Elucidating the Mechanism of Multispecific Antibody Aggregation through Subunit Analysis

Area of Science:

• Biopharmaceutical development
• Protein characterization
• Analytical chemistry

Background:

• Multispecific antibodies offer improved therapeutic efficacy but present analytical challenges.
• Characterizing critical quality attributes like aggregation in these complex molecules is difficult.
• Existing analytical tools struggle to fully interrogate novel biopharmaceutical constructs.

Purpose of the Study:

• To develop and apply a novel native subunit analysis method.
• To investigate the mechanism of aggregation in a specific multispecific antibody.
• To identify the molecular origins of aggregation in complex antibody formats.

Main Methods:

• Utilized IdeS and IgdE enzyme digestion for native subunit analysis.
• Employed native size exclusion chromatography coupled with mass spectrometry (SEC-MS).
• Analyzed noncovalent interactions and subunit integrity post-digestion.

Main Results:

• Localized aggregation to single-chain fragment variables (scFvs) within the antibody.
• Identified a domain swapping mechanism as the cause of aggregation.
• Demonstrated the utility of IdeS/IgdE digestion for analyzing noncovalent interactions.

Conclusions:

• The novel native subunit analysis is effective for characterizing aggregation in multispecific antibodies.
• Domain swapping in scFvs is a key mechanism driving aggregation.
• This method provides critical insights into the quality attributes of complex biotherapeutics.