SEC-MALS

Absolute macromolecular characterization

No more assumptions

Not all macromolecules behave alike. Your samples may have a different conformation than molecular standards or exhibit non-ideal column interactions; relevant reference molecules may not even exist. As an absolute technique for determining molar mass (MW) and rms radius (Rg) in solution, SEC-MALS—the combination of size exclusion chromatography for separation with multi-angle light scattering for analysis—eliminates such assumptions and overcomes the limitations of standard analytical SEC/GPC/GFC.


SEC-MALS analysis does not depend on the column calibration curves obtained with reference molecules. It can directly indicate if a peak is homogeneous, as for the proteins above, or heterogeneous, as for the polymers below. Even if macromolecules with different MW elute at the same time (due to different conformation or column interactions), MALS provides correct MW.

Beyond MW and size

What’s more, SEC-MALS is the solution to characterizing complex macromolecules for which there are no standards. It goes beyond MW and size to characterize conjugation, conformation, branching and the absolute stoichiometry of macromolecular complexes, from peptides to proteins, nucleic acids to VLPs, and small oligomers to broadly heterogeneous polymers.

More productivity

With SEC-MALS, you no longer have to run daily column calibrations or worry about finding standards that match your analyte and mobile phase. Precise elution time, which can change as columns age, temperature varies or mobile phase is replaced, is inconsequential to MALS.

Versatile configurations

Found in biotherapeutic, protein, biopolymer and synthetic polymer characterization labs around the world, Wyatt’s DAWN® and miniDAWN® MALS instruments are valued for reliable and robust measurements. SEC-MALS systems—based on any industry-standard HPLC, GPC or FPLC pump, autosampler and detector modules—usually incorporate, in addition to MALS, an Optilab differential refractive index (dRI) detector as a universal concentration detector. For polymer analysis, a ViscoStar® differential viscometer, both from Wyatt Technology is also included. These instruments are controlled, and the data acquired and analyzed, by our powerful ASTRA® software. Need more than SEC? In many cases, the instruments can also be used with gradient chromatography such as ion-exchange or reverse-phase to determine the molar mass of eluting peaks.


A complete SEC-MALS system includes a standard HPLC stack connected to a DAWN or miniDAWN MALS detector, an Optilab differential refractive index detector, a ViscoStar differential viscometer (for polymers) and ASTRA software. A WyattQELS™ online dynamic light scattering module may be embedded in any Wyatt MALS detector.

More options, more characterization

A WyattQELS™ on-line dynamic light scattering is commonly integrated with the MALS instrument for orthogonal size determination of proteins and nanoparticles. Wyatt also offers MALS-guaranteed SEC columns for optimal analytical separations of proteins. Reductions in sample and solvent consumption, with faster runs and improved resolution, are possible with UHPLC and Wyatt’s microDAWN™, microOptilab™ and microViscoStar™ instruments.


miniDAWN and Optilab are often combined with popular FPLC systems in protein research labs that are more familiar with FPLC than with HPLC.

SEC-MALS Instruments

Adding MALS and other Wyatt detectors to HPLC or FPLC

Wyatt online detectors can be added downstream of an existing HPLC or FPLC with little effort, or combined with a new system. ASTRA software utilizes analog data from 3rd-party online UV and/or dRI modules.

Wyatt’s HPLC Service can control most Agilent HPLC modules (degasser, pump, autosampler and UV detector) directly from a desktop application, as well as from ASTRA sequences. If direct control is not available, ASTRA uses a contact-closure auto-inject signal to synchronize data acquisition. Please contact your Wyatt sales representative to learn how we can provide you with a complete SEC-MALS system.

Configurations for UHPLC

Wyatt’s microDAWN, microOptilab and microViscoStar are compatible with all major UHPLC platforms. As with HPLC, Wyatt software can directly control most Agilent UHPLC modules. Please contact your Wyatt sales representative for details.

SEC-MALS Instruments

MALS Detectors

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. Learn more.


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. Learn more.

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. Learn more.

