A Design of Experiments and Risk Management-Driven Analytical Platform for Charge Variant Analysis of Therapeutic...

The evaluation of monoclonal antibodies (mAbs) charge variants during their entire life cycle is crucial, as their profiles represent a critical quality attribute of biotherapeutics. While the current scenario still shows marked analytical non-uniformity in the evaluation of charge variants with imaged capillary isoelectric focusing (icIEF) with many "product-specific" methods, regulatory authorities are increasingly encouraging the utilization of horizontal standards, such as Platform Analytical Procedures (PAPs). A practical Analytical Quality by Design (AQbD) workflow is provided, emphasizing the Design of Experiments as a Quality Risk Management tool to develop a PAP based on icIEF, able to accurately measure charge variants pI values. Infliximab was chosen as the leading molecule. The cause-effect matrix, combined with an asymmetric screening design, identified key parameters exerting a critical impact on the Analytical Procedure Attributes. PAP quality measurements were ensured by a 10% risk acceptance level, employing response surface methodology and Monte Carlo simulation. The developed PAP was validated using three independent System Suitability samples and exhibited a low bias in the pI measurement (less than 2%), while maintaining satisfactory separation performance. Good intra-day and inter-day repeatability, combined with a robustness test and an exploratory application to real samples of three different therapeutic mAbs, confirmed its versatility. The study supports regulatory trends by demonstrating the successful application of AQbD in PAP development. This icIEF platform would ensure a systematically consistent analysis of charge variants, where pI is promoted to an objective tool, to be used as an additional reliable parameter in the Quality Control context.