The Analytical Ultra Centrifuge AUC is a quantitative technique, with reasonable resolution, but is time consuming. Dynamic Light Scattering is a comparatively fast test for the purity of a protein, and requires a very low sample volume, especially suitable for quick assessment of oligomeric state.
Analytical ultracentrifugation (AUC) is a versatile technique that can provide quantitative information about the sizes and shapes of macromolecules in solution. Samples are subjected to a centrifugal field, and experiments may take from an hour to several days. Dynamic light scattering (DLS) is a quick method to characterize the size of biomolecules in solution - in a matter of minutes. In this application note these two different methods are used to find the proportion of monomer in samples where other oligomeric species are present in equilibrium with the monomer. The example system under investigation consists of a number of different preparations of antibodies.
A set of antibody samples (IgG4, MW ~ 146 kDa) from different lots were analysed with two different techniques in order to quantify the amount of monomer in the prepared solutions. The analytical ultracentrifugation (AUC) data were taken on the Optima XL-A system (Beckman Instruments, Palo Alto, USA) with solute concentration distributions recorded as ultraviolet absorbance at 290nm. Sedimentation velocity runs were performed at high rotor speed (typically 42000 rpm) for at least 7 hours at 20°C.
Samples were prepared at various concentrations from 0.6 to 1.7 mg/mL in 0.1M ammonium hydrocarbonate buffer (pH not adjusted), no filtration was used.
For the light scattering results samples were measured in a 12 μL cuvette (ZEN2112) in a Zetasizer Nano S. The instrument was used for sizing using DLS to determine the z-average molecular size in solution. All experiments were performed at a temperature of 25°C.
The distribution function of apparent sedimentation values g*(s) was modelled as a collection of Gaussian distributions for individual species according to Dam and Schuck (2003)1. All samples exhibited various amounts of monomer, with typical sedimentation coefficients of the main component at about s20,w ~ 6.7 Svedberg. The suffix 20,w indicates that the parameter has been corrected to standard solvent conditions - the density and viscosity of water at 20.0°C. This corresponds to the MW expected for these samples. Results predicted from the centrifugation are shown in Table 1.
Table 1: Fitting Results from the Analytical Ultracentrifugation, expressed in terms of volume % of monomeric species present.
|Sample||Conc. (g/L)||Main species s20,w (S)||AUC %mono-mer (by mass)|
Typically, more than 80% of the antibody molecules were found to be present in monomeric form. Clearly, the majority of the sample (by mass) was monomeric.
Dynamic Light Scattering
Light scattering measures the diffusion of molecules in solutions. Just as the sedimentation coefficient may be used to find the molecular size, so can the diffusion coefficient. The result may be obtained from fitting the decay of the correlation function g(τ). Assuming a Gaussian distribution, a simple exponential fit produces the z-average (or cumulant) size. As the measured property is the intensity of scattered light (rather than the %mass as in UV absorbtion), it is necessary to take the high sensitivity to larger sizes (intensity ~ size6) into account when converting to the volume distribution. It is possible to calculate the contribution from the monomer when a monomer-dimer equilibrium is assumed to contribute to the measured light scattering result. Figure 1 shows this effect of the conversion to %intensity, %mass and %number, for the example of the antibody used in this lot comparison. The monomer is expected to show a hydrodynamic diameter of 10.1 nm, the dimer would then be 13.5 nm.
Figure 1: Monomer contribution versus measured hydrodynamic size, by intensity (blue), by mass (red), by number (yellow). Example: 11.7nm would correspond to 75% monomer by mass.
Figure 1 can be used to find the amount of monomer for any measured hydrodynamic size. The measured size for the different sample lots is shown in Table 2, also listed are the monomer contributions by mass as predicted by this technique.
Table 2: Fitted Results from dynamic light scattering, expressed in terms of volume % of monomeric species present.
|Sample||Hydrodynamic Diameter (nm)||DLS %monomer (by mass)|
All values are close to or above 80%, in good agreement with the AUC data. The largest deviation was seen for Lot4-3 where the analysis by ultracentrifugation shows a 7% higher monomer presence.
Light scattering may be used to quickly establish whether a protein is a monomer or oligomer in its native state. The presence of aggregates in solution is easily detected. As shown here, with good data quality the amount of monomer may be quantified and was shown to agree well with analytical ultracentrifugation. Light scattering may not match the accuracy of ultracentrifugation but certainly provides a useful prediction of the sample composition in a matter of minutes.
1 Dam, J.; Schuck, P. Meth. Enzymol. 384 (2003), 185-121