Quadrupole Dalton-Based Controlled Proteolysis Method for Characterization of Higher Order Protein Structure

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Higher-order structure (HOS) of proteins is crucial for biological drug efficacy and stability.
• Current HOS characterization methods have limitations in sensitivity, specificity, and accessibility.
• Regulatory agencies require thorough HOS characterization during drug development.

Purpose of the Study:

• To develop a novel, sensitive method for detecting HOS changes in proteins.
• To establish a controlled proteolysis-based approach for HOS analysis.
• To support biological drug development, formulation, and stability testing.

Main Methods:

• A novel controlled proteolysis-based liquid chromatography-quadrupole detection (LC-QDa) method was developed.
• Proteins were digested without denaturation or reduction to preserve HOS information in peptides.
• Signature peptides were identified to monitor HOS changes in antibody domains.

Main Results:

• The method identified 13 signature peptides capable of detecting HOS changes.
• The technique demonstrated higher sensitivity than near-UV circular dichroism (CD) for tertiary structural changes.
• A streamlined, high-throughput method with five signature peptides was established.

Conclusions:

• Controlled proteolysis-based LC-QDa is a sensitive method for detecting protein HOS changes.
• The established method can support process and formulation development for biological drugs.
• This approach has potential applications in stability testing of biopharmaceuticals.