Many interesting and useful macromolecules consist of two chemically and physically distinct species, conjugated covalently. This can make it quite difficult to characterize the essential properties of molar mass and size by size-exclusion chromatography since no calibration standards exist to represent the elution behavior of these composites. Often one or both of the primary constituents is not amenable to mass spectrometry.
The combination of multi-angle light scattering (MALS), UV and refractive index (RI) detection with size exclusion chromatography, plus ASTRA's Conjugate Analysis algorithm, offers the solution to the characterization of conjugated macromolecules such as:
- Block co-polymers
- PEGylated proteins
- Membrane proteins solubilized in detergent micelles
- Protein-polysaccharide vaccines
- Antibody-drug conjugates
Combining MALS-UV-RI analysis with size exclusion chromatography enables the analysis of conjugated proteins.
Conceptually, a complex consisting of a protein (containing a UV chromophore) and a modifier such as poly-ethylene glycol, or PEG (which does not), can be analyzed by combining information from two distinct measurements. UV absorption determines the protein concentration, while RI determines total macromolecular concentration. The modifier concentration is just the difference between these two (actually, an accurate calculation accounts for the extinction coefficients and differential refractive indices of both species). These two measurements are sufficient to determine the ratio of the protein and modifier in the complex. However, this analysis cannot determine the total mass or oligomeric state of the species.
The addition of a MALS detector provides the answer to full conjugate characterization. Since light scattering is proportional to the product of molar mass and concentration, the combination of these three signals is sufficient to determine not only the ratio but the actual molar mass, and hence oligomeric state, of each constituent. This information is critical in assessing aggregation of conjugates, measuring the molar mass of block co-polymers, or deciding whether or not a detergent-solubilized membrane protein is present as a native oligomer.