Absolute Size Exclusion Chromatography

Size exclusion chromatography (SEC) and dynamic light scattering (DLS) are common technologies in protein laboratories. SEC is used routinely for the purification, identification, and quantification of protein mixtures, while batch DLS is used for pre-column size and polydispersity measurements, along with aggregate quantification. Until now, coupling of the two technologies has been difficult—largely because the eluted protein sample concentrations from SEC are often well below the detection limit of most DLS instruments. The recent introduction of a chromatography flow mode for the high-sensitivity Zetasizer® Nano particle characterization system (Malvern® Instruments, Westborough, MA) changes this situation. By overcoming the problem of detection limits, it makes the coupling of SEC and DLS technologies a practical and attractive option.

Combining the high resolution of chromatography with the ease of data acquisition from DLS has the advantage of allowing rapid peak identification. It also confers the ability to perform absolute size exclusion chromatography (ASEC), whereby protein size can be measured in real time, under flow, as the samples are eluted from the column, rather than relying on column calibration or additional data from a second detector. Good estimates of molecular weight can then be computed rapidly on the basis of protein size. This has the practical advantage of negating the need for molecular weight standards with conventional SEC in order to identify oligomeric species or determine protein purity. In short, ASEC allows:

  •  Confirmation of oligomeric state—monomer, dimer, etc.
  •  Purity determination
  •  Quantification of mixtures
  •  Confirmation of protein identity
  • Detection of low-molecular-weight impurities.

As shown in the examples that follow, ASEC provides a valuable new tool for protein laboratories.

Instrument setup

Figure 1 - Setup for absolute size exclusion chromatography.

In standard size exclusion chromatography, after the column, the eluent flows through one or more detectors, typically monitoring ultraviolet light absorbance and refractive index in order to detect the presence of molecular species. For absolute size exclusion chromatography, a DLS instrument, namely a Zetasizer Nano, simply needs to be connected after the last detector (Figure 1). The absolute nature of the measurement means that no column calibration is required and the software makes the entire measurement process simple and straightforward. For the majority of protein separations in aqueous media, no extra parameters are required. Size is plotted in real time as material is eluted from the column, and the molecular weight of each peak is calculated automatically at the end of the measurement.

The other detector outputs can be displayed simultaneously with the light scattering data, and by determining the volume delay between detectors during the setup procedure, the data output from all detectors is aligned.

The conversion of molecular size to molecular weight using DLS is particularly simple and accurate for globular materials such as proteins, as demonstrated by the examples that follow.

Experimental

Protein samples were prepared in an appropriate buffer and loaded onto a Superdex 200 column (GE Healthcare, Uppsala, Sweden). Each measurement used a flow rate of 0.5 mL/min. Dynamic light scattering measurements were accumulated continuously and analyzed every 3 sec. The Zetasizer Nano software recorded and stored all correlation functions and intensity values.

Bovine serum albumin

Bovine serum albumin (BSA) of expected molecular weight 67 kDa was prepared in 0.1 M phosphate buffered saline at a concentration of 8 mg/mL; 500 μL (4 mg of protein) was loaded onto the Superdex 200 column. Such a quantity of BSA slightly overloaded the column, leading to nonideal separation of the component oligomers.

Figure 2 - Hydrodynamic diameter (z-average, nm, in blue) and light scattering intensity (kilo counts per second, in green) versus elution volume for BSA, injected onto a Superdex 200 column.

Figure 2 shows the z-average size (DLS) and the light scattering intensity trace. The monomer peak eluted between 15.2 and 16.0 mL, corresponding to a hydrodynamic diameter of 7.4 nm. This is very close to the expected size for this molecule. On the basis of this size, the computed molecular weight prediction was 72 kDa, slightly larger than the 67 kDa expected. Nevertheless, this estimate clearly confirms that this material is the BSA monomer, rather than any other oligomer.

