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.

MicroCal PEAQ-DSC

MicroCal PEAQ-DSC Automated

OMNISEC

Zetasizer Advance Range

MicroCal PEAQ-DSC MicroCal PEAQ-DSC Automated OMNISEC Zetasizer Advance Range

Gold standard protein stability analysis for research applications

Gold standard protein stability analysis for the regulated environment

The world’s most advanced multi-detector GPC/SEC system

Light Scattering for every application

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Technology
Differential Scanning Calorimetry (DSC)
Size Exclusion Chromatography (SEC)
Gel Permeation Chromatography
Dynamic Light Scattering
Electrophoretic Light Scattering
Non-Invasive Back-Scatter (NIBS)
Multi-Angle Dynamic Light Scattering (MADLS)
Characterization of biopharmaceutical stability with Differential Scanning Calorimetry

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