SEC-MALS

Not all macromolecules behave alike. Your samples may have a different conformation than molecular standards or exhibit unexpected column interactions. As an absolute technique for determining molar mass and size in solution, SEC-MALS - the combination of Size Exclusion Chromatography with Multi-Angle Light Scattering – offers an advanced characterization technique and overcomes the many limitations of column calibration.

What is SEC-MALS?

SEC-MALS combines multi-angle light scattering with size-exclusion chromatography. It is a powerful technique for characterizing macromolecules in terms of:

  • molar mass
  • size
  • conformation
  • conjugation ratio.

In SEC-MALS, the SEC column serves to separate molecules by size; retention time is not used to determine molecular weight. After exiting the HPLC or FPLC system, the molecules pass through a MALS detector and are probed by a laser beam.

The MALS signals, along with UV absorbance and/or differential refractive index (dRI) signals, are analyzed to quantify the analyte’s physical properties and distributions thereof. In polymer analysis, a differential viscometer is often added to measure intrinsic viscosity. In protein and nanoparticle analysis, an online dynamic light scattering detector is often added to measure hydrodynamic radius.

Why SEC-MALS?

Assumptions of analytical SEC

Size-exclusion chromatography separate molecules by size (hydrodynamic radius). However, conventional analytical SEC relates molar mass with retention time by assuming that the analyte elutes in accord with a column calibration curve. For column calibration to work correctly, these assumptions must all be true:

  • Same molecular conformation
    • protein samples are perfectly globular, like the globular protein standards
    • polymer samples are perfectly linear random coils, like the linear polymer standards
  • Same density / specific volume
  • Same column interactions
    • Electrostatic charges of samples and standards are equal
    • Hydrophobicity of samples and standards are equal

In addition, SEC columns age over time, requiring frequent re-calibration.

SEC-MALS is absolute

SEC-MALS provides a first-principles analysis that is independent of the assumptions inherent in the column calibration curve.

  • Basic physical equations connect the molar mass with scattered intensity (measured by MALS) and concentration (measured by UV or dRI)
  • The analysis does not depend on molecular shape/conformation and is not impacted by non-ideal column interactions.
  • The molar mass, and (if the molecule is large enough its rms radius as well), are measured at each elution volume, typically once per second.

SEC-MALS determines molecular weight from 200 g/mol to 1 billion g/mol. It can also determine molecular size – the rms radius, or radius of gyration Rg – from 10 nm to 500 nm and beyond. By combining molar mass and size, it can also assess molecular conformation. And MALS detectors only require annual calibration.

Who uses SEC-MALS?

Molar mass, size and conformation are essential physical properties of macromolecules. Scientists who will not settle for ‘an answer’, but require ‘the answer’ to their characterization challenges, look to Wyatt MALS instrumentation and software for reliability and confidence.

In academic, government and other labs carrying out fundamental research, SEC-MALS is used extensively in basic protein and polymer characterization.

In the biopharmaceutical and advanced materials industries, SEC-MALS is used across R&D for evaluation of solution properties, stability testing, process development, regulatory filing and quality control.

A basic SEC-MALS set-up is comprised of a standard HPLC or FPLC system, SEC column, a concentration detector such as UV or RI depending on the application, a Multi-Angle Light Scattering instrument and a computer with ASTRA software for data acquisition and analysis.


How does SEC-MALS work?

In SEC-MALS, the function of the SEC column is to separate solution components by hydrodynamic size (due to column interactions, some components may elute in the opposite order, and components of the same size may elute at different times).

As each component passes though the detectors, its concentration and light scattering properties are measured every second or so. In UHP-SEC-MALS, the peaks are much narrower, so measurements are performed roughly 10 times per second.

The MALS detector incorporates between 3 and 18 photodiodes positioned at different angles θ relative to the laser beam in order to measure the scattered light. At each data point:

  1. the plot of scattered intensity vs. angle is fit to determine R(0) (the y-intersect at angle θ = 0) and the slope.
  2. molar mass, M, is calculated from the ratio of R(0) and the concentration
  3. radius of gyration, Rg, is calculated from the slope.

