Coupling Light Scattering with Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) is a common analytical method used for the analysis of proteins and other biopolymers. Adding light scattering (LS) to SEC provides many benefits. It is possible with different LS detectors to determine the absolute molecular weight without the need for a reference standard, the conformation, the size (oligomerization/aggregation state), and other key characteristics of a protein. In addition, problems with the SEC column not identified when using standard ultraviolet (UV) detectors are often made apparent when light scattering is used in conjunction with SEC.

Challenges in combining SEC with LS, including the need to understand how the light-scattering signals are converted to molecular weight data, the additional user knowledge required to analyze the data, particle shedding from SEC columns, the need for good concentration measurement, and the switch to ultra high-pressure chromatography, are being addressed with new software, SEC columns, and detector technologies.

Why combine LS with SEC?
Molecular weight is a defining parameter for proteins and biomolecules; therefore, reliable measurement is crucial, particularly during the development of new products, but also in quality control. “For many biomolecules, especially those that are novel, there are no relevant reference standards with which to calibrate the separation performance of an SEC column; light-scattering detectors bring value by eliminating this requirement,” says Paul Clarke, industrial portfolio manager at Malvern Instruments.

The main advantage is the ability to determine the absolute molecular weight of the analyte in solution regardless of its shape, conformation, density, or non-ideal (e.g., hydrophobic) column interactions, according to Michelle Chen, head of analytical services with Wyatt Technology. Multi-angle, static light scattering (MALS) decouples molar mass analysis from retention time and provides a first-principles determination of molar mass. “In addition,” says Chen, “with SEC-MALS, the determination of protein properties that cannot be measured using UV detection with SEC is possible. MALS data combined with UV and differential refractive index (dRI) signals from an SEC separation can be used to measure the stoichiometry of protein conjugates, assess SEC peak heterogeneity, and estimate protein conformation.

Molecular size (radius) can also be determined using SEC with LS. While static light scattering (including MALS) is appropriate for molecules that are sufficiently large (typically >10 nm radius), dynamic light scattering (DLS) combined with SEC enables the measurement of molecular size across a highly applicable range for biopharmaceutical research, according to Clarke. In a typical SEC-MALS/DLS setup, according to Chen, MALS can measure radii from ~10 nm to 500 nm, and DLS can measure ~1 nm to 250 nm. “Hence, DLS is most appropriate for characterizing the size of proteins and small oligomers while both techniques are appropriate for larger aggregates, virus-like particles, liposomes, etc. MALS is more sensitive and hence better suited to characterizing trace quantities of large aggregates,” she says.

The combination of MALS and DLS is also useful for assessing conformation or shape, and in addition, the molecular weight data can be used to differentiate oligomers from aggregates in a sample. The use of light scattering with SEC is particularly beneficial for the analysis of proteins and protein conjugates, including membrane protein detergent complexes (PDCs), according to Clarke. It is in fact crucial, according to Chen, that the SEC-LS combination be applied whenever there is a need to characterize protein aggregates, oligomeric state, conjugates/complexes, binding, and conformation.
Clarke also notes that the characterization of polymeric drug-delivery components including protein encapsulation technologies is a second important application area for SEC combined with LS techniques. Furthermore, Chen points out that the addition of LS to SEC often reveals problems of the SEC method that were invisible using UV detection alone, such as contaminated SEC systems, inadequate separations, column interactions, the presence of micelles, and low sample recovery. “This sensitivity can be extremely helpful in detecting a less than optimal chromatography set-up,” says Clarke.

A few limitations
In general, the limitations of the SEC-LS method stem mainly from the limitations of SEC itself and include the potential removal of large aggregates as well as sample property changes due to dilution and solvent exchange during separation, according to Chen. One issue of note for Clarke is the concentration sensitivity of light scattering detectors, which is directly proportional to both molecular weight and concentration. As a result, measuring small amounts of low-molecular-weight biomolecules can be a challenge. A separate issue is the additional user knowledge required to analyze the data produced when using SEC combined with light scattering.

