Quantitative Laser Diffraction Mediated Detection of Submicron and Subvisible Aggregates (100 nm–10 μm) in Biotherapeutics: Need for Regulatory Attention To this Neglected Particle Size Range

Abstract

Objective

Immunogenicity caused by aggregates in biotherapeutics, particularly peptides and monoclonal antibodies (mAbs), is a significant concern. Aggregates formed during manufacturing and storage may exist in the nanometer (1–100 nm), submicron (100 nm–1 μm), and subvisible (> 1 μm) size ranges, contributing to immunogenicity. Currently, the United States Pharmacopeia (USP) guidelines focus on particles ≥ 10 μm, while there are no specific regulations for monitoring or quantifying submicron and subvisible particles (SMPs and SVP1, 100 nm–10 μm) despite their known immunogenic potential. This study aims to highlight the need for regulatory guidelines by investigating the occurrence of such particles under stress conditions using a quantitative laser diffraction (qLD) technique.

Methods

The study evaluated the formation of SMPs and SVP1 in four representative biotherapeutic molecules under pH and temperature stress. A relatively newer analytical tool, quantitative laser diffraction (qLD), was employed to detect and quantify these particles. Additionally, case studies, including Peginesatide’s market withdrawal due to hypersensitivity reactions linked to SMPs and SVP1, and research demonstrating higher immunogenicity of submicron particles in mice, were reviewed to support the study’s objectives.

Results

The qLD analysis revealed a significant tendency for aggregate formation in the SMP and SVP1 size ranges under stress conditions. The findings reinforce existing evidence that these particles contribute to immunogenic reactions and highlight their potential impact on biotherapeutic safety and efficacy.

Conclusions

This study underscores the critical need for establishing regulatory guidelines for monitoring and quantifying SMPs and SVP1 in biotherapeutic products. Improved monitoring practices will enhance drug safety and efficacy, ultimately protecting patients from adverse immunogenic reactions.