Characterization of a Monoclonal Antibody with Wyatt Technology’s MALS and RI Detectors and a Bio-Inert HPLC

Multi-angle light scattering (MALS) represents a uniquely powerful technique to determine the absolute molar masses and sizes of proteins in solution. MALS detectors cover a wide detection range of sizes and masses and enable the characterization of protein molecules as monomers, dimers, or higher order aggregates.

The plurality of detectors at different angles improves the accuracy and the precision of the results. However, it is not just the number of angles that determines the quality of the measurements, but rather the engineering of the instrument, too. Superior signal-to-noise ratios and sophisticated algorithms for data management are pivotal features of the instrument and software design. As is shown below, these prerequisites are fulfilled by Wyatt Technology's DAWN detectors.

Materials and Methods

Different amounts (0.2 μg to 500 μg) of a monoclonal antibody directed against leukocytic antigens were separated using an Agilent 1260 Infinity Bio-inert LC coupled to a Superdex 200 SEC column. The LC consisted of a 1260 Infinity bio-inert quaternary pump (G5611A), a G5667A bio-inert autosampler, and a 1260 Infinity VLDiode array detector. Concentration was measured using a Wyatt Technology Optilab T-rEX differential refractometer (operating at the same wavelength of light as the DAWN MALS instrument). Light scattering measurements came from Wyatt's DAWN HELEOS II MALS detector. While in many applications no special liquid chromatography (LC) is required, for these experiments the Agilent 1260 Infinity Bio-inert LC was applied because it has a metal-free sample flow path. This prevents unspecific interaction of the analyzed molecule with instrument surfaces.

Results and Discussion

Figure 1 shows the signals obtained from the antibody injected into the separation system. The RI detector displays peaks of varying heights, as expected according to the various sample loads of the protein. In contrast, the MALS signal represents the product of the sample's molar mass times the concentration.

Figure 1: RI and LS signals of 11 measurements of the antibody.

Evaluation of Collected Data

The average molar mass of the antibody using Wyatt's ASTRA software was determined to be 153,650 Da, with a standard deviation of only 1.7%. As can be seen in Figure 2, molar masses were reliably determined over a huge dynamic range that varied by a factor of 2500. For antibody analysis this is highly advantageous. Frequently, there is only a small amount of precious sample available, so high sensitivity is essential to collect meaningful data. However, when performing quality control of therapeutic protein preparations, the solute concentrations are, of course, higher.

Figure 2: Comparison of sample load and molar mass determinations of the antibody by MALS.

Our data show that the Wyatt MALS and RI detectors combined with Agilent 1260 Infinity Bio-inert LC is a very useful approach that yields highly reproducible results over a wide range of protein injections. It represents the instrumentation of choice for the analysis of therapeutic proteins in numerous pharmacological settings.

Wyatt Technology Corporation
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