Step by Step Method Development and Analytical Validation of L929 Bioassay for rTNF-α Potency Evaluation in Manufacturing

Abstract

Introduction: The development of a cell based potency assay used for drug lot release is different from developing the same one used for drug discovery. Drug lot release requires the assay to be more robust and QC-friendly. In order to make the assay more stable with high accuracy and precision, not only the scientific design and experimental optimization, but also the assay format, plate uniformity & layout should be taken into consideration. Besides, assay performance should be tracked during the assay’s whole lifecycle to assure the test quality. Up till now, most publications about bioassay focused on developing technically new methods, while less were discussing the details about developing a practically robust bioassay in industry. Methods and Results: In this study, a L929 cell based potency assay of rTNF-α was developed and validated to show an example about the development of potency assay used for QC purpose in drug manufacturing. Scientific design of the assay is common. After carefully assessing plate uniformity, plate layout and other key parameters, the assay was well developed with 7-points 4-P curve format instead of the traditional 11-points 4-P curve format. The assay validation results indicated that it had good accuracy with recovery of 95%-105%, good fit (R2>0.98), low RSD (even<10%) of replicates and broad detection range of 50%-150%. Furthermore, the assay performance was also monitored by tracking and recording the internal control’s 4-P curve parameters using the control charts. Conclusions: This study represents a good example on how to develop a robust cell based bioassay in the industry of biotechnology. The well developed and validated L929 bioassay has not been reported before, and could serve as a reliable potency assay for rTNF-α lot release in industry. Keywords: Biological activity, potency, TNF-α, L929, bioassay.