The characterization of proteins involves identification of their complex chemical and physical structures and requires the use of a range of analytical techniques. Size exclusion chromatography (SEC) is generally used as a non-denaturing method that provides information on the apparent molecular weight, the presence of aggregates or higher molecular weight (MW) species, and the formation of degradation products, such as protein clips, and impurities. Today, SEC is predominantly used to measure aggregation, because the determination of protein molecular weight by calibrating the retention times against those of known molecular standards is not effective unless the proteins under study have similar conformations and column interactions as those of the standards. The combination of SEC with downstream light scattering analysis and the development of columns that can be used for ultra high-pressure liquid chromatography (UHPLC)-based SEC are overcoming many of the limitations of SEC and enabling more detailed protein characterization.
SEC and aggregation
SEC is most commonly used to characterize proteins by resolving monomers and aggregates and quantifying them to determine how much of the sample is aggregated, according to Mark Pothecary, product manager of Malvern Instruments’ Viscotek line. “Understanding the aggregate content is important because aggregates not only consume monomers and thus reduce the efficiency of the drug, they also are believed to cause immune responses. Thus, even at low levels, their presence can lead to antibody production against the drug, resulting in more rapid clearance and lower efficacy, and at higher levels, anaphylaxis,” he explains.
In addition, Pothecary says that characterization of oligomers, which are ordered assemblies of multiple proteins, is important because oligomerization controls protein activity. Measurement of molecular weight gives an indication of the oligomeric state.
SEC alone generally not recommended
Because SEC is an isocratic method, peak capacity can be an issue, according to Stephan M. Koza, principal applications chemist with Waters Corporation. “Column length can be increased to increase capacity, but a balance is often found between column housing, pressure tolerance, and efficient run times,” he notes. Robert Birdsall, a senior scientist with Waters, adds that the limited dynamic range of SEC often requires the use of columns that span zones of molecular weight ranges. “While this issue is not difficult to overcome, generally a range of SEC columns is required for screening samples, and when working on the extremes of the column (total exclusion for very big proteins vs. total inclusion for very small), resolution is affected much more significantly,” Birdsall observes.
The range of columns and buffers available for use in SEC, however, means that there are always improvements to be made in method development, according to Pothecary. “Alternative columns, different buffers, pH, or salt concentrations and in some cases even an organic modifier can all improve performance. Better separation always results in a better characterization,” he asserts.
Koza and Birdsall also note that SEC is rather insensitive to small changes in apparent MW, and non-specific interactions with the stationary phase can be encountered depending on the column chemistry, which can cause biased results. “In the latter case,” says Koza, “tailing or asymmetry in the chromatographic peaks may be a tell-tale sign, and this problem can often be addressed by increasing ionic strength.” Regardless, they both recommend that orthogonal methods be used to confirm any parameter of interest.
Band broadening between detectors also contributes to loss of resolution and subsequent sensitivity, according to Pothecary. While better connections between the tubing and shorter distances between cells can improve this situation, the best solution in this case is integration of all the detectors into a single unit. “Integration reduces the distances between the detector cells and minimizes temperature variations experienced by the sample, which improves the separation performance, and thus detection and characterization,” he states.
In addition to resolution, SEC analysis times are also an issue. “At 20 minutes to an hour for a measurement, it is a relatively slow technique,” says Pothecary.
When SEC alone is suitable
In certain purification or formulation tasks, it may suffice to determine only the fraction of aggregated protein relative to the unaggregated monomer, rather than the true molecular weights. “In this case,” says Daniel Some, director of marketing and a principal scientist with Wyatt Technology Corporation, “if the monomeric species is resolved from the aggregates and well-behaved in the mobile phase, SEC alone can quantify this fraction.”
Why downstream analysis
Most proteins are generally not globular, well-behaved, stable, and hydrophilic like available standards. They also often do not consist of discreet populations, such as monomers and low oligomers or fragments. “Real proteins may be partially or completely disordered, contain significant hydrophobic residues that interact with column packing material in a non-ideal way, or perhaps be continuously heterogeneous as a result of extensive glycosylation or PEGylation with extended conformations vastly different from those of well-folded globular proteins,” states Some. He adds that because SEC separates macromolecules by size, it provides an avenue for downstream characterization by other detectors that do not depend on elution time.
