Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography...

Area of Science:

• Biochemistry
• Protein Chemistry
• Analytical Chemistry

Background:

• Protein tyrosine sulfation is a post-translational modification (PTM) infrequently observed in recombinant therapeutic proteins.
• Sulfation introduces a negative charge, potentially altering protein interactions and antigen-binding activity, thus posing a critical quality attribute challenge.
• Controlling protein tyrosine sulfation is essential for ensuring the efficacy and safety of therapeutic antibodies.

Purpose of the Study:

• To develop and validate a robust method for separating and quantifying varying degrees of protein tyrosine sulfation in a therapeutic bispecific antibody.
• To investigate the impact of tyrosine sulfation on the antibody's structure-function relationship, specifically its antigen-binding activity.
• To establish tyrosine sulfation as a critical quality attribute and demonstrate an effective assay for its control.

Main Methods:

• Development of a unique hydrophobic interaction chromatography (HIC) method for high-resolution separation of sulfated and non-sulfated antibody species.
• Utilized mass spectrometry and fluorescence-linked immunosorbent assay (FLISA) for structure-function relationship studies of enriched sulfated antibody variants.
• Evaluated the HIC method's efficacy in controlling critical quality attributes related to protein tyrosine sulfation.

Main Results:

• The developed HIC method achieved baseline resolution of antibody species based on their degree of sulfation, independent of other PTMs.
• Enriched sulfated bispecific antibody species, particularly those with sulfation in the complementarity-determining region (CDR), exhibited reduced binding to their cognate antigen.
• The HIC assay proved effective in separating sulfated species, confirming its utility for controlling this critical quality attribute.

Conclusions:

• Tyrosine sulfation in therapeutic bispecific antibodies is a critical quality attribute that can negatively affect antigen-binding activity.
• The developed HIC method is a powerful tool for the separation, quantification, and control of protein tyrosine sulfation in therapeutic proteins.
• Stringent control of tyrosine sulfation is necessary to maintain the desired biological function and therapeutic efficacy of recombinant antibodies.