METHODS FOR ASSESSING BINDING AFFINITY OF AN ANTIBODY VARIANT TO THE NEONATAL FC RECEPTOR
FIG2B depicts exemplary flow rates and buffer solutions used for column 1 (an affinity chromatography column)  FIG2C depicts exemplary flow rates and buffer solutions used for column 2 (a reverse phase chromatography column)
An exemplary first buffer solution comprises 20 mM MES/HCl and 140 mM NaCl, adjusted to pH 5.5 (see FIG2B, column 1, buffer A). An exemplary second buffer solution comprises 20 mM TRIS/HCl and 140 mM NaCl, adjusted to pH 8.8 (see FIG2B, column 1, buffer B). An additional exemplary second buffer solution comprises 20 mM HEPES adjusted to pH 8.6. Another exemplary second buffer solution comprises 20 mM TRIS adjusted to pH 8.2. In one aspect, the time (minutes) for the pH gradient is set forth in FIG2B. Other appropriate times for the pH gradient will be understood by one of ordinary skill in the art. The first-dimension affinity separation was conducted on a stationary phase containing immobilized FcRn (Roche Diagnostics, Germany) using the buffers and conditions set forth in FIG2B (referred to as column 1). In particular, the sample was loaded in an acidic mobile phase using 80% buffer A (20 mM MES/HCl, pH 5.5, 140 mM NaCl) and 20% buffer B (20 mM Tris/HCl, pH 8.8, 140 mM). Antibodies and antibody PTM variants bound to the FcRn affinity column were eluted from the column using a positive pH gradient, which mimics the physiological FcRn-IgG binding and dissociation process, using the buffers and conditions set forth in FIG2B.  An exemplary first buffer solution comprises formic acid in water, e.g., 0.1% formic acid in water (see FIG2C, column 2, buffer A). An exemplary second buffer solution comprises formic acid in acetonitrile, e.g., 0.1% formic acid in acetonitrile (see FIG2C, column 2, buffer B). In one aspect, the time (minutes) for the reverse chromatography portion of the method is set forth in FIG2C. Other appropriate times will be understood by one of ordinary skill in the art. The eluted antibody species of interest was sequentially fractionated, desalted and further separated online with the second-dimension reverse phase column using the buffers and conditions set forth in FIG2C (referred to as column 2). In particular, the sample was loaded in a 99% buffer A (0.1% formic acid in water) and 1% buffer B (0.1% formic acid in acetonitrile), and eluted over time as set forth in FIG2C.
flowchart
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Advances in Immunology
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Natl. Acad. Sci. USA
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Natl. Acad. Sci. USA
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['1. A method for assessing binding affinity 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; and d) eluting the reverse phase chromatography in a mobile phase to obtain a second eluted sample, wherein antibody variants that elute ahead of a control sample are identified as having weaker binding affinity to FcRn than the control sample.', '8. The method of claim 7, wherein a first buffer solution comprises 20 mM MES/HC1, pH 5.5, 140 mM NaCl and a second buffer solution comprises 20mM Tris/HCl, pH 8.8, 140 mM NaCl.']