A convenient microscale colorimetric method for terminal galactose on immunoglobulins

Area of Science:

• Biochemistry
• Analytical Chemistry
• Glycobiology

Background:

• Accurate quantification of terminal galactose residues in immunoglobulins is crucial for characterizing glycoprotein structure and function.
• Existing methods may lack sensitivity or stability for precise determination of these critical glycans.

Purpose of the Study:

• To develop and validate a novel, sensitive, and stable method for the quantitative determination of terminal galactose (Gal) residues in immunoglobulins.
• To optimize conditions for exoglycosidase digestion and improve the ferricyanide colorimetric assay for Gal quantification.

Main Methods:

• Combined exoglycosidase digestion using Diplococcus pneumoniae beta-D-galactosidase with a modified ferricyanide colorimetric assay.
• Modified the ferricyanide assay by increasing sulfuric acid and sodium dodecyl sulfate (SDS) concentrations to enhance chromophore stability and solubility.
• Separated released Gal from immunoglobulins and enzyme via heat denaturation or ethanol precipitation prior to colorimetric measurement.

Main Results:

• The modified method demonstrated a linear range of approximately 11 to 111 microM Gal with a typical assay variation of about 5%.
• Successfully quantified terminal Gal content in a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) produced in Chinese hamster ovary (CHO) cells.
• The method was utilized to optimize digestion parameters (pH, time, temperature, enzyme concentration) for maximal Gal release from rIgG.

Conclusions:

• A robust and reliable method for quantifying terminal galactose residues in immunoglobulins has been established.
• The developed assay is suitable for assessing batch-to-batch consistency and optimizing production processes for glycoprotein therapeutics.
• This technique advances the analytical capabilities for characterizing immunoglobulin glycosylation patterns.