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SAXS

Small Angle X-Ray Scattering (SAXS)


Small Angle X-Ray Scattering (SAXS) is a powerful analytical method for determining the dimensional parameters and structure of nanoscale particulates. The method is based on the registration of X-rays scattered by the sample at very low angles with the use of dedicated instruments. SAXS method is applicable to any particulates exhibiting a contrast of electron density with the surrounding media, such as biopolymers in aqueous dispersions, nanoparticles in a solid or liquid matrix or nanopores in a solid matrix.


For the characterisation of biopolymers, we offer the range of analytical methods outlined below. While implemented within the lab scale, our SAXS methodology is largely based on cooperation with biological beamline community, particularly the group at EMBL Hamburg. Some of our projects devoted to new SAXS methodologies are listed at the Research page.


Reconstruction of Higher-Order Structure of Proteins


The atomic-level structure of biopolymers in crystalline state is traditionally determined using monocrystal X-ray diffraction. While this is a powerful and established approach, the drawback is the necessity of a crystallisation step. This can be a very challenging task for some pharmaceutically-important molecules as for example in the case of membrane proteins.

The structural properties of a biopolymer in a biological environment such as its higher-order structure may be investigated by SAXS and often become the subject of independent interest. The reason for this is related with the fact that the actual structure of a biopolymer in solution may differ from the crystalline structure due to the conformational flexibility and tendency to form oligomers or aggregates.


SAXS is recognised as the tool of choice for determinimg the tertiary and quaternary structure of a biopolymer in a close-to-natural aqueous environment. With solution-NMR and cryo-microscopy, together these are the only available methods to visualise the actual higher-order structure of a biopolymer in the native environment.

The ab-initio or model-based reconstruction of low-resolution envelope biopolymer structures by SAXS may be possible with as little as a 0.25 wt% formulation of the biopolymer in the appropriate buffer - as in the example depicted below:



Example 1. Left - Envelope structure of Immunoglobulin IgG1, reconstructed from solution scattering experiments - 0.25% wt. in a buffer (DANNALAB B.V. ® 2011) right - high-resolution crystallographic structure deposited as 1IGY entry in Protein Data Bank (www.pdb.org)


Investigation of Peptides and Flexible Systems


For peptides with the defined 3D structure in dispersion, the methods of ab-initio reconstruction may be useful to access dimensions, oligomeric state and approximate molecular mass - as in example of figure 2:


Example 2. Left - High-resolution HIV GP41 core structure (1AIK entry in Protein Data Bank) shown versus the SAXS reconstructed structure of trimeric HIV fusion inhibitor in phosphate buffer (5mg/ml at 0 hours) shown on the right.
(Published by permission of IWT project team of Johnson&Johnson Pharmaceutical Research & Development, more examples available at this link - (TIDES 2012)).

Together with the envelope structure, the useful structural invariants such as radius of gyration and excluded volume are determined as well.


For flexible system as for example - naturally unfolded proteins - we use an alternative approach (following Bernado and co-authors, 2007) where similar to solution NMR, the actual system is presented as an ensemble of the most probable conformations determined by the criterion of best match with experiment - as in the example below:


Example 3. alpha-Synuclein is a naturally unfolded protein with two domains (1-60, 61-95) connected by a flexible linker and third flexible domain (96-140). In the experiment, the coexisting conformers of alpha-Synuclein in the presence of different concentrations of SDS (simulating binding to membrane) has been investigated by SAXS. As the result, the selected set of conformers (out of a collection of thousands) is found to be representative of the observed scattering data. The identified conformers have been clustered to select few different co-existing clusters or "types". The members of one of the clusters are shown above after 3D alignment. The advent of this "U-shape" cluster within the total ensemble of conformers is suspected to be a precursor for alpha-Synuclein neurotoxicity. (DANNALAB B.V. ® 2011, NanoNextNL project);

Often important information may be obtained from the different types of analysis as for example from the reconstruction of the PDDF function. PDDF analysis yields information about particle shape and dimensions even when the complete ab-initio shape reconstruction is not possible.



Determination of Size and Shape of Nanoparticulates


Small angle scattering allows reconstruction of the size and shape of any particulate, including specific variations of electron density contrast and different dimensional invariants. This method is used to determine the dimensional parameters of core-shells, lammelas and sphere, rod or disc-type particles. It may also be useful for characterisation of the nanoscale morphology of API and excipients, particularly in nanoscale amorphous domains.


Another area of application for this methodology is in the investigation of drug delivery systems based on nanoparticles, micelles, vesicles or liposomes. The analysis of scattering data can provide details about carrier binding with API, the position of API in the system and the size and shape of the drug-loaded carrier.


Size Distribution and Specific Surface


SAXS is a powerful non-destructive method for accessing the distribution of nanoparticles or nanopores by size. Particle-size distribution and specific surface parameters (total surface per unit of volume) are the process parameters influencing processability and reactivity, solubility and bioavailability of the drug substance.

More information is available at Nanoparticles and Drug delivery pages.