Resolving resolution issues
The most common challenge a user faces while using SEC-LS methods is particle shedding from the SEC columns, according to Chen. “These particles increase the LS baseline noise and reduce detection sensitivity,” she observes. At the same time, there has been growing interest in the use of ultra high-pressure (UHP) SEC methods for high-speed and high-efficiency SEC separations, which have a peak volume of roughly 100 µL or less depending on the column dimension. “Because the combined dispersion volume of some current LS and differential refractive index (dRI) detectors is generally more than 50 µL, loss of resolution between aggregate or fragment peaks will occur when these detectors are used, making them incompatible for a UHP-SEC system,” says Chen.

Fortunately, the recent introduction of LS-friendly SEC columns with greatly minimized shedding is helping to reduce particle shedding noise. In addition, the use of light scattering and RI detectors with smaller flow cells is enabling UHP-SEC-LS.

Concentrating on concentration measurement
Another hurdle for some users of SEC combined with light scattering is the need for good concentration measurement, according to Clarke. “In many instances, biopharmaceutical researchers using SEC rely on a UV detector for concentration. UV detectors provide relative concentration measurements based on the amount of chromaphore present in a sample, rather than an absolute measure of concentration. When adding a light scattering detector to SEC, it may, therefore, be necessary to simultaneously add an RI detector in order to be able to obtain concentration measurements of the required quality,” he explains.

Training and advanced software systems
The lack of familiarity with LS instruments and software can be a challenge for some users of this analytical method. It is, however, crucial to understand how the light-scattering signals are converted to molecular weight data to successfully select, apply, and exploit light scattering-technology within the context of SEC, according to Clarke. Proper training is, therefore, imperative. In addition, advanced software can be helpful with respect to the setting of appropriate baselines and integration limits.

Cleaning up the column
Because light-scattering results are sensitive to the presence of particulates, the cleanliness of the mobile phase and column is important. Bacterial growth in an SEC system will similarly compromise data quality, according to Clarke. Importantly, where an SEC system is less than optimal, light scattering can be a useful troubleshooting tool because of its ability to sensitively detect and reveal chromatography issues. “More generally,” Clarke notes, “it is vital to recognize that adding a light scattering detector to a poor SEC set-up will not solve the problems. A light-scattering detector may, however, provide sensitive, useful insight into the issues responsible for poor SEC performance.” In most cases, according to Chen, contamination and other SEC issues that are highlighted by LS detection can be dealt with using a regimen of cleaning and maintenance or mobile phase modifications, guaranteeing better overall SEC performance.

Making the right choice
The key to successful implementation is to choose the best type of light-scattering detector for the application, according to Clarke. To do so, however, requires an understanding of how light-scattering signals are converted into molecular weight data. “In many instances, a right-angled light-scattering (RALS) detector, the least expensive and simplest light-scattering detector available, will be more than adequate for protein analysis,” he says. Chen notes that a three-angle MALS detector can be just as inexpensive as a RALS detector, yet provide more reliable data. “Only by acquiring measurements from at least three well-spaced detector angles can you truly differentiate between a clean protein signal and noise due to column shedding,” she asserts. Low-angle light-scattering (LALS) and MALS detectors are particularly useful for the characterization of larger molecules and the determination of radius of gyration. In this application, MALS is also effective for differentiating between real signals and particle noise, according to Chen. “Understanding how molecular weight is calculated is also critical when it comes to identifying possible sources of errors with a chosen detector,” Clarke says.

Clarke also recommends that other data that might become accessible with light scattering in place be considered to maximize the positive impact of an investment in light-scattering technology. By adding a viscometer to the detector array of an SEC system in combination with a light-scattering detector, for example, it becomes possible to generate intrinsic viscosity and hydrodynamic radius data for proteins or polymers, which is useful for detecting structural differences or changes. Also useful is a dynamic light scattering module that integrates directly into a MALS detector to simultaneously determine molar mass, radius of gyration, and hydrodynamic radius, according to Chen. “The shape factor--the ratio of the two radii--and the specific volume--the ratio of the molar mass to the hydrodynamic volume--are important biophysical characteristics of biomacromolecules,” she explains.