Most of the constants used to determine M and Rg are related to system optical properties such as laser wavelength and solvent (mobile phase) refractive index. In addition, the analyte’s dn/dc value (specific refractive index increment) in the mobile phase must be known or measured (this is easier than it sounds!). For more details on the theory of MALS, please see our MALS Theory page.

What constitutes a complete SEC-MALS system?

Basic

For basic SEC-MALS, all you need are a standard HPLC or FPLC system including concentration detector (UV for proteins, dRI for polymers), SEC column, a MALS instrument and a computer with ASTRA software for data acquisition and analysis.

  • HP/FPLC-SEC-MALS uses a DAWN or miniDAWN MALS instrument; UHP-SEC-MALS requires a microDAWN.
    • DAWN has 18 angular detectors; it covers 200 - 109 g/mol and 10 - 500 nm with maximum sensitivity.
    • miniDAWN and microDAWN have 3 angular detectors; they cover 200 - 107 g/mol (106 g/mol for linear polymers), and 10 - 50 nm.
  • MALS is plumbed downstream of the UV detector or upstream of the dRI detector
  • Two wired connections are made from the LC system to the MALS instrument: analog output from the concentration detector, and auto-inject for synchronizing ASTRA collection with the sample injection.

Better

Most SEC-MALS users find it beneficial to include an Optilab dRI detector, or microOptilab for UHPLC.

  • Protein analysis - including an Optilab is very useful since nearly all proteins have the same dRI response (dn/dc) - it is not necessary to know the extinction coefficient of each peak.
  • Bioconjugates - MALS, UV and dRI are combined for analysis of glycoproteins, AAVs, protein/DNA complexes, membrane proteins embedded in surfactants or lipids, and other dual-component biomacromolecules. The same analysis is suitable for many copolymers.
  • Polymer analysis - replacing the HPLC’s dRI detector with an Optilab affords many benefits including direct digital data acquisition, matched MALS and RI wavelengths for more accurate molar masses, and concentration range up to ~ 20 mg/mL for convenient and accurate dn/dc measurements.

Full control

ASTRA software can directly control select HPLC instruments, eliminating the need to synchronize the ASTRA method with native HPLC software. Please contact info@wyatt.com for the current list of supported HPLC and UHPLC components.

Extended

Additional Wyatt products enable extended characterization capabilities:

  • Proteins and nanoparticles – most proteins and some nanoparticles fall below the lower size limit for Rg (this does not prevent molar mass determination). A WyattQELS dynamic light scattering module is embedded in the MALS detector to determine hydrodynamic radii down to 0.5 nm, which is sufficient for most proteins and peptides. Alternatively, a NanoStar or Mobius may be connected to the MALS flow cell by optical fiber to serve as the online DLS detector.
  • Polymers – many polymers are also too small for Rg analysis, even though MALS has no difficulty in determining their molar mass. For analysis of size, conformation and branching of such small molecules, a ViscoStar differential viscometer is added (microViscoStar for UHPLC). SEC-MALS-IV determines hydrodynamic radius for conformation analysis and Mark-Houwink-Sakurada parameters.
  • Difficult polymers - ViscoStar may be used without MALS, in a SEC-IV configuration, for analysis of polymers that are not amenable to light scattering such as highly fluorescent lignins, materials that absorb strongly at the MALS wavelength, or polymer-solvent systems with no scattering (dn/dc = 0).

The four key application areas of SEC-MALS are biotherapeutics proteins biopolymers and synthetic polymers. Below you will find a select set of application notes and on-demand webinars for each area. The searchable Bibliography, containing thousands of references to peer-reviewed articles citing Wyatt instruments, is an excellent resource for researching specific SEC-MALS applications.

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

Dan to provide content

If you already analyze your samples by analytical SEC, you have probably overcome the most difficult part of SEC-MALS: finding the right column, mobile phase and other conditions that assure good separation and recovery. Now you are ready to add MALS for absolute characterization and deeper understanding.

