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Biophysical Characterization

The WuXi Biologics Forensic Analysis & Biophysical Characterization Center of Excellence (CoE) provides high quality particle identification and biophysical characterization to support our clients in pursuit of superior product quality and safety. Leveraging world-class equipment, the Biophysical Characterization group performs analytical methods for biophysical analysis of proteins including higher-order structural characterization, monomer/aggregates species determination, free-sulfhydryl content determination, amino acid analysis (AAA), extinction coefficient determination and thermal stability characterization. Below are additional details on the methods and their intended purpose.

  • APPLICATION: Characterization of protein secondary and tertiary structures.

 

  • DESCRIPTION: Circular dichroism (CD) measures the differential absorption of left- and right-handed circularly polarized light in an optically active substance. Far-UV CD (190-250 nm) can reveal the protein secondary structure characteristics. Near-UV CD (250-350 nm) provides information on the protein tertiary structures. By comparing the CD spectra of two protein samples, the similarity between the samples can be evaluated.

  • APPLICATION: Characterization of protein secondary structures.

 

  • DESCRIPTION: Fourier Transform Infrared (FTIR) spectroscopy can be used to obtain information about the vibrational states of molecules. The major IR bands for proteins and polypeptides include the amide I (C=O stretching) and amide II (C-N stretching) vibrations. Different secondary structures (α-helix and β-sheet) exhibit characteristic frequencies and intensities in the amide I band region due to the differences in the hydrogen bonds in these structures. FTIR methods can provide the percentage of secondary structures more accurately and easily.

  • APPLICATION: Molecular weight determination of protein monomers and aggregates.

 

  • DESCRIPTION: Size exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS) detector can separate proteins based on size and then measure the molecular weight of the separated monomer and aggregates via MALS detector. This method is able to detect molecular weight across a wide size range and with high sensitivity.

  • APPLICATION: Determination of protein molecular weight and aggregation states in native solution.

 

  • DESCRIPTION: Analytical ultracentrifugation (AUC) is a technique for the characterization of protein homogeneity, determination of aggregates and protein-protein interactions. Sedimentation coefficient is the key parameter for the aggregation analysis from both sedimentation velocity (SV) and sedimentation equilibrium (SE) methods. Multiple detection methods may be used in this technology including absorbance and interference.

  • APPLICATION: Determination of concentration of amino acids in cell culture medium or from degraded protein/peptide via hydrolysis.

 

  • DESCRIPTION: Waters AccQ-Tag Pre-column derivatization method is used for amino acid concentration determination with high accuracy and sensitivity (pmol level).


  • APPLICATION: Determination of protein/polypeptide extinction coefficient (EC).

 

  • DESCRIPTION:

 

    • Amino acid analysis method: Protein absorbance can be measured by ultraviolet spectrophotometry at 280 nm, and protein concentration (C) can be determined by quantitative Amino Acid (AA) analysis. The extinction coefficient (ɛ) of a protein can be determined based on Beer-Lambert’s Law.

 

    • Two detectors method： The extinction coefficient (ɛ) of a protein can be determined by obtaining data using an SEC-MALS system with both an ultraviolet (UV) detector and a refractive index (RI) detector. The coefficient determination is based on Beer-Lambert’s Law


  • APPLICATION: Determination of free sulfhydryl content in protein samples.

 

  • DESCRIPTION: Disulfide bonds play a key role in protein structural stability. Mismatched disulfide bonds and improper folding of protein may occur due to  free SH groups in the proteins. Free sulfhydryl content may be measured using a commercial kit (Measure-iT ™ Thiol Assay Kit).
  • APPLICATION: Determination of particle size, polydispersity index (PDI) and Zeta-potential of proteins.

 

  • DESCRIPTION: Dynamic light scattering (DLS) measures particle size by illuminating the particles with a laser and analyzing the intensity fluctuations in the scattered light. We use the Zetasizer Nano instrument to provide DLS analysis and provide information on particle size, PDI and Zeta potential.

 

WuXi Biologics Particle Size Distribution Analysis

  • APPLICATION: Characterization of protein thermal stability.

 

  • DESCRIPTION: DSC is a thermal analysis technique that measures the excess heat capacity of a sample relative to a reference sample during a thermal transition (temperature change). Thus, sample thermal stability can be assessed and characterized.

WuXi Biologics Characterization of protein thermal stability.

Contact us to learn more about our Biophysical Characterization services and our other Centers of Excellence.