Light scattering a fruitful approach
Downstream characterization by means of multi-angle light scattering (MALS) provides first-principles determination of molar mass through the combined measurement of light scattering intensity and sample concentration, according to Some. In addition, the range of molar mass measured by a good MALS instrument far exceeds that of soluble proteins. Therefore, absolute characterization of the distribution of molar masses, at least those that are resolved by SEC chromatography, is possible. In addition to measuring the absolute molecular weight with light scattering analysis, it is possible to distinguish between monodisperse (i.e., single population) and polydisperse (i.e., poorly separated mixed populations) peaks, giving even greater insight into the exact composition of the sample, according to Pothecary.
Some also notes that for proteins conjugated to a modifier such as glucose, polyethylene glycol (PEG), or surfactant micelles for which no standard SEC reference molecules exist, the combination of MALS with two concentration detectors--usually ultraviolet (UV) and refractive index (RI)--can be used to determine the molar mass of each constituent to assess the oligomeric state and the overall molar mass. “Light scattering analysis with a combined UV and RI detector can provide information on whether the modification occurred and to what extent (i.e., how many PEG molecules are attached). The compositional information can be deconvolved to indicate the percentage of protein and PEG, which when combined with the absolute molecular weight measurement, provides the mass of the complex and its composition, which in turn can be used to determine the number of PEG molecules and the protein oligomeric state,” he explains.
The characterization of membrane proteins is also done in the same vein, according to Pothecary. “Membrane proteins make up something like 60% of all drug targets, but they have proven difficult to purify and crystallize. Today, however, the same compositional analysis can be done to a purified membrane protein to find out how much detergent it is associated with it and to see if there is any free detergent in the sample, both of which affect crystallization,” he says.
“In addition,” states Some, “certain protein oligomers exist in dynamic equilibrium with monomers or other subunits (‘reversible self-association’) as a function of protein concentration. Since MALS determines the weight-average molar mass at each elution volume, changes in this equilibrium across the chromatographic peak observed as the concentration increases and declines clearly indicate the presence of this phenomenon and can even be used to estimate the equilibrium dissociation constant (Kd).”
If a dynamic light scattering (DLS) detector module is incorporated into the MALS detector, the fluctuation spectrum of light scattered by proteins based on their Brownian motion can be used to determine the diffusion coefficients of the separated components, which can then be related to their effective sizes. “Comparison of molar mass and size is a good indicator of conformation, such as whether the protein is well-folded, denatured, or partially disordered,” Some says.
Greater throughput with smaller particle sizes
Several evolutionary advances in SEC have had a positive impact in recent years. Smaller flow cells and improved cell design, for example, have made it possible to connect more detectors together without the negative consequences of significant peak broadening and tailing. “As a result, a single run can generate more and more data, which makes the entire process more efficient,” says Pothecary.
UHPLC, on the other hand, offers a step-change in measurement time, speed, and resolution, but is still a new technology with respect to SEC, according to Pothecary, and its promises still need to be fully realized. In particular, he states that the amplification of peak broadening between detectors is due to the extremely narrow peaks that come from UHPLC. Over the past few years, however, smaller particle sizes have led to greater sample-throughput capabilities for SEC, according to Koza. “Improvements in high-performance liquid chromatography (HPLC)/UHPLC design with respect to system dispersion and sensitivity and advances in particle technology will further improve chromatographic performance in the future,” he notes.
Some agrees that UHPLC-based SEC has begun emerging in the past few years as the next step in SEC technology for proteins, offering faster separation with better resolution and much reduced consumption of sample and mobile phase. He notes, though, that until 2014, MALS detectors did not keep pace by decreasing the dispersion to match the narrower eluting peaks. The µDAWN MALS detector introduced in March 2014 by Wyatt Technology, along with the Optilab UT-rEX RI detector introduced in 2013, is designed for UHPLC and maintains the ultra-low dispersion required for UHPLC with no loss in sensitivity, according to Some. “In combination with UHPLC UV and IR detectors and an upgraded DLS module, the µDAWN extends all of the capabilities of MALS to the realm of UHPLC,” he remarks
SEC also has the potential to be combined with other downstream measurements to further enhance protein characterization. The measurement of intrinsic viscosity (IV), for example, gives information on protein density and, therefore, structure. “Non-globular proteins with either elongated or open structures can be readily identified using IV, which then makes size calculation possible. Theoretically, combining measurements of size from DLS and IV can give even more information on shape,” says Pothecary. Some adds that tagging proteins with fluorescent dyes offers the potential for better resolution of protein-protein complexes. “A third-party inline fluorescence detector may be combined with Wyatt’s MALS and RI instruments and software to determine unequivocally the molar masses of such complexes. This ability to combine different detectors is one of the benefits of a modular system as opposed to a fully integrated system,” he says.
About the Author
Cynthia A. Challener is a contributing editor to BioPharm International.