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PCR/Thermal Cycler Calibration

Case Study 103 PX

PyroCAD/Proj.ID: TCC-20050118


Objective:

The objective of this case study is to demonstrate the capabilities of PyroDisk-XT temperature calibration system and the importance of temperature calibration/calibration verification of the PCR/thermal-cyclers.
Major components of the study are:

  • Demonstration of the temperature calibration/verification protocol set up
  • Demonstration of the temperature calibration/verification graphical output
  • Demonstration of the temperature calibration/verification evaluation relative to specification, including:
    • Temperature accuracy
    • Block temperature uniformity
    • Temperature overshoot & undershoot

  • Demonstration of the temperature & time performance evaluation relative to the cycling protocol, including:
    • Temperature reproducibility
    • Temperature repeatability
    • Time accuracy

Benefits of PCR/Thermal-Cycler calibration/verification:

  • 21 CFR Part 11 compliance - the calibrations are performed with a compliant system
  • Product yield and purity can be correlated to temperature accuracy & uniformity
  • Performance Qualification of PCR/thermal-cyclers can be monitored
  • Reliable Preventative Maintenance Scheduling can be defined
  • Improved Compliance Management
  • Calibration scheduling relative to Calibration Needs Analysis
  • OOS, OOT, & CAPA investigations support
  • Highest Return-On-Investment

Definitions

Calibration of the PCR/Thermal-Cycler is the process of temperature measurements according to a pre-defined temperature cycling protocol against a reference standard for the determination of the adjustment factors needed to ensure that the results of temperature measurements by the calibrated PCR/Thermal-cycler are within the required specification.

Calibration verification of PCR/Thermal-Cycler is the process of temperature measurements according to a pre-defined temperature cycling protocol against a reference standard to verify that the temperature output of the calibrated PCR/Thermal-cycler remain within the required specification.

NOTE: The difference between calibration and calibration verification is that during calibration we determine the factors needed for future temperature measurements to be within specification, whereas during calibration verification we simply verify that the factors used in the measurements provide results to be within the required specification.

PyroLogger-XT is a 24-channel high performance Self-powered temperature data-logger. Specification characteristics of PyroLogger-XT:

  • Temperature resolution is ± 0.002 °C
  • Temperature dynamic range is -50 °C to 150 °C
  • Accuracy is better than ± 0.05 °C
  • Reproducibility (between sensors) is better than ± 0.02 °C
  • Precision (within sensor) is better than ± 0.02 °C

PyroDisk-Pro is a 21 CFR Part 11 compliant application program, which administers and integrates the various functions for temperature calibration, temperature validation, and temperature monitoring. PyroLogger-XT and PyroDisk-Pro communicate through the built in RS 232 interface, providing support for Self-powered operation based on user defined time and data acquisition parameters.

NOTE: PyroLogger-XT and PyroDisk-Pro have been designed for ad hoc measurement of temperatures (i.e., the current case study) and for the thermal evaluation of microplate readers/incubators/PCR/thermal-cyclers.

21 CFR Part 11 defines the requirements for Electronic Records and Electronic Signatures. Its purpose is to ensure that systems, which are subject to FDA regulation for authentication, processing, or storage of electronic data, are equivalent to paper-based data sources and evidence systems.

Out of specification (OOS) results includes all suspect results that fall outside the specifications or acceptance criteria established in new drug applications, official compendia, or by the manufacturer.

Out of trend (OOT) results includes all suspect results that fall outside the trend (e.g., control chart) specifications or acceptance criteria established in new drug applications, official compendia, or by the manufacturer.

Corrective & Preventive Action (CAPA) shall mean a set actions to correct nonconforming product and other quality problems and to eliminate the cause of potential nonconforming product and other quality problems. Specifically CAPA actions shall include:

  • Correction: correct nonconforming product and other quality problems
  • Corrective action: prevent recurrence of nonconforming product and other quality problems
  • Preventive action: eliminate the cause of potential nonconforming product and other quality problems

NOTE: 50% of all FDA Warning Letters is due to non-compliance to CAPA requirements.

Materials & equipment:

  • Thermo Hybaid Px2 PCR/Thermal-Cycler , S/N: 200962-1053083
  • PyroLogger-XT 24-channel high performance temperature data-logger ("PL-XT" S/N: 0233098632) having the following specification:
    • Temperature resolution is ° 0.002 °C
    • Temperature dynamic range is -50 °C to 150 °C
    • Accuracy is better than ° 0.05 °C
    • Reproducibility (between sensors) is better than ° 0.02 °C
    • Precision (within sensor) is better than ° 0.02 °C
    • PyroLogger-XT has been calibrated against NIST reference prior to use

  • External adapter, S/N: 0263599722
  • PyroDisk-Pro software
  • 8 x Thermowell Tubes, 0.2 ml, Costar, Cat.No.6531, Lot No. 08402007
  • 8 x stopper for Thermowell Tube
  • Distilled water
  • Computer - HP Laptop

Procedure Summary:

1. Insert the external sensors to the Thermal Cycler wells

2. Close the PCR/Thermal-Cycler lid

3. Program the PCR/Thermal-Cycler


4. Program the PyroLogger-XT (use the same protocol as above for the PCR/Thermal-Cycler)

5. Simultaneously start the PCR/Thermal-Cycler program & switch on PyroLogger-XT

6. When Thermal Cycler program stops, switch off PyroLogger-XT

7. Open the Thermal Cycler lid and remove the external sensors from the PCR/Thermal-Cycler

8. Download the data from PyroLogger-XT into PyroDisk-Pro program

9. Evaluate the data

Results:



Evaluation of the 3rd cycle by PyroDisk-Pro program




Interpretation:

  1. Temperature accuracy, temperature block uniformity, and temperature overshoot during temperature cycling are significantly out of specification during the PCR/Thermal-cycler process.
  2. The above deviations could greatly influence the product yield and purity.




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