Correct higher order structure (HOS) is critical to ensure biopharmaceutical products' proper functionality, activity, and stability. The ICH Q6B guideline for testing procedures and acceptance criteria for biologics states that HOS characterization is a key component in defining critical quality attributes (CQAs) for proteins or peptides. As experts in therapeutic proteins and peptides, CD Formulation uses a variety of analytical techniques, including mass spectrometry, chromatography, spectroscopy, and more, to fully evaluate the higher-order structure of proteins and peptides, paving the way for your next steps.

HOS & Why Perform HOS Characterization?

HOS stands for higher-order structure, which refers to the general term for the secondary, tertiary, and quaternary structures of proteins. It describes the overall structure and distribution of protein subdomains or individual structural components and involves the spatial conformation, topological structure, and combination of functional regions of proteins. HOS is very important for understanding the function and activity of proteins, as well as for designing protein engineering and drug development.

Changes in HOS can affect the quality, stability, safety, and efficacy of protein or peptide biopharmaceutical products and increase the potential for immunogenicity and loss of biological function. Therefore, such analyses are needed to measure and monitor secondary, tertiary, and quaternary structures during early and late characterization and as part of comparability studies. Therefore, such analyses are needed to measure and monitor secondary, tertiary, and quaternary structures during formulation development and process manufacturing, which is also part of comparability studies.

Fig.1 Protein higher-order structures (HOS). (CD Formulation)

Explore Our Higher-Order Structures (HOS) Proteins & Peptides Characterization Services

At CD Formulation, our experienced protein scientists can apply a variety of analytical methods to obtain high-level structural data about proteins and peptides, which can guide subsequent formulation development, process development, stability studies, etc. Our extensive high-level structural characterization services include:

Secondary Structure Characterization

Protein or peptide secondary structure refers to the specific conformation formed by the backbone atoms of the polypeptide chain circling or folding along a certain axis, that is, the spatial arrangement of backbone atoms of the peptide chain, without involving the side chains of amino acid residues. α-helix and β-fold are the main forms of secondary structure.

Characterization Technologies: Circular dichroism (CD) spectroscopy, fourier transform infrared (FTIR) spectroscopy.

Fig.2 Protein secondary structure. (CD Formulation)

Tertiary Structure Characterization

The tertiary structure refers to the relative spatial position of all amino acid residues in a single polypeptide chain, that is, the three-dimensional spatial structure of the entire peptide chain, which usually includes α helix, β sheet, random coil, and loop. The formation and stability of the tertiary structure depend on the hydrophobic bonds, salt bonds, disulfide bonds, hydrogen bonds, etc. between the amino acid side chains.

Fig.3 Protein tertiary structure. (CD Formulation)

Characterization Technologies: X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (cryo-EM), hydrogen-deuterium exchange (HDX)-MS.

Quaternary Structure Characterization

The quaternary structure refers to a more complex protein complex with a complete structure formed by the interaction of multiple polypeptide chains. It mainly describes the spatial arrangement of protein subunits and the connection and interaction between subunits and doesn't involve the internal structure of subunits. The formation and stability of the quaternary structure are similar to the tertiary structure and are mainly formed by hydrophobic interaction, hydrogen bonding, ionic bonding, etc.

Fig.4 Protein quaternary structure. (CD Formulation)

Characterization Technologies: Similar to tertiary structural characterization, including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (cryo-EM), and hydrogen-deuterium exchange (HDX)-MS.

Protein/Peptide Biosimilar HOS Characterization

As part of biosimilarity study, protein or peptide biosimilar HOS data is one of the key information to demonstrate its similarity and equivalence to the reference product. These data include the amino acid sequence, three-dimensional structure, secondary structure, and possible post-translational modifications of the drug. Regulatory agencies require that protein or peptide biosimilar development must provide these data to ensure that the generic drugs they produce are structurally similar to the original reference product and do not have major differences related to efficacy or safety.

Our protein expert team provides protein or peptide biosimilar HOS analysis services, combining multiple methods with expert data interpretation to facilitate the evaluation of comparative analytical characterization data of multiple batches of biosimilars and reference products to assess the variability of quality attributes.

Our Technology Platforms

A comprehensive HOS characterization method panel is an important part of the intact protein or peptide product characterization program. These methods can be used to compare a large number of identical products to ensure batch-to-batch consistency and comparability, as well as establish biosimilarity. Thanks to many years of experience and expertise in protein/peptide characterization, CD Formulation has established several first-class advanced technology platforms. Our pharmaceutical team and protein chemist team often combine multiple protein and peptide characterization strategies, including but not limited to:

Why Choose Us for Higher-Order Structures (HOS) Proteins & Peptides Characterization?

• Our team of scientists and experts have years of experience in the field of higher-order structures (HOS) proteins and peptides characterization, ensuring professional and accurate analysis.
• We offer a comprehensive range of analysis services for HOS proteins and peptides, including structural elucidation, stability studies, and aggregation analysis, allowing for a thorough understanding of your biomolecules.
• Our laboratories are equipped with state-of-the-art instruments, including mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy, etc., ensuring reliable and precise results.
• Our team has decades of experience supporting biopharmaceutical product development using protein and peptide characterization methods.
• Flexible experimental design and testing options can meet any customer's specific needs.

Publication

The ultimate goal of CD Formulation is to provide innovative protein or peptide HOS characterization solutions for the biopharmaceutical industry using advanced analytical techniques. We always work closely with our customers to provide customized solutions based on their needs and project goals, ensuring the accuracy and reliability of the results. Please feel free to contact us if you are interested in our services. We will provide you with the most professional advice and support to ensure the smooth launch and implementation of your project.