21 CFR Part 11 Compliant Temperature & Calibration System for Microplate Readers

Bela Jancsik, Gabor Klivenyi, Robert Bakos, Anita Istokovics, Gabor Danyi, Laszlo Bernatsky, Andras Fabula, and Zoltan Lukacs
OPULUS Ltd.

Abstract

We have developed an ultra-thin - 4mm thick - wireless data logger for temperature calibration & temperature verification for controlled temperature operations in microplate readers. The device, named PyroDisk, can be utilized in various ways to evaluate temperature accuracy & temperature uniformity of the incubating chamber and of the intra-well and inter-well characteristics of the microplates relative to the incubators' temperatures. The functionality of the device has been extended by the insertion of replaceable & modular calibrated photometric filters for wavelength accuracy or photometric accuracy calibrations. Additional inserts can be used for luminometry or fluorescence calibrations. 

The calibrator system has been integrated to 21 CFR Part 11 software with natural language SQL for SQC & SPC modeling. 

The measured results were evaluated statistically and the temperature gradient was mapped graphically. Various methods were tested for temperature compensation, gradient reduction, and improved heat transfer.

Experiments were conducted for the evaluation of temperature efficacy relative to standard microplates, glass microplates, silver coated microplates, & temperature compensator implants. 

Introduction

Temperature calibration is a core requirement in manufacturing processes, environmental monitoring, and analytical chemistry. In fact, temperature is a critical control point in kinetic measurements. In spite of this, incubating-kinetic microplate photometers, which support photometric calibrations, do not provide meaningful solution for temperature calibration or temperature verification of the readers. Why? Let us explore some of the challenges, that must be "tackled" in the creation of a "meaningful" temperature calibrator:
1. Create a suitable wireless datalogger for real-time & in situ temperature measurements;
2. Integrate multiple - at least 6 - sensors to provide gradient analysis of the measurements;
3. Develop an NIST traceable reference calibration of the calibrator;
4. The system must be enabled for electronic record and electronic signature, and must be compliant to 21 CFR Part 11;
5. Integrate flexible and easy-to-use SQL decision support for PQ via SQC & SPC. 

Materials and Methods

Equipment
- Reference Standard Temperature Calibrator - NIST 934/Serial No. 9213442: Fig. 3
- Secondary Reference Standard - PyroSet Opulus Cat. No. 80-PDE-004-01: Fig. 3
- Microplate reader calibrator - PyroDisk Opulus Cat. No. 80-PDE-003-01: Fig. 1a, 1b, 1c
- Microplate reader - Anthos hTIII: Fig. 6

Material
- Polystyrene-96 well microplate - Corning Costar Cat. No. 3585 (hereinafter 3585), Lot. 08899016: Fig. 7d
- Aluminum block insert for 3585 - PyroStore Opulus Cat. No. 80-PDE-003-02: Fig. 7c
- 3585 (Lot. 25197019) with PyroStore inserted: Fig. 7c
- 3585 (Lot 08899016) with silver coating: Fig. 7b
- Glass-96 well microplate - Cambrex Cat No. 25-302: Fig. 7a
- Calibrator fluid - Distilled water Lot. 022003-01: Fig. 2

Calibration & Evaluation Sequence
1. Calibrate PyroSet against NIST Reference Standard: Fig. 5
2. Calibrate PyroDisk with PyroSet Secondary Reference Standard: Fig. 4
3. Calibrate microplate reader with PyroDisk: Fig. 6
4. Execute the calibrations with PyroDisk 21 CFR Part 11 software: Fig. 8
5. Evaluate the calibration data and PQ with EQUAL decision support subsystem: Fig. 9

Experimental Design (Fig. Design Qualification)

Objectives
1. Evaluate the design and qualify the system.
2. Evaluate the incubator's suitability relative to temperature accuracy (true temperature - displayed temperature) & temperature gradient (as determined by inter-sensor reproducibility.
3. Evaluate the effect of mixing (via shaking) on temperature measurement within the filled wells.
4. Evaluate the heat transfer efficiency and temperature distribution homogeneity relative to various materials, as measured by accuracy and reproducibility.
5. Propose best practices, generically and specifically, for qualification, calibration, and method development of microplate readers, relative to temperature.

In the present work the following parameters of the equipment were determined:

Heat transfer efficiency: The parameter was characterized by the average of the temperature values measured by the sensors at the end of the 80 minute runs.

Accuracy: Temperature accuracy of the microplate photometer used in our experiment is defined by the difference of the temperature determined by the PyroDisk (taken as the true value) and the temperature readout (temperature set) of the the microplate photometer.

Variability: the maximum of the difference between any two temperature values measured by different sensors at the same time.

For the determination of the two latter parameters, the last 5 minutes of the 80-minute measurement runs were evaluated.

Design of Experiments

Results

1. From Fig. 9a can be concluded, that takes almost 80 minutes to attain the steady state temperature.
2. The Al-inserted microplate has the best variability however its deviation from the set temperature is greater than that of 3585. (Fig. 9b, 9c)

Conclusion

From the existing experiments we have been able to conclude the following:

- DQ specifications are necessary and sufficient,
- The requirement for temperature calibration & temperature verification has been proven,
- Additional studies are needed to establish best practices.