(M0930-02-11) Investigation of Abnormal Trend for Reduced CE-SDS and Continuous Method Performance Monitoring in Quality Control Testing of Monoclonal Antibody

Purpose: The purpose of this study was to investigate the abnormal trend for reduced CE-SDS assay that HC% and LC% are fluctuating in opposite directions overtime during quality control testing, and identify the potential root cause by analyzing historical data and evaluating multiple contributing factors. Method lifecycle management was focused for improving assay consistency and robustness, and ensuring reliable test results for product release.

Methods: A retrospective analysis of historical data was conducted to assess the abnormal trend in HC% and LC% results. The investigation included a thorough evaluation of potential contributing factors, including method execution, analyst, equipment, cartridge usage, and critical reagent. Aperture used in different CE-SDS cartridges was examined. Visual inspections of the apertures were performed to detect any observable deterioration. A controlled experiment was then conducted to test the hypothesis that aperture swapping and re-use among different cartridges was a possible root cause of the abnormal trends. Continuous method performance monitoring was conducted after corrective action was implemented.

Results: Preliminary assessment showed abnormal trend for reduced CE-SDS that HC% and LC% are fluctuating in opposite directions overtime. LC% showed an upward trend, while HC% showed a downward trend approaching the lower specification limit (Figure 1). Reference standard HC% and LC% showed a similar trend. The root cause analysis (RCA) revealed that the aperture used in CE-SDS cartridges exhibited variability, which was visually observable during inspections. It was found that the practice of swapping and reusing apertures among different cartridges at the testing lab contributed to the abnormal trends in the assay results (Figure 2). The controlled experiment confirmed that the aperture variability directly influenced the assay outcomes. HC% with the old aperture was 65% using capillary A and capillary B, while HC% with the new aperture was 68% using capillary A and capillary B. The differences between apertures on LC% results were statistically significant (3% for both capillary A and capillary B). Furthermore, continuous monitoring of method performance highlighted the importance of managing the number of injections and cartridge usage period to minimize the risk of abnormal results. Corrective action was implemented by ensuring no aperture replacement during the release testing. Results showed that no trend was observed for the same capillary without aperture replacement after 1 year usage and after 250 injections.

Conclusion: Our investigation confirmed that the aperture variability and the practice of reusing apertures among different cartridges were the primary contributing factors to abnormal trend in heavy chain % and light chain % results in the reduced CE-SDS assay. The study highlighted the importance of standardizing cartridge usage and aperture handling in order to improve method consistency and reduce the risk of assay failure. Continuous performance monitoring has shown to be an effective approach in identifying appropriate cartridge and aperture usage period and ensuring reliable assay results during quality control testing.

References: 1. Sänger-van de Griend, C. E. Electrophoresis 2019, 40, 2361− 2374
2. Beck, A., D’Atri, V., Ehkirch, A., Fekete, S., Hernandez-Alba, O., Gahoual, R., Cianférani, S. Expert Review of Proteomics 2019, 16(4), 337–362.

Acknowledgements: The authors declare no competing interests.


Figure 1. Abnormal trend for reduced CE-SDS that HC% and LC% are fluctuating in opposite directions overtime during quality control testing.


Figure 2. Investigation and root cause analysis at the testing lab. HC % CPA decreased overtime with the same CE-SDS cartridge.


Figure 3. Continuous method performance monitoring. A). Total corrective peak area (TCPA) monitored before and after corrective action was implemented. B). Average TCPA versus days since the initial use monitored after corrective action was implemented. C). Average TCPA versus cumulative injections monitored after corrective action was implemented.