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. Learn more.

microOptilab™ - The first RI detector specifically designed for use with all UHPLC systems. Learn more.

optilab t-rex, sec-mals, size exclusion chromatography, protein aggregation, polymer analysis, polymer molecular weight

Viscometers

ViscoStar® - A highly sensitive, on-line differential viscometer used in conjunction with SEC-MALS to determine the size and conformation of all types of biopolymers, synthetic polymers and even proteins and peptides.

The ViscoStar incorporates multiple novel technologies to provide the highest sensitivity, stability and solvent compatibility of any available viscometer for GPC. Its ease-of-use and serviceability make it the perfect companion for Wyatt's DAWN® light scattering and Optilab® refractive index detectors. Temperature controlled from 4°C to 70°C. Learn more.

microViscoStar™ - Similar to the ViscoStar but designed specifically for use with UHPLC/APC. Learn more.

Online DLS

WyattQELS - dynamic light scattering module that can be embedded in any Wyatt MALS instrument for on-line DLS.

DynaPro® NanoStar® - stand-alone dynamic light scattering detector, may be connected via optical fiber to the flow cell of any Wyatt MALS instrument for on-line DLS.

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Biotherapeutics

Target, candidate and reagent proteins

SEC-MALS characterizes proteins and other biomolecules for solution properties, including molar mass of the molecule and its soluble aggregates, regardless of conformation or non-ideal column interactions. This is beneficial in quickly identifying optimal purification conditions and can be a key indicator of the viability of reagent proteins used in various assays.

The importance of SEC-MALS for quality control of ELISA reagents is investigated in AN1606: Assessing ligand-binding assay reagent proteins.

Aggregation, characterization and quality attributes

SEC-MALS is the preferred method for characterizing soluble aggregates of biopharmaceuticals in regulatory filings. It is well-established in comparing lots from different processes as well as comparing biosimilars to originator biologics.

To learn more about the basics of SEC-MALS analysis of proteins, download WP1615: SEC-MALS for absolute biophysical characterization.

Gene therapy: RNA, VLPs and AAVs

Small viruses such as AAVs, as well as virus-like particles and nucleic acids that are amenable to separation by SEC, may be characterized by SEC-MALS for critical quality attributes such as molar mass, aggregation, physical titer and ratio of nucleic acid-bearing to empty capsids.

View AN1617: AAV critical quality attribute analysis by SEC-MALS and AN1616: SEC-MALS methods for characterizing mRNA for essential SEC-MALS applications in small viral vectors.

Oligomerization

Some biologics, such as insulin, are required to form well-defined oligomeric states for optimal stability and efficacy. SEC-MALS unambiguously identifies the oligomers that are present in different formulations.

SEC-MALS analysis of insulin oligomerization in different formulations is explored in AN1605: Identification of insulin oligomeric states.

Conjugation

Post-translational modifications such as glycosylation or PEGylation are often applied to proteins or peptides in order to improve stability and increase physiological retention time. Protein-polysaccharide vaccines are another common type of conjugated biotherapeutic. Triple-detection SEC-MALS combines UV, MALS and RI instruments to determine the degree of glycosylation or PEGylation, or the molecular weight distribution of conjugated polysaccharides and the number of proteins per molecule. In some instances SEC-MALS may be used to calculate the drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs).

See AN1612: Protein PEGylation processes characterized by SEC-MALS to read how this technique is used in developing PEGylation processes.

Therapeutic biopolymers

Heparin, hyaluronic acid and other polymers derived from animals can be highly variable and must be characterized carefully by SEC-MALS to establish quality metrics related to molar mass distributions and conformation.

AN6301: Discriminating heparin from chondroitin sulfate by charge:mass ratio explains how SEC-MALS is combined with charge analysis to ensure product identity and quality.

Proteins & Biomacromolecules

Protein identification and quality

Molar mass is the key to identifying proteins, their oligomers or complexes, yet all too many researchers rely on potentially invalid analysis of molecular weight by native PAGE or traditional size exclusion chromatography (SEC). These techniques invoke assumptions of conformation and ideal matrix interactions that may lead researchers to fundamentally inaccurate interpretation of their data for scientific publications. SEC-MALS constitutes a rigorous, first-principles analysis of molar mass that does not rely on retention time or calibration with reference molecules. The only function of the SEC column is to separate molecules by size, while MALS determines molar mass of eluting proteins independently.

