New Agilent AdvanceBio SEC column simplifies mAb/ADC analysis
Linda Lloyd Agilent Biocolumns Product Manager
M. Sundaram Palaniswamy Agilent Biocolumns Application Scientist
Therapeutic proteins are subjected to aggregation and degradation during the production processes, such as expression, refolding, downstream processing, formulation, sterilization, and storage. As part of the development of a new biotherapeutic, the probability of aggregation occurring must be determined and the amount of aggregation quantified for all stages of production. The structure and form of the monoclonal will impact the likelihood of aggregate formation and so, with the attachment of hydrophobic payload to form the ADC (antibody drug conjugate), there will be a change in the aggregation profile as the modification enhances hydrophobicity-driven aggregation. Although aggregates or degradants are present in low concentrations, they may have a big impact on the quality of biologics. Thus, small soluble aggregates can lead to immune reactions, and large insoluble aggregates, particulates, can cause adverse events upon administration.
Size exclusion chromatography (SEC) is the standard method for characterizing protein aggregation. However, for successful SEC quantitation of aggregation, monomer, dimer, and higher aggregates, it is vital that the mobile phase does not affect sample composition. As environmental conditions can change the level of aggregation, it is important that the SEC separation be done in aqueous mobile phases at neutral pH and with low levels of salt. Most published methods for SEC analysis of ADC that used aqueous phase revealed poor peak shapes and incomplete resolution of aggregate from the monomeric conjugate. This effect was due to nonspecific interaction of the hydrophobic payload with the stationary phase.
Breakthrough technology from Agilent
Agilent scientists have developed a new biocolumn that overcomes the deficiencies of standard SEC for the separation and quantification of therapeutic mAbs and ADCs. The Agilent AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm column has been designed and manufactured around an innovative silica particle and unique bonding chemistry.
Figure 1. Chromatogram of native (red trace) trastuzumab overlaid with pH/heat-stressed trastuzumab using an Agilent AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm column.
Figure 2. Chromatogram of native (red trace) ADC overlaid with pH/heat-stressed ADC using an Agilent AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm column.
This breakthrough technology delivers resolution and size separations over a wide range of sample types, without the need to add an organic modifier to the mobile phase. Another advantage is that you can run ADCs and mAbs with the same mobile phase and column, without the need to switch, as is the case with traditional SEC approaches.
You can see the benefits of using SEC to monitor degradation in a comparison of native and pH/heat stressed trastuzumab (Figure 1). The monomer aggregates a dimer and a small amount of higher aggregates. Figure 2 shows the same comparison for the ADC sample. It is evident that the new column resolved the aggregates when the samples were subjected to pH/heat stress and revealed their different profiles. Using the Agilent AdvanceBio SEC column and the same chromatographic conditions provides critical information on the level of aggregation and the size of the aggregates before and after mAb modification.
A simple and single method for biopharma QA/QC
The chromatograms of pH/heat-induced aggregates indicate that the AdvanceBio SEC column was able to separate and detect aggregates, as well as degraded trastuzumab and ADC. Intact aggregates and degradants were distinctly separated from each other. Such a simple and reproducible method, coupled with the bioinertness and corrosion resistance of the Agilent 1260 Infinity Bio-inert Quaternary LC, makes this solution suitable for QA/QC analysis of monoclonal antibody/ADC for the biopharma industry.
Full details of this work are freely available in Agilent publication 5991-6303EN.
Agilent offers a wide range of biopharma solutions to ease your workflows
Agilent offers many resources and products to help you separate, identify, and quantify aggregates and fragments from biopharmaceutical products.
For example, Agilent BioHPLC columns for SEC offer fast, reliable, accurate performance for your biopharmaceutical analysis. These columns are easy to integrate into your workflow and are available in a wide range of pore sizes and dimensions. Agilent has been a leading manufacturer of SEC columns and instruments for more than three decades. Explore our family of biologics solutions and LC biocolumns, then contact your Agilent Representative today and see how our solutions can speed your workflows and reduce your time to market for new biotherapeutics.
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