During development and manufacture, protein therapeutics are monitored for several critical quality attributes (CQAs) to ensure the efficacy and safety for use of these complex, heterogenous biopharmaceuticals. One of the most important CQAs is aggregation. Understanding and controlling aggregation in therapeutic proteins, such as monoclonal antibodies (mAb), is essential; aggregation can impact both therapeutic efficacy and, lifetime. In this article, learn why size exclusion chromatography (SEC) is the method of choice for protein aggregate and fragment analysis, discover top tips for choosing the best SEC column, and explore the range of columns from Agilent and how they can help you achieve high-resolution, high-throughput aggregate analysis of protein biopharmaceuticals.
Protein aggregation is very common and occurs in response to stress conditions, such as chemical, environmental, or mechanical stress, caused by changes in pH level, temperature, and shear force during processing or storage. Fragmentation can also occur under reducing conditions or by cleavage, for example the loss of a mAb subunit. Consequently, accurate measurement of aggregation and fragment levels by robust analytical methods plays a critical role in protein therapeutic characterization.
Size exclusion chromatography (SEC) is commonly used for the separation and quantitation of monomers, dimers and some higher order aggregates within the nm range to sub-micron range. This non-denaturing approach involves careful manual sample preparation that avoids altering the aggregate content, followed by LC separation based on relative molecular size in solution, and detection by UV or light scattering methods. Based on the absence of interaction between analytes and the column stationary phase, SEC enables separation and detection of intact proteins from contaminants including aggregates and fragments – larger molecules cannot permeate the column particles and elute first, while smaller molecules diffuse into the particle pores and diffuse later.
When selecting the right equipment to optimize your biotherapeutic aggregate analysis, there are several factors to consider. Here, we look at the top features to consider when choosing your next SEC column and the range of solutions available from Agilent that will help you streamline and accelerate aggregate analysis.
Agilent offers SEC columns in a wide variety of pore sizes to tackle all aggregate or fragment analysis challenges, from peptide analysis to protein conjugates and large molecules. Its versatile AdvanceBio SEC portfolio of 1.9 and 2.7 µm columns is available in a range of pore sizes and incorporate a unique hydrophilic coating that minimizes secondary interactions, enabling the analysis of challenging samples like ADCs with a simple phosphate buffer mobile phase. The Agilent Bio SEC-5 column is available in large pore sizes up to 2000Å, making it ideal for larger biotherapeutics such as adeno-associated viruses (AAVs), virus-like particles (VLPs), and other macromolecules. In combination with the biocompatible Agilent 1260 Infinity II Bio-Inert LC System, standards, and method validation kits, these columns can help you ensure accurate characterization of your biotherapeutic.
In a comprehensive downloadable compendium, learn more about Agilent solutions for aggregate/fragment analysis, discover detailed advice for successful biomolecule analysis covering the entire SEC workflow, and explore a range of methods to help you achieve a higher-throughput, high-resolution analysis of your biotherapeutics.
Methods detailed include:
Learn more about Agilent Technologies’ AdvanceBio SEC 1.9 and 2.7 µm columns and see examples of how they could help you gain confidence in your aggregate analysis.