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
- Glycoproteins, lipoproteins and nanodiscs
- PEGylated proteins
- Membrane proteins solubilized in detergent micelles
- Protein-polysaccharide vaccines
- Antibody-drug conjugates
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.
Abbas, S.; Lodge, T. P. Depletion interactions: effects of added homopolymer on ordered phases formed by spherical block copolymer micelles. Macromolecules 2008, 41, 8895-8902.
Bensaid, F.; du Boullay, O. T.; Amgoune, A.; Pradel, C.; Reddy, L. H.; Didier, E.; Sablé, S.; Louit, G.; Bazile, D.; Bourissou, D. Y-Shaped mPEG-PLA cabazitaxel conjugates: Well-controlled synthesis by organocatalytic approach and self-assembly into interface drug-loaded core-corona nanoparticles. Biomacromolecules 2013, 14, 1189-1198.
Berguig, G. Y.; Convertine, A. J.; Shi, J.; Palanca-Wessels, M. C.; Duvall, C. L.; Pun, S. H., Press, O. W.; Stayton, P. S. Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate. Mol. Pharm. 2012, 9, 3506-3514.
Peng, Y.; Zhang, L. Characterization of a polysaccharide-protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering. J. Biochem. Bioph. Meth. 2003, 56, 243-252.
Slotboom, D. J.; Duurkens, R. H.; Olieman, K.; Erkens, G. B. Static light scattering to characterize membrane proteins in detergent solution. Methods 2008, 46, 73-82.
Instrumentation for Conjugation Analyses
DAWN® - The most sensitive MALS detector available, anywhere. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 – 500 nm.
- Standard option: ambient temperature
- Heated/cooled option: -15°C to +150°C
- High-temperature option: ambient to +210°C
The DAWN offers special options to handle fluorescent samples: fluorescence-blocking filters and an infrared, 785 nm laser.
miniDAWN® - Second only to the DAWN in sensitivity. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 10 MDa and radii from 10 – 50 nm. Ambient only.
microDAWN™ - The first MALS detector for UHPLC, with interdetector dispersion as low as 1.5 µL. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.
Refractive Index Detectors
Optilab® - A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. Temperature controlled from 4°C to 65°C. The high-concentration option accommodates protein concentration up to 180 mg/mL.
microOptilab™ - The first RI detector specifically designed for use with all UHPLC systems.
Field-Flow Fractionation Systems
Asymmetric Flow Field-Flow Fractionation (AF4) and Hollow-Fiber Flow Field-Flow Fractionation (HF5) perform versatile separation of macromolecules and nanoparticles ranging from 1 nm to 10,000 nm in radius. Combined with MALS and DLS instrumentation, FFF separation is the ideal means for obtaining distributions of molar mass and molecular size, in solution.
Eclipse™ DualTec™ - Tip-injection FFF fluidics control system that integrates with your HPLC pump and autosampler to provide all the advantages of FFF without losing SEC capabilities. Supports switching between any two of AF4, HF5 and SEC.
Eclipse™ AF4™ - Center-downstream injection FFF fluidics control system, optimal when use with HF5 or SEC is not important. The AF4 is required for the fritless channel and semi-preparative FFF.
Eclipse Channels - The Eclipse system offers a wide selection of separation channels and membranes to accommodate a variety of analytical and even semi-preparative tasks.
ASTRA® - Our comprehensive software solution for MALS analysis in chromatography, AF4 or batch mode. ASTRA is available in a 21CFR(11) compliant version and offers additional options such as particle analysis and a research database.