

Various processing methodologies are routinely used to reduce volume and red blood cell content of umbilical cord blood (UCB) units collected for hematopoietic stem cell transplantation. There is limited information regarding effects of UCB processing techniques on clinical outcomes. Retrospective data analysis compared laboratory and clinical outcomes following single-unit UCB transplantation performed between 1999 and 2015. All UCB units were from St. Louis Cord Blood Bank and all were manually processed with either Hetastarch processed cord blood units (HCB) (n= 661) or PrepaCyte processed cord blood units (PCB) (n= 84). Additional sensitivity analysis focused on units transplanted from 2010 to 2015 and included 105 HCB and 84 PCB. There were no significant differences in patient characteristics between the two groups. Pre-freeze total nucleated and CD34+ cell counts, cell doses/kg of recipient weight, and total colony-forming units (CFUs) were higher in PCB compared with HCB. Post-thaw, the PCB group had a significantly better total nucleated cell recovery, while there were no significant differences in cell viability, CFU recovery, or CD34+ cell recovery. Primary analysis demonstrated faster neutrophil and platelet engraftment for PCB but no differences in overall survival (OS), whereas sensitivity analysis found no effect of processing method on engraftment, but better OS in the HCB group compared with PCB group. The UCB processing method had no significant impact on engraftment. However, we cannot completely exclude the effect of processing method on OS. Additional studies may be warranted to investigate the potential impact of the PCB processing method on clinical outcomes.

Transient warming affects potency of cryopreserved cord blood units

Background aims. Cryopreserved cord blood units (CBUs) can be exposed to transient warming events (TWEs) during routine banking operations, which may affect their potency. NetCord-FACT guidelines recommend removal of these CBUs from inventory. The objective of this work was to evaluate warming kinetics of frozen CBUs in different settings to determine the optimal working environment and define the impact of different TWE scenarios on CB post-thaw quality and potency. MethodsThe warming kinetics of frozen CBUs was influenced by both working surfaces and ambient working temperature, with cold plates providing better protection than vinyl or metal surfaces. Measurement of time for required operational activities revealed that CBUs are probably exposed to core temperatures greater than –150°C even when cold plates are used to reduce warming rates. ResultsOn the basis of the warming kinetics and observed operational activities, three TWE causing scenarios (control, typical, worst case) were investigated using a pool-and-split design and cell viability, recovery and potency (colony-forming unit [CFU]) assays were performed. TWEs were found to have little impact on the recovery of total nucleated cells or on the viability of CD34+ cells. In contrast, the viability and recovery of CD45+ cells in the smaller CBU compartments were reduced by TWEs. Moreover, the worst-case TWE reduced CFU recovery from CBUs, whereas the typical-scenario TWE had little effect. Conclusions. Our results demonstrate that the distal segment underestimates the viability and potency of CBUs and that TWEs can affect the post-thaw viability and potency of CBUs. Although TWEs are almost inevitable during cord-blood banking operations, their effects must be diminished by reducing exposure time, using cold plates and strict operational protocols, to prevent worst-case TWEs.

Effect of Long-Term Storage on Hematopoietic Progenitor Cell Transplant Product Quality

