Protein aggregation is a critical quality attribute that, if left unchecked, can compromise the quality, safety, and efficacy of antibodies. Quantification of aggregation is therefore vital in the development of biotherapeutics, and choosing the right analytical column is an important first step. Size exclusion chromatography (SEC) is the standard analytical method used to quantify protein aggregation, but researchers face several challenges when it comes to column performance such as slow separations, poor resolution and sample backlogs.
Monoclonal antibody aggregates
Large-scale manufacturing of biotherapeutics is a complex process that involves several stages. Ensuring the stability of a therapeutic protein is critical throughout the development and manufacturing process to prevent a reduction in therapeutic properties or immunogenic reaction, which could potentially endanger a patient’s health.
Monoclonal antibodies (mAbs) have proved to be a highly successful and important class of therapeutic product for the treatment of diseases such as cancer and multiple sclerosis. The aggregation process is influenced by the biochemical and biophysical properties of the mAb itself as well as different stages of the manufacturing process. Throughout the manufacturing, formulation and stability testing process, the ratio of aggregates to the free (monomer) drug are monitored and must remain at low levels and regulations such as ICH(Q6B) state that aggregates must be resolved from the desired product and quantified. Accurate and reliable analysis is therefore essential when characterizing monoclonal antibody aggregates to guarantee consistent levels of product quality.
Size exclusion chromatography for the analysis of biotherapeutic molecules
Size exclusion chromatography (SEC) is an important tool for monitoring aggregates and potential degradants in biotherapeutic protein samples, including monoclonal antibodies and their derivatives. SEC involves the chromatographic separation of biomolecules based on their size in solution and provides an ideal technique for the separation and analysis of intact proteins from contaminants including aggregates, excipients, cell debris and other impurities arising from degradation. These types of protein separations demand the highest levels of accuracy and speed.
Successful development of a mAb-based pharmaceutical requires assessment of aggregation and fragmentation resulting from forced degradation studies, which include physical and chemical degradation pathways. A significant challenge facing today’s researchers is identifying an SEC method that is capable of separating and monitoring such variants.
Optimum column performance where it matters
If the incorrect column is selected for quantification of monoclonal antibody aggregates, issues such as slow separations, poor resolution and sample backlogs can occur. There are a number of important considerations to achieve optimum column performance for the analysis of monoclonal antibody aggregates:
• Choosing the right column diameter
• Ensuring column robustness to prevent deterioration of stationary phase
• Use of appropriate protein standards for column calibration
• Carefully controlled separation temperature
Agilent has developed the AdvanceBio SEC column, packed with 2.7 μm particles of a unique, low binding, polymer coated silica stationary phase. This technology provides minimal nonspecific interactions, and gives improved peak shape for the analysis of biotherapeutic molecules such as monoclonal antibodies and antibody drug conjugates (ADCs). The AdvanceBio SEC column’s highly uniform particles provide excellent column efficiency providing maximum protein recovery with operating pressures that enable use on conventional 400 bar LC systems as well as newer ultrahigh-performance LC systems.
To keep up with the demands of biopharma discovery, development and manufacturing, analytical laboratories are under pressure to deliver results to strict deadlines. With so many samples to analyze, you may think you need more LCs and more columns to meet your deadlines. With AdvanceBio SEC columns the new silica particle provides the resolution and stability to run shorter columns at higher flow rates, giving you the right result in a quarter of the time.
A recent study demonstrated the ability of the AdvanceBio SEC column and Agilent 1260 Infinity Bio-inert Quaternary LC system to monitor the aggregation of commercial mAbs and ADCs. The column successfully resolved the therapeutic mAbs and ADCs according to their molecular mass, showing different retention times and demonstrating the suitability of this column for the analysis of these molecules. The retention time and peak area were less than 0.04% and 1% respectively, demonstrating the excellent reproducibility of the method and the precision of the system. The study can be downloaded here.
The Agilent AdvanceBio SEC 300Å columns are designed specifically for monoclonal antibody aggregate analysis. For smaller proteins and peptides, AdvanceBio SEC 130A is the column of choice. The columns provide optimum performance where it matters, regardless of the instrument used. To find out how the AdvanceBio SEC columns can help you break through the roadblocks to monoclonal antibody characterization download the AdvanceBio column information kit which includes comprehensive technical and compatibility details, a competitive review and application notes covering ADC aggregation, Rituximab aggregates/fragments, and more.
Agilent AdvanceBio columns are designed to enhance the accuracy and speed of your biomolecule characterization, including monoclonal antibodies (mAbs), other proteins, peptides and synthetic oligonucleotides. Learn more