Figure 3 - Eluted size distribution from BSA in PBS. In the analysis of the elution profile, the majority by intensity is found to be a monomer.

Results can also be interpreted in terms of size distribution to give an indication of the relative composition of the original sample. Figure 3 shows the size distribution of the BSA sample. The majority of the protein (60.2% by intensity) is contained in the monomer elution peak. The next peak is the dimer with 20% of the signal. This distribution is a much higher resolution than can be obtained by a “batch” measurement, i.e., measurement of the sample without separation by SEC.

IgG4 antibody

Figure 4 - Hydrodynamic diameter (z-average, nm, in blue) and light scattering intensity (kilo counts per second, in green) versus elution volume for IgG4, injected onto a Superdex 200 column.

An antibody (IgG4) of expected molecular weight around 150 kDa was prepared in 0.1 M ammonium bicarbonate buffer (pH not adjusted) at a concentration of 7 mg/mL; 100 μL (0.7 mg of protein) was loaded onto the Superdex 200 column.

Figure 5 - Hydrodynamic diameter (z-average, nm, in blue) and light scattering intensity (kilo counts per second, in green) versus elution volume for carbonic anhydrase.

The main monomer peak eluted between 13.3 and 14.2 mL, corresponding to a hydrodynamic diameter of 11.6 nm (Figure 4). This is close to the expected size for this molecule. A prediction of the molecular weight from the measured size leads to 200 kDa, rather more than the nominal 150 kDa expected. The higher size is due to the Y-shape of the IgG4 molecule—antibodies do not give an ideal fit with the globular protein family model. A typical compact globular protein of 150 kDa would have a size of 10 nm. Nevertheless, the estimate of 200 kDa clearly confirms the presence of the monomer.

Carbonic anhydrase

Carbonic anhydrase of expected molecular weight 29 kDa was prepared in phosphate buffered saline at a concentration of 5 mg/mL; 100 μL (0.5 mg of protein) was loaded onto the Superdex 200 column.

The single peak, eluted between 18.0 and 18.6 mL, had a z-average size of 4.81 nm diameter (Figure 5), corresponding to an estimated molecular weight of 26.2 kDa for a globular protein. This simple size measurement confirmed the main elution peak as that of the carbonic anhydrase monomer.

Conclusion

The latest generation of DLS systems, specifically the Zetasizer Nano, allow the coupling of dynamic light scattering and size exclusion chromatography in the form of absolute SEC. A powerful, easy-to-use protein characterization tool, ASEC has the advantage of providing a rapid and direct measurement of protein size without the need for costly and time-consuming column calibration. In addition, since size versus mass relationships for globular proteins are well known, DLS measurements of the hydrodynamic radius can also be used to calculate molecular weight. The same technique is applicable to other types of chromatographic measurement, such as affinity or ion exchange chromatography, thereby enhancing the experimental return.

Figure 6 - Zetasizer Nano.

About dynamic light scattering
Dynamic light scattering is a powerful technique for determining structural characteristics such as molecular weight, size, melting point, and charge (zeta potential) of particles in solution. One of its great advantages is its ability to measure a large population of particles in a very short time period, with no manipulation of the dispersing medium.The Zetasizer Nano (Figure 6) is a benchtop instrument that includes both the hardware and software for combined dynamic, static, and electrophoretic light scattering measurements. The system was designed specifically to meet the low concentration and sample volume requirements typically associated with pharmaceutical and biomolecular applications, along with the high concentration requirements for colloidal applications. With a size range of 0.6 nm to 6 μm, and a concentration range of 0.1 ppm to 30% w/v, its specifications for sample size and concentration far exceed those of other commercially available DLS instruments.

Dr. Nobbmann is Nanometrics Specialist, Malvern Instruments Inc., 117 Flanders Rd., Westborough, MA 01581, U.S.A.; tel.: 508-768-6400; fax: 508-768-6403; e-mail:ulf.nobbmann@malvern.com.

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