Abstract Hematopoietic progenitor cell (HPC) transplant product quality indicators after prolonged cryopreservation are variable. A recent study in Europe demonstrated wide variability of cryopreservation times, from 1 to >20 years, with most centers storing products for 5 or 10 years. Thawed HPC viability using various ex vivo means and in vivo by studying neutrophil and platelet engraftment has generally supported preservation of CD34+ viability with prolonged storage. However, sample size, methods, and timepoints varied, and clear guidelines currently do not exist regarding how long HPCs can be stored. This study’s objectives include an evaluation of HPC quality indicators over time and if HPC product age and/or viability correlates with HPC engraftment. Retrospective record review was conducted on 837 HPC products thawed between January 1, 2011, and December 31, 2018. HPC products were sampled and characterized at thawing by flow cytometry for total nucleated cell (TNC) recovery, CD34+ recovery, and CD34+ viability (by 7-AAD). Preestablished acceptability criteria for TNC recovery, CD34+ recovery, and CD34+ viability were >80%, >40%, and >60%, respectively. If available, products with ANC engraftment data were further analyzed to correlate data with time to HPC engraftment. HPC products were stored between 3 and 5,943 days; 816 products were infused, ranging from 3 to 5,273 days in storage. Most (81%) HPC transplants occurred within the first year, 47 infusions occurred after storage >5 years, and 4 infusions occurred after storage >10 years. CD34 viability and percent recovery of TNC and CD34 were not significantly different as time in storage increased. Given sample paucity, 19 samples stored for >10 years were analyzed in individual years and grouped. In total, 96.5% samples stored for <1 year and 68% in samples stored >10 years met acceptability criteria for >60% CD34+ recovery; 98.9% samples stored <1 year met the >60% CD34+ viability acceptability threshold versus 90% of samples stored for >10 years. The oldest sample infused after storage of 5,273 days (14.4 years) showed 42% TNC recovery, 19% CD34+ recovery, and 84% CD34 viability; ANC engraftment data were not available. Sixty-five samples containing ANC engraftment data showed that time to ANC engraftment ranged from 9 to 15 days. No clear trend was present for viability or CD34 recovery when compared with days to engraftment based on limited data. Although rare, infusion of HPC products of extended duration occurs. This study suggests that HPC product quality may decrease after extended cryopreservation based on preestablished acceptability criteria. The oldest sample infused had preserved CD34+ viability despite significantly less TNC and CD34+ recovery, suggesting thawing might select for more robust HPCs. More studies with older samples are needed to determine the clinical impact of cryopreservation and if modification of current quality indicators could be useful clinically.

ABO-mismatched marrow processing for transplantation: Comparative results of 80 procedures performed with Cobe Spectra and Spectra Optia

Backgroundremoval of incompatible red blood cells (RBCs) or plasma is usually required to avoid hemolysis during infusion of ABO incompatible bone marrow (BM) allogeneic transplants. This process often involves separation of buffy coat (BC) by centrifugation in automated devices. We have evaluated the Spectra Optia™ (Optia) apheresis system to determine its effectiveness in BC concentration, volume reduction and RBCs depletion of ABO-incompatible BM compared with our previous method using Cobe Spectra™ (Cobe). Materials and methods28 processes were performed with Optia and 52 with Cobe. We compared volume reduction, RBCs depletion, and recovery of total nucleated cells (TNCs), mononuclear cells (MNCs), CD34+ and CD3+ cells in the final product. Hematopoietic engraftment was ascertained. We used Saphiro-Wilks and Kolmorgorov- Smirnov tests to test normality and Mann-Whitney’s U test to compare means between both groups. ResultsWe found statistically significant differences favoring Optia versus Cobe in TNCs recovery (62% vs. 37%), CD34+ cell recovery (98 vs 84%), volume reduction (91 vs 84%), and RBCs depletion (99 vs. 97%), but not in processing time or time to engraftment. ConclusionOptia achieves high RBCs and volume depletion of BM, while providing excellent CD34+ recovery in clinical routine. Some parameters compare favorably with Cobe Spectra.

Abbreviated T-Cell Activation on the Automated Clinimacs Prodigy Device Enhances Bispecific CD19/22 Chimeric Antigen Receptor T-Cell Viability and Fold Expansion, Reducing Total Culture Duration

