Methods for assessing binding affinity of an antibody variant to the neonatal fc receptor

A method for assessing binding of an antibody variant to the neonatal Fc receptor (FcRn), or a fragment thereof, using an online two dimension liquid chromatography system, the method comprising:

• a) contacting a first sample comprising antibody variants to an affinity chromatography stationary phase at an acidic pH, wherein the affinity chromatography stationary phase comprises immobilized FcRn or a fragment thereof, to thereby bind the antibody variants to the stationary phase;
• b) eluting the first sample from the affinity chromatography stationary phase using a positive pH gradient, to obtain an eluted sample;
• c) contacting the eluted sample to a reverse phase chromatography stationary phase;
• d) eluting the reverse phase chromatography in a mobile phase to obtain a second eluted sample;
• and
• e) analyzing the second eluted sample using mass spectrometry (MS),
• wherein antibody variants that elute ahead of a control sample are identified as having weaker binding to FcRn.