Researchers recently developed a harmonized, validated way to use imaged capillary isoelectric focusing (icIEF) to evaluate the charge heterogeneity of monoclonal antibodies (mAbs). The new method is based on guidelines from the European Pharmacopoeia and harmonized with those of the ICH Q2(R2)—the recent revision to the International Council for Harmonizations of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines regarding the validation of analytical procedures.
Applications include routine quality control assessments during manufacturing and throughout a mAb’s life cycle, as well as to help identify counterfeit antibody drugs when reference standards are unavailable. Importantly, the procedure has the potential to be used for multiple antibody drugs within a particular pH range, according to Alessandro Ascione, PhD, research scientist, National Center for the Control and Evaluation of Medicines, Instituto Superiore di Sanita, senior author of the recent paper, and colleagues.
Isoelectric point is key
Basically, they explain, this optimized protocol treats the isoelectric point as a key identity parameter for mAbs and enhances the accuracy and precision of outcomes.
“This method is based upon a new assortment and ratio of carrier ampholytes to achieve an improved linearity across the mAb-relevant pH gradient of available calibration software integrated in icIEF instruments,” Ascione and colleagues note.
Specifically, the carrier ampholyte range was narrowed to five to eight, replacing the European Pharmacopoeia’s generic, broad pharmalyte range of three to ten. This overlaps the narrow pharmalyte range of 8 to 10.5 and allows the full capillary tube to be used to separate antibody variants while focusing the gradient on the range most relevant to mAbs. The concentration of sacrificial agents was also adjusted to enhance pH stability at the capillary tips.
Further optimization involved narrowing the isoelectric point makers flanking the region of analysis to 6.14 and 10.17, compared to the Pharmacopoeia’s 5.85 and 10.17. The ratio of anodic and cathodic spacers changed to 1.67:1, from the Pharmacopoeia’s 1:1, to extend the separation space and stabilize pH. The separation settings were extended to 11.5 minutes, up from the Pharmacopoeia’s eight minutes, to “optimize focusing the charge species across the entire pH gradient.”
The optimized method reported isoelectric points that were closer to those of the certified reference than those achieved by the European Pharmacopoeia procedure.
“The method [is] a new benchmark for the quality control of biopharmaceutical products for both manufacturers and regulatory authorities,” Ascione and colleagues assert, “addressing the growing demand for horizontal standards concerning mAbs charge heterogeneity analysis.”