Introduction: As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy expand, cell manufacturing incorporating closed-system, automated instruments are supplanting traditional open-system, labor-intensive culture methods. At our institution and others, the CliniMACS Prodigy (Miltenyi Biotec), a closed-system automated device, has demonstrated success in the production of CAR T-cells from T-cell enrichment, activation, viral transduction, and expansion to downstream harvest for cryopreservation/fresh infusion. However, the duration of T-cell activation/viral transduction, and total T-cell culture duration are variable across centers (2-5 days and 7-13 days) and merit evaluation prior to routine use.Methods: Following the opening of our clinical protocol (Clinicaltrials.gov NCT03448393), CD19/CD22 Bispecific CAR T-cell products were manufactured on the Prodigy for 4 patients (Original Method, OM) but CAR T cell manufacturing was felt to be suboptimal. Consequently, we investigated a modified processing method (Modified Method, MM), for the manufacture of clinical grade products using the Prodigy. Specifically, TransAct CD3/CD28 reagent mediated T-cell activation/stimulation and lentiviral transduction (MSCV-CAR1922-WPRE; Lentigen Inc.) was terminated with a wash step at Day 3 (instead of the wash step at Day 5, as in the OM). Overstimulation of the relatively more sensitive patient cells was proposed as a likely cause of suboptimal cell viability and expansion in the OM runs. Final cell harvest was planned between culture days 7-12. A total of 4 apheresis products were evaluated using this MM and compared with the 4 prior runs using the OM. All products were obtained from live or deceased patients with disease profiles similar to patients on the clinical trial. Other process parameters (enrichment for CD4/CD8 subsets, in-process media changes with GMP-TexMACS Medium supplemented with human IL-2 (200IU/mL) and 3% human AB serum) were kept unchanged across the 2 methods. Transduction by Protein L expression, viability and cell phenotype (CD3, CD4/CD8) were measured by flow cytometry.Results: From ~0.1x109 CD4/8 enriched T-cells placed into the Prodigy culture chamber on day 0, the mean viable Total Nucleated Cells (TNC) obtained in the final product was 1.93x109 ± 0.27x109 in the 4 MM runs. This cell dose was accomplished by culture Day 7. In contrast, in the OM runs, the mean viable TNC obtained in the final products between Days 9 and 12 was 0.8x109 ± 0.7x109 (Figure 1a). Viable CAR transduced CD3+ fold increase was calculated for days 0-7 of the MM cultures and 0-9 and 0-12 of the OM cultures depending on day of harvest and for the 4 MM products the average fold increase was 15.3 ± 4.2 by Day 7 (Figure 1b) which was ~3 fold greater than OM products harvested on day 9 or 12. Viability of transduced cells was >80% throughout MM culture. In contrast, viability was about 31% during manufacturing of one of the OM products (Figure 1c). On the day of harvest, >99% of the cells were CD3+ T-cells for all 4 MM products (Figure 1d) with no remaining CD19+CD22+ cells. The CD4/CD8 ratio was as expected and favored CD4 T-cells over CD8 T-cells (Figure 1e). Transduction efficiency based on Protein L binding was >70% for the MM products and passed clinical release criteria (Figure 1f). A head-to-head comparison of the 2 methods from the same starting fraction in one patient product (Figure 1, 3A, 3B) also confirmed all findings above.Conclusions: Our data demonstrate that the modified CD19/CD22 Bispecific CAR T-cell manufacturing method (MM) which terminated T-cell activation/transduction by culture Day 3, resulted in reproducible and robust CAR T cell production, even in the relatively more sensitive patient cells. Viability, Viable TNC recovery, CD3% and Protein L expression were consistently higher with the MM compared to the OM. All final products in the MM met product release criteria. In addition, final product dose requirements were consistently met by culture Day 7 when using the MM, augmenting process efficiency. Consequently, we have adopted the MM for the manufacture of clinical CD19/CD22 Bispecific CAR T cells. However, to determine if this change effects CAR T cell potency, studies have been initiated to compare differences in T-cell subsets, activation/exhaustion/senescence and differentiation markers, and the metabolic activity of cells manufactured by the 2 methods. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Validating and implementing the use of an infusion pump for the administration of thawed hematopoietic progenitor cells-a single-institution experience.

Direct thaw and administration of previously cryopreserved peripheral blood stem cell products is a commonly used practice and should be performed rapidly to reduce cellular damage caused by dimethyl sulfoxide exposure. Cells are typically thawed at the bedside and infused by gravity through a high-flow-rate central venous catheter. An existing nontunneled catheter is occasionally used instead and often results in a slower infusion rate. To ensure expedient and consistent infusions, we validated and implemented the use of an infusion pump for thawed peripheral blood stem cells. Validation was performed in two phases: in vitro simulation and in vivo clinical assessment. Total nucleated cell recovery and viability plus progenitor cell viability and potency were compared in vitro between two cryopreserved peripheral blood stem cell units that were either passed through a preset infusion pump or drained by gravity. The infusion rate, adverse events, and engraftment times were retrospectively compared between patients who received infusions by infusion pump (n = 35) and by gravity (n = 38). No significant differences were observed in vitro between the infusion methods for all measured variables. Overall infusion rates were similar in vivo for both groups but were significantly lower for patients who had nontunneled catheters that delivered the infusion by gravity. The time to neutrophil and platelet engraftment was similar for both groups. This is the first study to assess the use of an infusion pump for stem cell transplant. The use of an infusion pump for peripheral blood stem cell infusion is safe, provides a reliable and consistent infusion method, and can mitigate the effect of the type of venous access line used.

Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method

Background aimsUmbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. MethodsIn this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. ResultsUsing Sepax, we observed higher recovery of total nucleated cells (TNC), CD34+ cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34+ viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 – 175 minutes versus manual method: 180 – 290 minutes). The use of DNAse and MgCl2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DiscussionWe optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system.

Cord Blood (CB) Stability and Potency Evaluation: Consistent, Predictable Recovery of Hematopoietic Progenitor Cells (HPC) and High CD34+ Cell Viability in Stored Cord Blood Units (CBU) of the National Cord Blood Program (NCBP)

Cryopreserved HPC, CB products are maintained in long-term storage for future clinical use. To evaluate the impact of cryopreservation and storage over time on the quality and potency of the CBU as well as fulfill the FDA requirements for licensed products, stability studies are performed annually. These in vitro studies compare CBU stored for several years to recently processed ones with respect to HPCrecovery, potency, bacteriology, identity and integrity. This analysis summarizes the 2010-2015 annual stability evaluations including45 clinical CBU randomly selected among those processed by NCBP during the period 2006-2015 and stored inBioArchivefreezers.Pre-cryopreservation results, i.e., tests performed prior to freezing after CBU processing with the AXP system, are compared to post-thaw values of the CBU bag, and those of the CBU segment. We also present the quality assessment of segments (N=1924) of CBU processed during the same period that were released for transplantation.Methods:CBU bags were thawed and underwent albumin dextran reconstitution (dilution 1:7).Total nucleated cell (TNC) counts were measured in aSysmexXE2100 analyzer. CD45+/CD34+ counts and viability were evaluated using single platform, 3-color flow cytometry with 7-AAD, and ISHAGE gating strategy.CFU assays were evaluated using the NCBP CFU strategy (Albano et al, ASH 2008).The same assays were used for segment evaluation. Recovery was expressed as the ratio of post-thaw to pre-cryopreservation values. The segment "yield" was calculated as the ratio of segment results to the pre-cryopreservation or post-thaw bag values.Results: All CBU met acceptance criteria for identity, sterility and container integrity.Post-thaw CBU bag TNC recovery averaged 100.5% (SD: 6.2%). The range was 87%-114.5%; only one sample had recovery below 90%. TNC was not measured routinely in the segments.Average post-thaw bag CD34+ viability was 93.1% (SD: 3.2%). While the lowest value was 83.5%, 41/45 samples had viability above 90%. Segment post-thaw mean CD34+ viability was 94.7% (SD: 3.8%); 42 samples had values above 90%. The difference between segment and bag averaged 1.6% (SD: 2.5%; range: -3.1% to 7.5%). This change was statistically significant but too small to impact product quality.CD34+ and CFU recoveries (mean and SD) are shown in the Table. Post-thaw recovery of viable (v) CD34+ cells from theCBUbag ranged 60-133%, while the segment yield was 48-106%. For the 30 CBU with pre-cryopreservation CFU results, mean CBU bag recovery was 71% (range: 34%-120%) and segment CFU yield was 75% (range: 23%-125%). Segment and bag CD34+ and CFU results correlated well (p<0.01).In agreement with the stability studies, CD34+ viability evaluation of 1924 CBU segments showed average of 95.7% (SD: 3.5%; only 5% of the samples had CD34+ viability below 90%). These segments were evaluated prior to CBU release for transplant; median time in the freezer was 2.3 years (range: 0.1-9.5), and they represent 4% of the total AXP-processed CBU in the NCBP inventory.The segment yield of vCD34+ cells was 79% (SD: 25%). Further, a strong correlation was seen between pre-cryopreservation and segment vCD34+ counts (r: 0.87; p<0.01). CFU values of the segment and pre-cryopreservation also showed good correlation (r: 0.72; p<0.01) and the average CFU segment yield was 71% (SD: 24%).CBU processed with manual method (period 1993-2006) are also included in annual stability studies and have met acceptance criteria (data not shown). Further, analysis of 684 segments of manually processed CBU stored for a median of 10 years (range: 6.3-21) showed mean CD34+ viability 94.2% (SD: 4.3%; 11% with viability below 90%); results that compare favorably to those of recently processed CBU.In conclusion, systematic evaluation of NCBP CBU processed in different periods demonstrates that quality/potency can be maintained with storage over many years. The stability studies for the AXP-CBU (2006-to date) and the pre-release segment evaluation show high CD34+ viability and consistently high recovery of HPC, indicating that the process is under control, and set the standard for future studies and other potency assays.The strong correlations between post-thaw bag and segment results demonstrate that the segment is a representative sample of the cryopreserved CBU and its evaluation can predict reliably the potency of the thawed product. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Direct immunomagnetic depletion of RBC without hypotonic lysis or sedimentation.

