During the development of proteins for use as biologics, the primary structure (amino acid sequence) is important in defining protein activity. Due to the complex nature of protein drugs, it is important to characterize the protein’s higher-order structure (HOS) to understand its stability, folding, structure, and functional activity.

Protein structure can be characterized into different levels:

  • Primary Order Structure
    The sequence of amino acids in the polypeptide chain. The primary sequence of a protein defines its structure and function.
  • Secondary Order Structure
    This includes the localized structures within the protein backbone. The most common types of secondary structures are the α helix and the β pleated sheet, held in place by hydrogen bonds.
  • Tertiary Order Structure
    The three-dimensional shape of a protein.
  • Quaternary Order Structure
    This is the structure of a multi-protein complex such as a dimer or trimer.

Secondary, tertiary and quaternary structure is often collectively termed as the higher order structure (HOS) of a protein. HOS is responsible for the correct folding and three-dimensional shape of a biologic drug. This can be affected by different formulations, which in turn can affect protein activity. The folding and shape of the protein impacts directly on the functionality of the protein drug.

Is Higher Order Structure right for my application? 

Incorrect higher order structure can also raise safety issues - if the overall folding and therefore 3D shape of a protein is incorrect, immunogenic epitopes can be exposed and protein aggregation can occur. HOS characterization is a critical component of biologics development and should be performed alongside functional analysis and primary structure characterization to allow a full understanding of the overall protein structure.

HOS is characterized by a variety of our biophysical solutions including:

  • Mass spectrometry (MS)
  • Circular dichroism (CD)
  • Fourier transform infrared spectroscopy (FTIR)
  • Raman spectroscopy
  • X-ray crystallography
  • Nuclear magnetic resonance (NMR)
  • Near-UV CD
  • Size exclusion HPLC, and SEC-MALS
  • Fluorescence
  • Static and dynamic light scattering (SLS and DLS)
  • Differential scanning calorimetry (DSC)
  • Analytical ultracentrifugation (AUC)

With complimentary and orthogonal techniques, HOS data can be used to make decisions as to which drugs to move forward within development, how to formulate the drugs, and/or as quality control and biocomparability studies.

What Higher Order Structure (HOS) solutions do Malvern Panalytical Offer?  

Several instruments in our characterization toolbox are used in HOS characterization of biologics, including: MicroCal PEAQ DSC and PEAQ-DSC Automated systems, the Zetasizer series of light scattering instruments, and OMNISEC for size-exclusion chromatography (SEC) including SEC-MALS.

Système MicroCal PEAQ-DSC

Système automatisé MicroCal PEAQ-DSC


Gamme Zetasizer Advance

Système MicroCal PEAQ-DSC Système automatisé MicroCal PEAQ-DSC OMNISEC Gamme Zetasizer Advance

Analyse de la stabilité des protéines comme référence absolue pour des applications en recherche

Analyse de la stabilité des protéines comme référence absolue pour l’environnement réglementé

Le système GPC/SEC multi-détecteurs le plus avancé au monde

La diffusion de la lumière pour toutes les applications

Plus de détails Plus de détails Plus de détails Plus de détails
Calorimétrie Différentielle à Balayage (DSC)
Chromatographie d'exclusion de taille (SEC)
Chromatographie par perméation de gel
Diffusion dynamique de la lumière
Électrophorèse laser Doppler
Non-Invasive Back-Scatter (NIBS)
Multi-Angle Dynamic Light Scattering (MADLS)
Caractérisation de la stabilité de produits biopharmaceutiques par calorimétrie différentielle à balayage


Knowledge center

Want access to free online webinars, white papers, user manuals and much more?

View resources