(P-BT-27) Qualification of a Cytomegalovirus qPCR Assay for Safety Testing of Human Cord Blood

Background/Case Studies: According to the Centers for Disease Control and Prevention (CDC), over half of the adults in the United States have been infected with Cytomegalovirus (CMV) by the age of 40. CMV testing of starting materials used to manufacture biotherapies is performed to ensure the safety of the products. Cord blood, rich in stem cells, plays a significant role in the pharmaceutical industry, primarily through its use in stem cell transplantation for various blood disorders and as a source material to produce cell therapies. This study evaluated the suitability of a CMV qPCR assay on CPD plasma from cord blood by determining key analytical parameters such as sensitivity, inter- and intra-assay precision, accuracy, stability, specificity, and robustness.   

Study

Design/Methods: The RealStar™ CMV kit protocol was utilized for all experimental runs. Sensitivity or Limit of Detection (LOD) was determined by probit analysis using data from five CMV concentrations, 24 replicates at each concentration, and performed over 3 days. Intra-assay precision was evaluated by testing six replicates of two known concentrations of CMV within a single independent run. Inter-assay precision was evaluated by testing a known concentration of CMV over ten days by two independent operators, for 20 replicates. Accuracy was evaluated by testing two known concentrations of CMV in duplicate across 5 independent runs utilizing two independent operators. To evaluate sample stability, samples containing 3 x LOD of CMV were prepared with one, two and three freeze/thaw cycles. Specificity was evaluated by using Epstein–Barr virus (EBV) and CMV spiked samples. Robustness was evaluated through sample stability by one to three freeze/thaw cycles of purified DNA, purified DNA stored at 2-8°C for 24 and 48 hours. Moreover, robustness was examined through critical steps in the assay such as the timing, combination of lysis buffer and Proteinase K, and the timing combination of the PCR polymerase with DNA.

Results/Findings: The LOD of the CMV assay in CPD cord blood plasma was determined to be 232 IU/mL. Samples tested for inter- and intra-assay precision and accuracy resulted in 100% positive correlation to expected results. EBV showed no interference on the CMV testing. The maximum freeze/thaw of purified DNA was determined to be 3 cycles. The maximum 2-8°C storage time was shown to be 48 hours. The maximum combination time for lysis buffer with Proteinase K was 51 minutes and the maximum combination time for PCR enzyme with DNA was shown to be 30 minutes.

Conclusions: The comprehensive qualification data generated in this study demonstrated that the CMV qPCR is both reliable and accurate for the testing of CPD cord blood plasma. This data provides confidence that the results generated by this assay are consistent, reproducible, and can be used to determine the safety of cord blood.