» MYCOPLASMA
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The baseline noise occurred sporadically in one of the duplicate test wells, resulting in false positive results in one well and negative results in the other well. This conflicting data interpretation resulted in an invalid test rate of 29%, which was too high to be useful for lot release of autologous cell therapy products, requiring final results on the same day as product assembly.
A statistical analysis predicting an equal percentage of false positive results and false negative results provided a rational Ct value to include in the results interpretation criteria for determining positive results. The analysis independently confirmed the Ct cutoff recommended by the test manufacturer in updated product documentation. Using the new criteria reduced the invalid test rate from 29% to 5%, which was reasonable for routine testing. The revised interpretation criteria required an assessment of the potential impact to the validation results. After reanalyzing all of the validation results, the report determined that using the revised interpretation criteria did not impact the results of the validation.
Although this situation was not ideal, it provided invaluable insight into the capabilities of the assay prior to implementation. Though not always feasible, these results recommend introducing as much sample variability into the assay development process as possible, especially for individualized treatments such as autologous cell therapy products.
Lessons Learned – Technology Transfer
Technology Transfer of the assay from the development laboratory in the United States (sending unit) to the production facility in Europe (receiving unit) occurred during the regulatory approval process after successful validation.
Technology Transfer and ongoing training faced similar challenges to validation logistics due to banning viable Mycoplasma cultures in the entire production facility, including the test lab. As a result, Technology Transfer and training needed to find a substitute to demonstrate the ability of the analysts to recover organisms without using viable Mycoplasma. Demonstrating proficiency in the full assay procedure was not possible using Discriminatory Positive/Extraction Control (DNA plasmid) spikes alone because the first step in the sample preparation involves centrifugation of an aqueous cell culture medium sample. Since DNA is soluble in water, it will primarily reside in the supernatant after centrifugation and will go undetected by the assay. Fortunately, the test manufacturer uses Escherichia coli (E. coli) to express the Discriminatory Positive/Extraction Control plasmid and they will provide this E. coli strain to their customers. The assay will not detect E. coli DNA, but will detect the Mycoplasma DNA sequence in the plasmid. Using this E. coli, which centrifuges similarly to Mycoplasma, it was possible to develop a working E. coli suspension for spiking at approximately 10 CFU/mL to mimic the assay detection limit and verify each analyst’s performance at that level.
After solving the technical issues surrounding demonstration of proficiency, there were also some positive lessons from the Technology Transfer exercise. First, it was critical to eliminate conflicting priorities at both the sending and receiving units prior to initiating the study. Second, performing intensive analyst training at the sending unit in the US, away from the daily routine at the receiving unit in the EU,
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reduced the amount of time required for training and allowed focused time to develop analyst expertise.
Conclusion
Rapid detection of Mycoplasma is essential for autologous cell therapy products with short shelf lives. Although product contamination is infrequent, the ability to detect potential contaminants prior to implantation is a critical component of product safety to ensure patient protection. This is likely one reason that final product testing for Mycoplasma is a regulatory requirement for cell therapy products. Developing, validating, and transferring a rapid Mycoplasma test based on Real-Time PCR presented many unique challenges. Validation challenges were either logistical, related to banning viable Mycoplasma cultures in the production facility, or technical, related to appropriate reference standards and detection criteria. Technology Transfer challenges related to demonstrating proficiency given the constraints on use of viable cultures. Proactively addressing each challenge was critical to successful validation, technology transfer, and implementation as a routine lot release test.
Author Biography
John Duguid is a Principal Process/Analytical Scientist at Genzyme, a sanofi company, responsible for developing and implementing rapid microbiological assays. Previously, he managed QC cell therapy operations. Mr. Duguid received his BS in Chemistry from the University of Michigan. Prior to Genzyme, he worked as an analytical
chemist at Abbott Laboratories and Arthur D. Little.
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