Isoelectric point determination by icIEF as quality control for structural and functional characterization of HPV16...

Human papillomavirus virus-like particles (HPV-VLPs) are widely used as vaccine antigens and research models due to their structural similarity to native virions. Their production relies on the use of an expression system and involves complex molecular and biochemical steps. Slight variations in these steps can lead to batch-to-batch differences. However, process consistency and conformational integrity are critical to ensure their biological functionality. There is a great need for a tool to assess the structural integrity and batch consistency of HPV16-VLPs.

In this study, we developed and validated a robust imaged capillary isoelectric focusing (icIEF) method to assess the isoelectric point (pI) of intact HPV16-VLPs as a sensitive quality control (QC) metric. Key method parameters, namely surfactant and urea content, together with focusing time were optimized to maintain VLP stability during icIEF analysis. The method was validated according to ICH Q2(R2) guidelines and demonstrated high specificity, precision, and accuracy. Analysis of multiple production batches revealed two distinct VLP populations with pIs around 6.4 and 7.5, respectively. While conventional QC tests could not discriminate between these batches, functional assays (performed using bio-layer interferometry and affinity capillary electrophoresis) showed an altered binding capacity to laminin 332 receptor for the high pI VLPs. A disassembly/reassembly process restored the lower pI and binding capacity, suggesting conformational differences as the underlying cause.

This work highlights icIEF as a powerful orthogonal tool capable of detecting subtle structural and functional changes in complex biotherapeutics. It supports the implementation of this method into routine QC strategies to enhance both the reliability of research outcomes and the safety and efficacy of VLP-based vaccines.