»


MYCOPLASMA


» Table 1. Validation Study


Validation Parameter


Specifi city Detection Limit


Robustness Repeatability


Ruggedness Equivalence *values from development Species


Table 2. Mycoplasma Solution GC/CFU Ratio Strain ATCC


Number


Acholeplasma laidlawii Mycoplasma arginini Mycoplasma fermentans Mycoplasma hyorhinis Mycoplasma orale


Mycoplasma pneumoniae PG8


G230 PG18 GDL


ATCC 23206 841 ATCC 23838 784 ATCC 19989 1022 ATCC 23839 778


CH19299 ATCC 23714 704 PI1428


ATCC 29085 763 Samples Un-Spiked Mycoplasma DNA Mycoplasma DNA Mycoplasma ≤10 CFU/mL Acceptance Criteria No Mycoplasma Detection


Mycoplasma Detection in Spiked Samples


Mycoplasma Detection in Spiked Samples


Mycoplasma Detection in Spiked Samples


Development Data from Test Manufacturer Un-Spiked


Mycoplasma DNA Analyst to Analyst


Instrument to Instrument Reagent Lot to Reagent Lot Laboratory to Laboratory Mycoplasma ≤10 CFU/mL


Un-Spiked Clinical Trial Samples


All Replicates Negative All Replicates Positive Δ(Average Ct)<3* Δ(Average Ct)<2* Δ(Average Ct)<3* Δ(Average Ct)<4*


NAT Positive ≥ PTC Positive NAT Results = PTC Results


which to perform this portion of the study in their non-cGMP training facility, three-thousand miles away. Considerable challenges related to equipment, assay materials, Mycoplasma reference standard solutions, and training existed.


First, the equipment in the facility was not part of a cGMP metrology program and subject to routine calibration. The equipment used in this validation fell into the following categories: Real-Time PCR Instrument, centrifuges, heat blocks, pipettors, and timers. The Real- Time PCR Instrument was the most critical piece of equipment used in the validation. Executing an Installation Qualifi cation Protocol and Operation Qualifi cation and Instrument Performance Verifi cation Protocol during the week prior to performance of the validation experiments mitigated the risk associated with using the Real-Time PCR Instrument because the protocols confi rmed that the instrument was operating within its tolerance range during the validation period. For the remaining equipment, assessing the risks of false positive and false negative results from the use of out-of-tolerance equipment found that assay controls in place mitigated these risks.


Average Titer (CFU/mL) GC/CFU


18


0.5 3


0.1 7 4


Mycoplasma genome size of 1,000 kbp. Table 2 contains the average titer and GC/CFU ratio for each species used in the validation study.


The low GC/CFU ratios provided additional supporting data indicating that the enumerated organism stock solutions obtained from the contract testing laboratory and used for spiking were of high viability (not biased toward PCR) and were appropriate for use as calibrated reference standards to compare the performance of the Real-Time PCR test with the performance of the offi cial method. Although this approach yielded favorable results in this case, use of well- characterized reference standards is preferable when designing and executing validation studies.


Logistics


Introducing viable Mycoplasma cultures into an autologous cell therapy manufacturing facility producing thousands of lots per year presented an unacceptable contamination risk. Most cell culture facilities choose to eliminate any risk from test lab personnel contaminating production cultures by banning viable Mycoplasma cultures in the entire facility, including the test lab. This presented signifi cant logistical challenges for validation. Performing the portion of the validation protocol requiring use of viable organism reference standards off -site was the most straightforward solution. Working closely with the test manufacturer identifi ed a one-week window in


100 | | September/October 2013 - 15TH ANNIVERSARY ISSUE


Second, interaction of computerized purchasing systems initiated shipments for all of the assay materials from the test manufacturer to the development laboratory, only to ship back to the test manufacturer training facility upon receipt. The reference standards were viable Mycoplasma organism solutions requiring storage at -80°C. Obtaining these solutions at the training facility without compromising the cold chain required coordinating the interactions of the contract manufacturer and test manufacturer as a third party to ensure appropriate storage at all stages (initial storage at contract manufacturer, shipping on dry ice, timely receiving, and storage at the training facility).


Finally, training for the analyst performing the validation experiments occurred in the development laboratory using familiar equipment. Executing the validation study in a non-cGMP training facility using unfamiliar equipment turned out to be a signifi cant challenge. Adding a time constraint to experiments comfortably completed in a longer period only added to the stress. As it happened, the analyst successfully completed the experiments in the time available by executing multiple experiments simultaneously.


Again, while the approach yielded favorable results, the ideal situation would have been to execute the entire validation in the development laboratory. When this is not possible, using an off -site cGMP lab with adequate time for training, material qualifi cation, and protocol execution is preferable.


Detection Criteria


Original detection criteria for positive samples subjected all samples exhibiting an amplicon having a Tm in the correct range for Mycoplasma to additional testing in a conservative attempt to minimize the number of false negative results. Analysis of spiked media samples and samples from reference chondrocyte cultures confi rmed this as being a feasible approach to take into validation. During validation for equivalence, however, analysis of 78 retain samples from a clinical trial of an autologous cell therapy revealed baseline noise in the melting curve having a Tm in the correct range for Mycoplasma. Testing using the conventional growth-based method had previously found these samples to be negative for Mycoplasma.


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