How do I use SEC-MALS?

Running a SEC-MALS measurement is quite similar to standard SEC. It involves

  1. Sample prep (minimal)
  2. Column equilibration
  3. Loading the sample(s) into an injection loop or autosampler
  4. Injecting the sample and collecting data until the included column volume is complete
  5. Analyzing the data

If you have an autosampler, sequences may be set up for dozens of automated injections that can proceed throughout the workday and overnight.

The are two main differences in workflow relative to standard SEC: the use of ASTRA for data analysis, and the extra precautions taken to remove particulates from the sample and mobile phase for high-quality light scattering measurements.

Sample and system preparation

Light scattering is especially sensitive to particulates, which are usually the main source of noise in the MALS signal but are entirely absent from UV and dRI signals. Eliminating particles from sample and solvents is not difficult, but it does require some diligence and adaptation relative to standard HPLC/FPLC protocols.

  • Samples should be filtered to the smallest pore size that does not remove the analyte of interest. For proteins and small polymers, that often means using 0.02 µm syringe-tip filters.
  • Mobile phase should be filtered to 0.1 µm, though acceptable results might be obtained with the more common 0.22 µm filters. In addition, a 0.1 µm filter is added to the system between the pump and injection loop. In UHP-SEC-MALS the level of cleanliness required for UHPLC systems is sufficient for good MALS results and additional filtration is not needed.

Is my system clean?

After flushing and equilibrating the SEC column, it may not be obvious if the system is sufficiently particle-free for high-quality MALS measurements. The System Health Monitor on the front panels of the DAWN, miniDAWN and microDAWN let you know if the noise level is below the recommended maximum for acceptable data.

ASTRA

ASTRA software is built in many ways like typical chromatography software. However, since it acquires and analyzes MALS data rather than retention time, there are important differences in the procedures and calculations.

  • Calibration – the response of the MALS detector is usually calibrated annually, using only pure toluene. The Optilab and ViscoStar require no calibration.
  • Band broadening correction and normalization – these procedures determine certain system parameters that are required for multi-detector SEC and light scattering. The parameters are determined simultaneously by running a known monodisperse sample with Rg < 10 nm, preferably with a known Rg value; the molar mass and concentration are not needed. These procedures are typically run every month or two if the mobile phase is maintained (most aqueous solvents can be considered the same for this purpose).
  • Bridge balancing – the ViscoStar’s capillary bridge is balanced using mobile phase alone. Balancing is fully automated and takes only a few minutes; typically it is done daily or weekly. In daily operation, all that remains is to set baselines, select peaks and report the results. Baselines and peak selection can even be automated by ASTRA, relieving users of this effort when multiple files are to be analyzed. ASTRA will also gather key user-specified results from multiple runs in tables and graphs for convenient reporting.

The final report may be customized to meet different needs. For those in GMP/GLP-regulated environments, ASTRA’s Security Pack add-on enables 21CFR(11) compliance including full audit trails and multiple signatures.

ASTRA offers many advanced analyses that are somewhat more involved than the basic analysis outlined above. Wyatt Technology offers several avenues for supporting novice and advanced users, including technical notes in the online Wyatt Support Center, phone support and Light Scattering University. Site visits may be arranged for IQ/OQ, service and preventive maintenance as well as group training.

Wyatt offers the most advanced instruments for SEC-MALS and its variants, SEC-MALS-DLS and SEC-MALS-IV. Here you can find basic descriptions of these products and links to pages with detailed descriptions and specifications.

MALS

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.

dRI

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

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.

IV

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.

Delve deeper to learn how SEC-MALS works and performs comprehensive macromolecular characterization. Explore these resources, all available on the Wyatt web site.

Theory

Please see the following pages for details of SEC-MALS theory:

These pages provide additional information on specific analyses performed in SEC-MALS:

Bibliography

An extensive searchable bibliography of publications citing Wyatt SEC-MALS instruments is available at www.wyatt.com/Bibliography. Just open the Advanced Search and click the Multi-Angle Light Scattering box, then enter your search terms below.

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