Successful Use of SEC for Protein and Peptide Aggregate Analysis
Pharmaceutical companies throughout the world are increasingly developing protein and peptide-related therapeutics. Consequently, the U.S. Food and Drug Administration and international regulatory agencies require companies to submit reliable analytical data to establish and confirm the efficacy and safety of biotherapeutic drug entities. Size exclusion chromatography (SEC) is well positioned to provide such data for protein biotherapeutics.
In fact, the primary application area for SEC is the biotechnology industry. For protein biotherapeutics, both the intermediary stages of production and the final parenteral therapeutic must be monitored for critical quality attributes, including the presence of dimer and higher multimer forms of aggregation. Although a broad range of techniques is used to assess protein aggregation, SEC has emerged as the most widely used method for routine validated analyses because of its speed and reproducibility. The limitation of SEC is that some large subvisible aggregates may not be observed; therefore, the result observed by SEC is typically confirmed by orthogonal methods, such as sedimentation velocity analytical ultracentrifugation (SV-AUC).
In SEC, compounds in solution traverse a medium typically composed of particles with controlled pore diameters, packed in a column. These pores are not uniform in diameter throughout their depth; they are wider near the surface of the particle than near the interior. As a result, when smaller compounds enter the pores, they can diffuse further into the pores than larger compounds and are therefore retained longer on the column. Compounds that are too large to enter the pores elute in the total exclusion volume of the column, and compounds that are small enough to diffuse throughout the entire volume of the pore elute in the total permeation volume of the column. The extent to which an analyte is retained is related to its size and shape, represented as a hydrodynamic radius (RH), not its absolute molecular weight. However, in general, SEC columns used for protein analysis are defined by the apparent molecular weight range of globular proteins that can be optimally separated.
As with other types of chromatography, a variety of factors, such as column length and flow rate, determine how well analytes are resolved by SEC. Here are a few other key considerations when selecting a column and when running an SEC method:
Select appropriate pore size
The optimal apparent protein molecular weight range that an SEC column is suited for is fundamentally dependent on the diameter of the pores into which these macromolecules can diffuse. Apparent molecular weight calibration curves are typically derived by plotting the log of the molecular weights of a series of proteins against their retention times. As a first approximation, the analyst should select a column for which the molecular weight of the protein falls closest to the midpoint of the pseudo-linear portion of that column’s calibration curve. Upon testing the selected column with the protein to be analyzed, a smaller-pore-size column may be warranted if the protein is eluted near the total permeation volume of the column; a larger pore size may be needed if the protein is eluted near the total exclusion volume. The use of two columns, with different pore sizes, in series also can be used to optimize or extend the pseudo-linear molecular weight range of an SEC separation.
The Effect of Particle Size on SEC Resolution: Shown is a comparison of the chromatograms obtained for the SEC separation of insulin from its covalent dimer and fragment forms on 1.7 µm and 10 µm particle-size columns of equivalent pore size (125 Å). The 10 µm particle-size column was run according to the USP and EP monograph methods (100 µL injection volume and 0.5 mL/min flow rate) and the 1.7 µm particle size column was run at 0.4 mL/min with an injection volume of 1 µL. Both chromatograms show comparable UV absorbance sensitivity, however, the 1.7 µm particle size column provides increased resolution, shorter analysis times, and significantly lower mobile phase use.
Better separation in less time
Innovations in both packing material and instrumentation have enabled development of Ultra High Performance Liquid Chromatography (UHPLC)-based size-exclusion separations. These sub-2 µm particle diameter-based UHPLC columns, when combined with low-dispersion, high-pressure UHPLC chromatographic systems, provide higher resolution and higher sensitivity SEC separations with much shorter run times than HPLC-based SEC separations.
Optimize SEC mobile phase
Electrostatic and hydrophobic interactions can occur between proteins and the packing material or hardware of an SEC column, causing prolonged retention and peak tailing. Conventional particles made from silica contain acidic sites that attract basic proteins. To counteract these ionic interactions, particle surfaces are commonly modified with functional groups such as diol. In addition, often it is necessary to use high-ionic strength eluents to offset these secondary interactions. However, increasing the ionic strength too much may increase hydrophobic interactions between the protein, or more commonly the peptide, being analyzed and the diol-modified particle surface. Mobile-phase optimization to minimize both of these potential sources of secondary interaction is an important part of method development.
Proper instrument configuration
SEC is an isocratic separation. Consequently, extra column dispersion can be introduced in the autosampler, detector and tubing set used to connect the column to the system. Unlike most other modes of separation, such as ion-exchange or reversed-phase separation, the entire SEC separation occurs in a single column volume; therefore, it is critical to minimize the extent of extra-column dispersion to reproducibly achieve high-resolution separations that will yield highly reproducible analytical results. To facilitate the development, transfer and troubleshooting of SEC methods, laboratories should establish extra-column dispersion metrics for their chromatographic systems.
Keep it clean
SEC mobile phases that are used for protein analysis (approximately neutral pH and low ionic strength) are vulnerable to growth of organisms such as bacteria and molds, which can prematurely shorten column life. It’s therefore important to use mobile-phase additives that will inhibit biological growth or to sterile-filter buffers and replace them frequently. In addition, blending SEC mobile phases from acidic, basic and high ionic strength concentrated buffers using a quaternary mobile-phase pump; keeping the mobile phase protected from sunlight; and using a continuous UV sterilization device inside the mobile-phase container will support trouble-free operation.
Stephan M. Koza, Ph. D., Principal Applications Chemist, Waters Corporation
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