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

EXPERIMENTAL APPROACH


Figure 1: Spectrophotometer screening, selection, and qualification experiments. (ALL FIGURES COURTESY OF THE AUTHORS)
Figure 1 shows the multiphase experimental strategy used to screen, select, and qualify the new spectrophotometer. The first set of experiments determined the linear range of each spectrophotometer. Linear range testing was performed in three parts, including: instrument linear range determination, spectrophotometer accuracy and precision testing, and process linear range determination.

The instrument linear range was determined by measuring NIST standard solutions over the normalized range of 0.2–12.0 OD, regressing the measured OD to the NIST OD standard values, and reducing the range until the statistical lack-of-fit test against a quadratic model had a p–value >0.05. A calibration model (see below) was then performed using inputs from the instrument linear range testing to assess the accuracy and precision of each spectrophotometer in the measurement of NIST OD standards over the instrument linear range.

Process linear range was determined by measuring a variety of dilutions of fermentation culture broth samples taken from multiple time points across the fermentation. The process linear range for each sample point was estimated by visual inspection of calculated actual OD (dilution factor times dilution OD) linearity when plotted against the dilution OD range. The output of these experiments led to setting an overall linear range for each spectrophotometer acceptable for use in microbial fermentation processes.

In the second set of experiments, the accuracy and precision of spectrophotometer models B, C, and D were assessed in comparison to those of Model A using a calibration model. The inputs of the calibration model included culture broth dilution OD sample measurements from each spectrophotometer from multiple time points across fermentations. The output of the accuracy and precision testing was the basis for selecting the replacement spectrophotometer.

Following spectrophotometer selection, bench-scale fermentations were conducted to confirm that process decisions based on the replacement spectrophotometer resulted in similar process performance as did the Model A spectrophotometer.

Calibration model analysis


Figure 2: Graphical representation of the calibration model.
The accuracy and precision of the spectrophotometers relative to either the NIST OD standard or to the Model A OD were assessed using a calibration model (1). This analysis was performed by regressing the measured value of a sample from a given spectrophotometer (Y) against the true standard value (either the NIST OD standard value or Model A value, X) (see Figure 2). From this model, both the accuracy (bias) and the precision can be estimated for each spectrophotometer using a 95% confidence interval.


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