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The solution-based analysis of macromolecular conjugates is vastly simplified by light scattering.

Conjugation

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

Conjugation

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.

Selected references

  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.

 BIBLIOGRAPHIC SEARCH

Application notes

Instrumentation for Conjugation Analyses

  • MALS Detectors

    dawn-heleos-II

    DAWN HELEOS II - 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.

    • Heated/cooled option -15 °C to +150 °C
    • High-temperature option to +210 °C

     

    miniDAWN TREOS - Second only to the DAWN in sensitivity. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.

    µDAWN - The first MALS detector for UHPLC, with interdetector dispersion as low as 2 µL, the µDAWN is also optimal for hollow fiber FFF. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.

  • Field-Flow Fractionation Systems

    eclipse-dualtec

    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 > 1000 nm in radius. Combines with MALS and DLS instrumentation, FFF separation is the ideal means for obtaining distributions of molar mass and molecular size, in solution.

    Eclipse DualTec - The complete 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 - The basic FFF fluidics control system, optimal when switching automatically between SEC and AF4 is not important. The AF4 is required for the fritless channel, and optimal for semi-preparative FFF.

  • Refractive Index Detectors

    optilab-t-rex, instrument

    Optilab T-rEX - A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. The high-concentration Optilab accommodates protein concentration up to 180 mg/mL.

    Optilab UT-rEX - The first RI detector specifically designed for use with UHPLC systems. Temperature controlled from 4°C to 50°C, and is compatible with all UHPLC systems.

  • Accessories

    Orbit, accessory

    Orbit - In a CG-MALS system, the Orbit is used to automatically select wash solvents in the post-experiment cleaning cycle.

    COMET - An ultrasonic transducer that cleans MALS flow cells automatically by agitating the fluid and loosening particles from the glass surfaces. This module may be integrated into a DAWN, miniDAWN or µDAWN.

    RI Calibration Kit - Pre-made RI calibration standards are available to speed up RI calibration. Can be used to calibrate any Optilab or third-party differential RI instrument.

    Solaris - Prevents growth of bacteria, fungi or algae in solvents with high efficiency and protects your system from down-time due to contamination.

  • Software

    ASTRA 6, software

    ASTRA - Our comprehensive software solution for MALS analysis in chromatography, AF4 or batch mode, included with each MALS, RI and viscosity detector. ASTRA is available in a 21CFR(11) compliant version and offers additional options such as particle analysis and a research database.

   

For essential macromolecular and nanoparticle characterization—The Solution is Light™

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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