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High-Order Structure

The higher-order structure (HOS) of a protein refers to the three-dimensional arrangement of the protein’s secondary, tertiary and quaternary structures. These structures are crucial for the protein's biological function, as they determine how the protein interacts with other molecules, its stability and its ability to trigger the desired therapeutic effect.
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Key levels of protein structure include:

Secondary structure

Localized folding into structures such as alpha-helices and beta-sheets, stabilized by hydrogen bonds

Tertiary structure

The overall three-dimensional shape of a single polypeptide chain, including all its secondary structure elements 

Quaternary structure

The assembly of multiple protein subunits into a functional complex (e.g., antibodies, enzymes)

A protein’s HOS can be sensitive to changes in environmental conditions, such as temperature, pH or ionic strength, which can affect its biological activity and stability. Therefore, understanding and controlling HOS is critical in the production of safe and effective protein-based therapeutics. 

In addition to characterizing the primary structure of protein biologics, thorough analysis of a protein’s HOS is vital for:

  1. Monitoring biological activity: Changes in HOS can lead to altered protein function, reducing its efficacy or causing unwanted immunogenicity. For therapeutic proteins and monoclonal antibodies, the protein's HOS must remain consistent and functional throughout the development and manufacturing process.

  2. Stability and storage: Protein stability is heavily influenced by its HOS. Stability testing at various stages of development, including during storage and transport, closely monitors and identifies if the protein has undergone unwanted conformational changes that could affect its therapeutic properties. 

  3. Comparability and consistency: When producing biologics across different manufacturing sites or in different production batches, maintaining the HOS is critical for meeting regulatory requirements and demonstrating product comparability.

  4. Regulatory compliance: Regulatory agencies like the FDA, EMA, and ICH require a detailed characterization of the protein’s HOS as part of the CMC package for biologics. A robust HOS analysis helps fulfill these regulatory requirements, supporting the approval of the product.

  5. Immunogenicity risk assessment: Alterations in protein structure can potentially expose immunogenic epitopes, increasing the risk of immune responses. Analyzing HOS can identify such risks early in development, allowing for formulation or process adjustments to mitigate immune reactions.

Determination of the HOS is an important part of biopharmaceutical drug development. Assessment of the HOS, using a panel of analytical techniques, forms a critical component of characterization studies, as well as formulation development and comparability studies. This can ensure the consistent manufacture of quality biologics and confirm the optimal formulation buffer is selected for the protein stability and efficacy.

HOS analysis assists with determining the structure-function relationship of protein therapeutics

With Labcorp, you receive a comprehensive HOS characterization package using a range of methods, including:

  • Differential scanning fluorimetry (DSF) 

  • Size exclusion chromatography (SEC) 

  • Size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS)

  • Dynamic light scattering (DLS)

  • Near or far-UV circular dichroism (CD)* 

  • Fluorescence spectroscopy* 

  • Nuclear magnetic resonance (NMR)*

  • Fourier transform infrared spectroscopy (FTIR)*

  • Analytical ultracentrifugation (AUC)* 

  • Cryogenic electron microscopy (CryoEM)* 

*Available via Labcorp university partnerships

Resources

Info Sheet

Setting the foundation of success in your drug development

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