Abstract Isolation of specific cell types for functional studies frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of samples may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride (NH4Cl) is often ineffective, leaving samples that still contain many RBC. We have developed an immunomagnetic method to deplete RBC that is rapid, can be used with a variety of sample types, and leaves untouched total nucleated cells (TNC) ready for downstream assays. RBCs were removed from four different types of samples (whole blood, leukopak, buffy coat and bone marrow) either magnetically using the EasySep™ RBC Depletion Reagent or by NH4Cl lysis. To perform magnetic RBC depletion, samples were diluted 1:1, the reagent added, and the sample placed in an EasySep magnet. Magnetically labeled RBC were held in the magnet and TNC were poured or pipetted off. The reagent addition, magnet incubation and removal steps were repeated a second time, and in some cases the magnet incubation and removal steps were repeated a third time with whole blood and buffy coat samples. RBC contamination of the TNC fraction was &amp;lt;2% for whole blood (n=8), leukopak (n=2), and bone marrow (n=5) samples treated with EasySep™, and 12 ± 10% for buffy coat (n=4) samples treated with EasySep™; in all cases this was equivalent to or significantly lower than the RBC contamination in the same samples treated with NH4Cl. TNC recovery from samples RBC-depleted by either EasySep™ or NH4Cl lysis was similar. RBCs can be immunomagnetically depleted from samples in 6–15 minutes, depending on the sample type, and TNC are immediately ready for downstream assays.

Cord blood collection and processing with hydroxyethyl starch or non–hydroxyethyl starch

BackgroundCollection and processing characteristics influencing quality of cord blood (CB) units play an essential role to cord blood banks (CBBs). At many CBBs, volume reduction is performed using hydroxyethyl starch (HES) and the Sepax (Biosafe) automated cell processing system. Due to the withdrawal of HES from the European market, a validation of the nonHES protocol was performed. MethodsThis partially retrospective study identified CB characteristics such as gestational age and CB volume/cell count correlated with higher quality. For the nonHES validation, CB was analyzed for total nucleated cell (TNC), mononuclear cell (MNC) recovery, hematocrit (HCT) and colony-forming units (CFUs). Viabilities of CD34+ and CD45+ cells were determined by 7-aminoactinomycin D (7-AAD) and AnnexinV (AnnV) staining and compared for 21 mL and 42 mL buffy coat (BC) samples applying the HES/nonHES protocol. ResultsFactors affecting the potency of CB transplants were the gestational age and the volume reduction to a defined BC volume. High initial cell counts and CB volumes correlated negatively with post-processing TNC recovery for lower BC volumes. Post-processing HES and nonHES results were comparable, but nonHES revealed a significantly lower post-thaw recovery of viable CD34+ cells measured by 7-AAD/AnnV (21 mL: 45.4 ± 16.4%; 42 mL: 67.3 ± 14.5%) as compared with HES (21 mL: 72.7 ± 14.4%, P = 0.0164; 42 mL: 83.4 ± 14.7%, P = 0.0203). DiscussionDue to the lower post-thaw CD34+ cell viability (AnnV−/7-AAD−) for nonHES samples, the use of HES is recommended, ideally combined with a high BC volume. The post-processing HCT has no statistically significant impact on the post-thaw CD34+ cell viability (AnnV−/7-AAD−).

Increasing Post-Processing Total Nucleated Cell (TNC) Recovery in Cord Blood Banking: Hespan Addition in cGMP Environment

