Considerations in Identification and Qualification of Spectrophotometer Equipment for Microbial Fermentations - The authors outline qualification procedures for a process-critical piece of equipment

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Considerations in Identification and Qualification of Spectrophotometer Equipment for Microbial Fermentations
The authors outline qualification procedures for a process-critical piece of equipment.


BioPharm International
Volume 24, Issue 5, pp. 27-35

RESULTS AND DISCUSSION

Instrument linear range determination


Figure 3: Regression of measured optical density (OD) versus National Instiutes of Standards and Technology (NST) standard OD used to determine instrument linear range.
Figure 3 shows the regression of measured OD versus NIST Standard OD for each spectrophotometer. Using the reduced regression models, the instrument linear range was set for each spectrophotometer (see Figure 3). The instrument linear range of Models A, B, and C were comparable, whereas the linear range of Model D was reduced.

Accuracy and precision of NIST OD standard measurement


Figure 4: Calibration model of each spectrophotometer to National Institute of Standards and Technology (NIST) OD standards performed over the normalized linear range of 0.2–4.0 dilution OD.
Fermentation samples are more variable than standard OD solutions. Therefore, it was necessary to determine the fermentation process linear range using each spectrophotometer. A calibration model assessed the accuracy and precision of the spectrophotometers to measure NIST OD standards over the common instrument linear range of 0.2–4.0 OD. Figure 4 shows the precision and accuracy of each spectrophotometer relative to the NIST OD standard values. Each spectrophotometer precisely measured the NIST OD standards over the linear range, as represented by small vertical bars. However, the measurement accuracy to the NIST OD standard values varied across the linear range. Model A had the highest accuracy and best matched the value of the NIST OD standard values. Model B and C had slightly less accuracy, and the predicted values were slightly higher than the NIST OD standard values at the high end of the linear range (2.8–4.0 OD). Model D diverges from the value of the NIST OD standard values at 1.2 through the linear range up to 4.0 OD.

Process linear range determination


Figure 5: Process linearity assessment using whole broth samples.
Process linear experiments were performed on samples at the start of feed, start of induction, and final OD. Figure 5 shows a representative example of this analysis. For visual reference, Figure 5 shows the average Model A data within the current historical linear range (0.2–1.6 OD 1 standard deviation). These data show an expanded actual OD linear range (0.2–6.0 dilution OD) for Models A, B, and C. Model D showed a reduced actual OD linear range (0.2–4.0 dilution OD), and an offset of measurement accuracy compared with the other spectrophotometers tested.

Linear range summary

Overall spectrophotometer linear range was set based on the instrument and process linear ranges. Spectrophotometer Models A, B, and C had high precision, accuracy, and similar process linearity from 0.2 up to 6.0 dilution OD. These spectrophotometers also had high instrument linear ranges up to 11.2 OD. As a result, the overall range for these spectrophotometers was conservatively set to 0.2–4.0 dilution OD which was greater than the historical Model A linear range of 0.2–1.6 dilution OD.

Model D had high precision and acceptable process linearity over the range of 0.2–4.0 dilution OD. However, the accuracy of this spectrophotometer to measure NIST standards and whole broth samples was lower. Therefore, the Model D linear range was conservatively set to 0.2–1.2 dilution OD. For consistency, all spectrophotometers continued to be assessed over the expanded linear range of 0.2–4.0 dilution OD.

New spectrophotometer selection


Figure 6: Calibration model of the test spectrophotometers to the Model A dilution OD over the normalized linear range of 0.2–4.0 dilution OD.
Figure 6 shows the precision and accuracy of each test spectrophotometer compared with the Model A spectrophotometer. Model B had the highest precision and accuracy relative to Model A measurements over the 0.2–4.0 dilution OD range. Model C had similar precision to Model B, but had lower accuracy compared to Model A from 1.6–4.0 dilution OD. If the process linear range of 0.2–4.0 dilution OD was used, Model C would need a correction factor as part of the sample measurement, which is not desired. However, operating at a reduced process linear range of 0.2–1.2 dilution OD would be acceptable as it would not require a correction factor. Finally, the precision and accuracy of Model D relative to Model A were poor across the entire dilution OD range tested. The poor performance for both precision and accuracy of this spectrophotometer made it an undesirable option for replacing the Model A spectrophotometer.

The output of the calibration model of the test spectrophotometers compared to the Model A spectrophotometer showed that the Model B spectrophotometer best matched the precision and accuracy over an expanded linear range of 0.2–4.0 dilution OD. Therefore, Model B was selected as the new spectrophotometer to replace the Model A spectrophotometer.

Bench-scale Fermentation Confirmation Runs


Figure 7: Bench-scale production fermentation growth profiles comparing Model A and Model B spectrophotometers.
Bench-scale fermentations were performed to test the ability of the Model B spectrophotometer to mimic the Model A spectrophotometer during a fermentation run. Figure 7 shows that similar growth profiles were achieved when the fermentation was controlled using the Model A and B spectrophotometers. Additionally, the production operating parameters, time-to-feeds, start, and induction, were comparable and indicated equivalent performance.


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