A Risk-Based Approach to Transferring a Mature Biopharmaceutical Process - The authors present risk-evaluation and mitigation strategies for transfer of the manufacturing process of a recombinant gly


A Risk-Based Approach to Transferring a Mature Biopharmaceutical Process
The authors present risk-evaluation and mitigation strategies for transfer of the manufacturing process of a recombinant glycoprotein.

BioPharm International
Volume 25, Issue 2, pp. 41-49

Gap example # 2: bioreactor pH

Another gap that was identified during the facility fit analysis was that both the bioreactor inline pH probes and the bench-top pH meters were different between the sending and receiving sites. The inline pH probes measure the culture pH in the bioreactors. The probe signal is used to control the culture pH at a target set point via additions of both base and acid (or CO2 gas for bicarbonate-buffered media). During the set-up of a bioreactor, the glass pH probes are calibrated with two pH standards. They are then inserted into the bioreactor and steam sterilized in place along with the bioreactor, before the cell culture medium is transferred through sterilizing-grade filters into the bioreactor. As the steam-sterilization process affects the glass membranes of the inline pH probes and causes the probe-sensing signal to drift, the pH probes are typically calibrated again after the medium is batched into the bioreactor (11). This calibration is performed by taking a medium sample from the bioreactor, measuring the sample's pH using a bench-top pH meter, and then adjusting the pH probe reading to match that of the bench-top pH meter. Therefore, the differences in both the inline pH probes and the bench-top pH meters between the sending and receiving sites could result in an unintended shift in the culture pH. Culture pH is known to affect cell culture growth performance, cell specific productivity and glycosylation patterns for recombinant proteins produced in CHO cells (3, 12). For this reason, this gap in pH instruments presented a high risk to the success of the process transfer.

To mitigate this risk, a two-pronged approach was taken. First, because the bench-top pH meters were used to calibrate the bioreactor inline pH probes, it was important to understand if there was any offset in the pH readings between the different bench-top pH meters. Second, a study was performed to expand the understanding of the pH effect in the current state of the process. As mentioned, this process was approved more than a decade ago and several process improvements have been made since then. It was therefore important to understand the current state of the process when evaluating potential process changes.

Figure 2: pH offset between the receiving site and sending site bench-top pH meters (pH offset=receiving site pH meter–sending site pH meter). Open diamonds represent cell free media samples and closed circles represent cell culture fluid samples. The dashed line represents the average pH offset of all samples.
A side-by-side comparison of the two different pH meters used at the sending and receiving sites was performed. Both cell free media samples and cell culture fluid (i.e., cell containing) samples were used in the comparison and an offset of about 0.1 pH unit was found (see Figure 2). A larger variability was observed in the pH readings of the cell culture fluid samples compared with the cell free media samples. This was not unexpected because cells continued to metabolize during the period between when the cell culture fluid samples were taken from bioreactors and when the pH readings were obtained from the pH meters, and cell metabolites such as lactic acid can change the sample pH.

Figure 3: Bivariate fit of cell specific productivity (Qp) by pH set point (p=0.04).
In the study to expand the understanding of the effect of pH, four different pH set points were compared (e.g., low-low, low, control, and high). The range of pH set points studied encompassed the control set point of the current process at the receiving site and the offset observed in the pH meter comparison study. A scale-down model was used for the study, the performance of which was previously demonstrated to be comparable to the full-scale process performance (data not shown). Cell culture performance indicators such as total cell mass, final cell viability and final product titer were evaluated for all four conditions. The culture fluid was purified so that product-quality analysis could be also performed. There was no significant effect of culture pH on total cell mass or final cell viability (data not shown). As shown in Figure 3, there was a significant effect of culture pH on cell specific productivity, where a higher pH set point resulted in lower specific productivity (p <0.05). Several product-quality attributes were measured for all tested conditions. The analysis of one glycosylation attribute, an indicator of glycoform distribution of the product protein, is shown in Figure 4 as an example. This glycosylation attribute was not significantly affected by pH, similar to the other product-quality attributes that were evaluated.

Figure 4: Oneway analysis of glycosylation attribute by pH set point. Red lines represent the bulk product release specification.
Based on the results of the pH meter comparison study and the small-scale pH set-point study, it was determined that the bioreactor pH control set-point at the receiving site should be adapted to account for the offset from the pH meters. This correction would ensure that the actual culture pH at the receiving site matched that at the sending site. Furthermore, the small-scale study on different pH set-points may be leveraged to widen the proven acceptable pH set-point range for the production culture operation. In conclusion, the potential risk from the pH instrument gap was mitigated by adapting the pH control set-point.

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