A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Monomeric purity is crucial for therapeutic monoclonal antibodies but often unknown for research samples.
• Variations in monomeric purity significantly impact quantitative antibody-based techniques like immunoassays.
• Accurate assessment of antibody integrity is essential for reliable experimental outcomes.

Purpose of the Study:

• To develop a novel multiplex technique for estimating monomeric purity and antigen affinity in research-grade antibody samples.
• To provide a rapid quality control method for antibody samples, enhancing experimental reproducibility.
• To address the challenge of unknown monomeric purity in research antibodies.

Main Methods:

• Utilized light scattering to simultaneously measure antibody binding to antigen and Protein A/G (PAG) surfaces.
• Estimated initial monomeric purity by detecting mass deficits on PAG surfaces compared to a high-purity standard.
• Refined purity estimates by analyzing antigen-binding stoichiometry to antibody bound to PAG.
• Validated results using electrospray ionization mass spectrometry (ESI).

Main Results:

• The novel method rapidly estimated monomeric purity in under 15 minutes.
• NISTmAb and infliximab biosimilar showed high monomeric purity (93.5% and 94.7% by ESI) and expected antigen-binding stoichiometries.
• Research-grade antibodies exhibited lower monomeric purity (54-89%) and variable antigen-binding stoichiometries.
• The technique successfully differentiated antibody quality based on purity and binding characteristics.

Conclusions:

• The developed multiplex technique offers a rapid and reliable method for assessing antibody monomeric purity and antigen affinity.
• This assay can serve as a valuable quality control tool for both research and therapeutic antibody applications.
• Improved antibody characterization through this method is expected to enhance the reproducibility of antibody-based experiments.