To learn more about the basics of SEC-MALS analysis of proteins, download WP1615: SEC-MALS for absolute biophysical characterization.

Membrane proteins and glycoproteins

Membrane proteins solubilized with detergent are particularly difficult to analyze by traditional techniques or even by mass spectroscopy because of the surfactant micelle surrounding the protein. Denaturing SDS-PAGE dissociates native oligomers and precludes their identification, while cross-linked mass spectroscopy can create oligomers that do not exist in solution.

Heavily-glycosylated proteins cannot be represented by reference standards or common models for globular proteins, and so are not amenable to analysis by traditional techniques.

These challenges are met by SEC-MALS which can distinguish between a protein and its associated detergent or carbohydrate by combining data from three detectors: UV, MALS and dRI. ASTRA's Conjugate Analysis algorithm calculates the molar masses of both the proteinaceous component and the conjugated or micellar component. The true oligomeric or complexated state of the protein, as well as the degree of glycosylation, are determined unambiguously.

View AN1617: AAV critical quality attribute analysis by SEC-MALS and AN1616: SEC-MALS methods for characterizing mRNA for essential SEC-MALS applications in small viral vectors.

Purification & aggregates

Scientists carrying out detailed mechanistic studies of proteins and their biological function can't afford to work with poor quality material. SEC-MALS is a fundamental means of assessing the quality and purity of protein samples. It performs true separations with absolute molar mass measurements in order to understand just which proteins and degradants are present in solution.

SEC-MALS analysis of insulin oligomerization in different formulations is explored in AN1605: Identification of insulin oligomeric states.

Oligomerization and complex formation

Most biological oligomers are in a dynamic equilibrium with the monomer. The degree of oligomerization depends on the concentration as well as buffer pH and ionic strength, therefore proper identification of a native oligomer must be performed wholly in solution.

SEC-MALS provides an initial diagnosis of oligomerization when a protein's molar mass differs significantly from monomer sequence weight, or the mass varies over the eluting peak according to concentration. Verification may be obtained by a few additional SEC-MALS measurements consisting of different starting concentrations.

View these webinars for an in-depth understanding of how SEC-MALS analyzes protein oligomers and complexes:

Understanding Absolute Stoichiometry of Oligomeric Protein Complexes Using SEC-MALS

Applications of Analytical Light Scattering in a Biophysics Core Facility

Characterizing Protein-Nucleic Acid Interactions by Light Scattering

Applications of Light Scattering to HIV Integrase Structural Biology and Drug Discovery

Absolute Characterization of Glycoproteins and their Interactions with Proteins and Antibodies by Light Scattering

Biopolymers

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Synthetic Polymers

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Application Notes

Click here to view our SEC-MALS Application notes.

Webinars

Click here to view our SEC-MALS webinars.

Selected References

Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol. Biol.  2006, 328, 97-112.

Laurén, J.; Gimbel, D. A.; Nygaard, H. B.; Gilbert, J. W.; Strittmatter, S. M. Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers. Nature  2008, 457, 1128-1132.

Ren, X.; Kloer, D. P.; Kim, Y. C.; Ghirlando, R.; Saidi, L. F.; Hummer, G.; Hurley, J. H. Hybrid structural model of the complete human ESCRT-0 complex. Structure  2009, 17, 406-416.

Tarazona, M. P; Saiz, E. Combination of SEC/MALS experimental procedures and theoretical analysis for studying the solution properties of macromolecules. J. Biochem. Bioph. Meth.  2003, 56, 95-116.

Zhang, M.; Zhang, L.; Cheung, P. C. K.; Dong, J. Fractionation and characterization of a polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography. J. Biochem. Bioph. Meth.  2003, 56, 281-289.

BIBLIOGRAPHIC SEARCH

The basics of MALS

When a light beam is incident on a solution of macromolecules, a portion of that beam is scattered in all directions. The quantity of light scattered into any given direction, relative to the incident intensity, is related to the molar mass, size, and concentration of the molecules. Therefore by placing photodetectors around the sample, measuring the scattered light and analyzing the results, it is possible to learn about those properties.

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