Size

Coupling a MALS detector to a fractionation method such as size exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS) is the most common method for MALS characterization of size distributions of homogeneous or heterogeneous samples. MALS analysis provides the rms radius R_g of the analyte at each elution volume, independently of retention time. Since the detection volume of the MALS flow cell is much smaller than a typical monomeric peak eluting from SEC or FFF, MALS provides accurate size distributions limited in resolution only by the separation technique.

Applications

SEC-MALS and FFF-MALS determine the size distributions of:

• Biopolymers such as polysaccharides
• PEGylated proteins and large protein complexes such as virus-like particles
• Synthetic polymers
• Liposomes or exosomes
• Large protein aggregates

An unfractionated, or 'batch' measurement, provides the Z-average radius R_g,z of the entire analyte content in solution. Batch MALS for size measurements is carried out by injecting the sample directly into the DAWN® or miniDAWN® flow cell. Historically, batch measurements have been laborious and prone to operator error; the Calypso® automates batch MALS for accurate, reliable and rapid results.

Applications

Batch MALS is often used to analyze:

• Very large, unstructured polymers that are not amenable to fractionation
• Large protein assemblies that dissociate under the dilution and shear inherent to fractionation
• Polymers that form gels or viscous solutions that may impede flow paths

DLS may be measured on-line in SEC or FFF by integrating a WyattQELS™ module into a DAWN, miniDAWN or microDAWN MALS detector, or by transferring light collected in the MALS detector via optical fiber to a batch DLS detector such as the DynaPro® NanoStar® or Mobius®. Depending on the collection angle, laser wavelength and flow rate, online DLS can analyze R_h from 1 nm up to ~250 nm.

Applications

• Quantum dots and metallic nanoparticles not amenable to MALS analysis because of size or anomalous refractive index
• Conformation of proteins and protein aggregates
• In conjunction with MALS size determination (R_g), conformations of biopolymers and other heterogeneous macromolecules, for example discriminating between filled and unfilled liposome drug delivery vehicles

In batch mode (no flow), DLS can measures particles as small as 0.2 nm and as large as 5000 nm in radius. Another valuable feature of batch mode DLS is its ability to extract a coarse size distribution, without any fractionation, by means of regularization analysis. While distributions calculated from batch DLS can only clearly separate populations which differ by about 3x-5x in radius, aggregates and other forms of heterogeneity may be assessed via the peak width, also known as polydispersity.

• Cumulants analysis estimates the polydispersity of a 'monomodal' distribution of particle sizes
• Through NNLS regularization analysis, DLS discriminates between populations of particles that differ in size by at least ~3-5x

Despite the low size resolution, DLS is quite sensitive to shifts in average sizes. The ability to sense size shifts as low as 1-2% is utilized in analyzing protein unfolding at the thermal melting transition, protein-protein interactions assessed via concentration or composition dependence of size, and even binding of lipids or oligonucleotides to nanoparticles.

The DynaPro NanoStar offers batch DLS in high quality quartz microcuvettes or disposable plastic cuvettes, while the DAWN and miniDAWN offer batch DLS in the flow cell ('microbatch') or in a quartz 10 µL microcuvette using the microCuvette Adapter Kit.

Applications

• Quick estimates of average size and polydispersity of organic and inorganic nanoparticles
• Rapid quality control of protein expression and purification, testing for large aggregates
• Simple, semi-quantitative assessment of oligomerization or aggregation states
• Protein melting and protein-protein interaction studies

While less sensitive than MALS to low sample concentrations, DLS is also less prone to interference by stray light and so may be carried out under less stringent optical conditions. The DynaPro® Plate Reader takes advantage of this particular capability to carry out automated DLS measurements directly in situ in industry-standard microwell plates, measuring hundreds or even thousands of samples and sample conditions per day.

Applications

• High-throughput screening of optimal formulation conditions for proteins and biotherapeutic complexes such as virus-like particles, based on criteria that include aggregation and the thermally induced onset temperature of aggregation
  
• Determination of protein melting temperature as a function of formulation conditions (excipients, pH, etc.)
  
• Characterization of non-specific protein-protein interactions via the diffusion interaction parameter k_D, evaluated from the concentration dependence of the diffusion coefficient

When polymers are too small to determine size distributions directly by online MALS, the addition of a ViscoStar® or microViscoStar™ to the SEC instrument stack offers an alternative solution. Hydrodynamic volume is directly related to the product of molar mass and intrinsic viscosity via the Einstein-Simha relation V_h=M[η]/(2.5N_A), and hydrodynamic radius may be calculated from the hydrodynamic volume.

Since both intrinsic viscosity and molar mass are determined without assumptions, the SEC-MALS-IV analysis provides an objective estimate of molecular size calculated independently of column interactions.

In addition to standard analytical applications of MALS, it may be used to develop, monitor and control manufacturing processes, directly in the pilot plant or on the manufacturing floor. The ultraDAWN™ along with OBSERVER™ software determine and report the z-average rms radius (R_g) of protein or polymer solutions passing through the instrument in real time; this is known as RT-MALS.

 pH Effects on Stability and Homogeneity of Protein Complex

 Thermal Stability as a Function of pH and Concentration

 Protein Aggregates Analyzed by the DynaPro Plate Reader

 Non-Specific Small Molecule Aggregation

 In Situ Analysis of Inorganic and Organometallic Clusters

 MORE APPLICATIONS

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Aichmayer, B.; Widermann-Bidlack, F. B.; Gilow, C.; Simmer, J. P.; Yamakoshi, Y.; Emmerling, F.; Margolis, H. C.; Fratzl, P. Amelogenin nanoparticles in suspension: deviations from spherical shape and pH-dependent aggregation. Biomacromolecules  2010, 11, 369-376.

Striegel, A. M. Architecture on the mechanochemical degradation of macromolecules. J. Biochem. Bioph. Meth.  2003, 56, 117-139.

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.

Wyatt, P. Submicrometer particle sizing by multiangle light scattering following fractionation. J. Colloid Interf. Sci.  1998, 197, 9-20.

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