Size Exclusion Chromatography (SEC) for Antibody Aggregation Analysis

Aggregation of monoclonal antibodies (mAbs) that are used as therapeutic agents can alter their biological activity and may also represent a safety concern for patients. As a result, it is required by regulatory agencies that aggregates should be analyzed and controlled at acceptable levels for mAb therapeutics. For this purpose, analytical methods are needed to quantify aggregate levels. A range of analytical tools is available at Creative Biolabs to analyze mAb aggregation. Especially, we offer size exclusion chromatography (SEC) for the characterization and quality control (QC) of mAb therapeutics.

Introduction to Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size or molecular weight. It is composed of the stationary phase and the mobile phase. A column of gel particles or porous matrix is used as the stationary phase while the solution of analytes is used as the mobile phase. As the solution travels down the column, large molecules pass by the pores because of their large size and elute first, while small molecules are trapped in the pores of the stationary phase and elute later. This technique involves gentle interaction with the sample, enabling high retention of biomolecular activity. SEC has been widely used in the biotech industry for various applications, including:

• Purification and separation of biomolecules, such as proteins and nucleic acids
• Determination of molecular size, analysis of purity, and study of interactions between proteins
• Molecular characterization of multivalent bioconjugates
• Analysis of biotherapeutics and their aggregates

Fig.1 SEC analysis of panitumumab aggregates. Peaks 1 and 2 are aggregated forms of the native antibody. (Fekete, 2014)

SEC for Antibody Aggregation Analysis

SEC is the standard method for aggregate and fragment analysis of mAbs in biopharmaceutical QC. Aggregates, monomers, dimers, and degradation products of monoclonal antibodies can be separated on size exclusion columns based on their molecular weights under native conditions. Moreover, we can combine the SEC with different detectors for different research purposes. Often, UV is used as the predominant mode of detection Especially, SEC coupled with multi-angle light scattering (MALS) offers a standard approach for characterizing the mass, overall shape, aggregation, oligomerization, interactions, and purity. SEC-MALS can provide information on high-order aggregates. Moreover, SEC is a great tool to monitor the forced degradation or stress testing of mAb products.

Antibody Aggregation Analysis Services Provided by Creative Biolabs

• Analyze the quantity and characteristics of antibody aggregates using an orthogonal approach
• Aggregation of mAb due to various types of stress factors can be studied in forced degradation studies
• Aggregation assessment in accordance with different regulatory requirements, e.g., ICH Q6B guideline

Besides, aggregation can be confirmed with other orthogonal methods, such as sedimentation velocity analytical ultracentrifugation (SV-AUC) and dynamic light scattering (DLS). If you are interested in our service, please contact us to discuss your requirements.

Reference

1. Fekete, S.; et al. Theory and practice of size exclusion chromatography for the analysis of protein aggregates. Journal of pharmaceutical and biomedical analysis. 2014, 101: 161-173.

For Research Use Only.