As shown in Figures 6a and 6b, although the total processing time was increased as a result of reducing the filtrate flow
rate, these modifications improved the filtrate turbidity profile of the third engineering run. The overall step yield and
product-quality results of the third engineering run were comparable to those of the typical runs from the sending site (data
not shown). Therefore, the performance of the TFF system at the receiving site using the modified process parameters was deemed
acceptable and the risk from the TFF equipment gap was successfully mitigated.
Figure 6b: Filtrate turbidity profiles of the engineering runs. Gray and back lines are typical profiles from the sending
Gap example # 4: viral clearance strategy
One risk that was identified early on during the transfer was not caused by facility fit constraints, but by the gap between
the licensed process and the current industry standards and regulatory expectations around viral clearance. This risk was
classified as a high risk. To mitigate this risk, two process improvements were made: improving the robustness of the acid-treatment
step for viral inactivation, and changing the virus-removal filter to improve the process capability for removing adventitious
The effectiveness of viral inactivation via acid treatment depends on temperature, pH, and duration of the treatment. Although
pH 3.8 inactivates model retroviruses, subsequent studies have found that the performance of viral inactivation is more robust
at pH 3.6 and below (17, 18). An exploratory study was performed at small scale to evaluate the effects of lower pH and longer
durations for the acid-treatment step. As expected, an inverse correlation was found between pH and the glycoprotein aggregate
level in the pH neutralized pool. It was observed that lower pH resulted in higher aggregate levels, as shown in Figure 7.
However, the subsequent chromatography steps were able to reduce the percent aggregate level in the purified pool well below
the limit for the bulk-product release specification. In a follow-up study, several product-quality attributes including the
aggregate level were analyzed to validate the acceptable pH range and duration for the acid-treatment step. Product-quality
analysis for the engineering runs also demonstrated that the full-scale performance of this step was acceptable.
Figure 7: Effect of the target pH for the acid-treatment step on aggregate levels in the pH-neutralized pool and purified
pool. The red line represents the limit in the bulk-product release specification.
In the past decade, more virus removal filters have become commercially available (19–21). The process transfer presented
an opportunity to change the virus removal filter. A small virus-retentive filter was selected to replace the current virus-removal
filter. This selection was based on considerations for the existing process capability for viral clearance, characteristics
of the product pools (e.g., protein concentration), and current industry standards. Sizing of the new small virus-retentive
filter was performed at small-scale. The performance of the viral-filtration step was also evaluated during the engineering
runs to confirm appropriate sizing of this filter and acceptable process performance at full-scale.
Overall, the potential gap in the viral-clearance strategy of the process was closed by improving process robustness and leveraging
current process technologies.