Purpose. Evaluate the effects of HESPAN (HES) addition on indices of cord blood unit (CBU) potency, stability and safety after automated volume reduction (reduction of erythrocyte bulk and plasma volume) using the AXP AutoXpressTM (AXP) processing system.Background and Methods. TNC recovery varies significantly and unpredictably after volume reduction during CBU processing. However, prompt engraftment of CBU allotransplants correlates with their TNC including hematopoietic stem/progenitor cells (HPC). As a result, TNC is important in selecting CBU for transplantation: for example, 75% of the National Cord Blood Program (NCBP) CBUs shipped during the period 2010-2013 had TNC >120 x 107. Therefore, minimizing TNC losses during processing improves the chances that a CBU will be useful for clinical transplantation.The first method to increase post-processing TNC recovery was the addition of HES to the centrifugal method for CB volume reduction [Rubinstein et al, PNAS (USA), 92:10119-10122, 1995]. Volume reduction consists of concentrating the buffy-coat (containing leukocytes and HPC) by centrifugal stratification and removal of bulk red cells and platelet-containing plasma. NCBP processed manually over 30,000 CBUs using HES during the period 1995-2006. In 2006, the AXP, a closed, automated and FDA-approved processing method, without HES, was implemented. AXP was designed to enhance the MNC and CD34+ cell recoveries but not those of granulocytes. This resulted in lower post- processing TNC counts. Additionally, CBUs with larger volume and TNC content have somewhat higher TNC losses during automated processing. Using the AXP method, NCBP’s HEMACORD® obtained FDA License approval on November 10, 2011. However, since TNC (not MNC or CD34+ count) remains the most commonly used indicator of CBU potency and engraftment ability, we describe here implementation of HES in AXP processing to augment TNC recovery, by adding HES to the CBU to a 1-2% final concentration.Results.The results of manual CBU processing showed that HES addition improves yield, without changes in cell viability after cryopreservation, freezing at -196°C and thawing, after 20 years (from NCBP continuing stability study). HES addition also preserved CFU numbers and CD34+ cell counts after thawing.1. Comparison of TNC recoveries without and with HES addition in the same CBU: A total of 25 CBUs were initially processed with the AXP platform without HES, as per routine procedure, and the TNC recoveries were calculated. Each CBU was then reconstituted after its initial processing into a new AXP bag set. HES was added aseptically to the reconstituted product and each CBU was processed in the same conditions as first time (same AXP device, centrifuge, etc.). The TNC recoveries after the second volume reduction process (AXP with HES) were ~20% higher on average than after the first (AXP without HES).2. Comparison of TNC recoveries in different cohorts of CBUs: Thirty clinical-grade CBUs, with volumes 80-156 mL and TNC counts 111-290 x 107, were processed with HES in the AXP system and the results were compared with those of AXP-processed CBUs without HES over an earlier six month period. TNC and CD45+ cell recoveries improved by 16 - 20% maintaining mean post-processing hematocrit at 30.6% (SD ± 2). CD34+ and CD45+ cell viabilities were unchanged: 99% (SD ± 0.7) and 96% (SD ± 2.7), respectively, while their mean recoveries were 95% (SD ± 18) and 94% (SD ± 6). In addition, the same consistent post-processing volume was obtained (mean 20.8 mL, SD ± 0.1). CFU - CD34+ correlation (R2) after processing with HES was 0.788 (not different than what was observed in the CBUs without HES). Six CBUs AXP-processed with HES, were thawed and tested, with very minor losses in cell count and viability, similar to results of thawed AXP-processed CBU without HES. Finally, HES addition did not result in microbial contamination in any of these AXP-processed CBUs.Conclusion. Adding HES to CBUs before automated AXP processing increased substantially the TNC and CD45+ recoveries without loss of viability, while the CD34+ recoveries remained basically unchanged, with a mean of 95%. The post-processing hematocrit was consistent and low. AXP-automated CB processing with HES addition can be performed in the GMP environment, results in higher post-processing TNC and therefore, increases the CB bank’s ability to store larger CBUs that are most useful to patients. DisclosuresNo relevant conflicts of interest to declare.

Impact of Long-Term Cryopreservation on Single Umbilical Cord Blood Transplantation Outcomes

Umbilical cord blood (UCB) may be collected and cryopreserved for years before use. In vitro and murine models suggest that the duration of storage does not affect UCB progenitor cell performance; however, the impact of UCB age on clinical outcomes has not been definitely defined. This study sought to determine the effect of UCB unit cryopreservation time on hematopoietic potency. We analyzed 288 single UCB units used for transplantation from 1992 to 2013, with unit cryopreservation time ranging from .08 to 11.07 years. UCB unit post-thaw characteristics were examined, including percent recovery of total nucleated cells (TNC). The number of years the UCB unit spent in cryopreservation had no impact on TNC recovery nor UCB unit post-thaw viability. Duration of cryopreservation also had no impact on neutrophil or platelet engraftment in single UCB transplantations. These results show that UCB units can undergo cryopreservation for at least 10 years with no impact on clinical outcomes.