Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range

• We present a method for the determination of high pI antibody charge variants.
• It is based on IEX chromatography in association with highly linear pH gradients.
• The quality of the separation depends on the linearity of the pH gradient.
• The linearity of the pH gradient depends on the linearity of the buffer capacity.
• The method allows the quantitation of charge variants as well as pI determination.

Recombinant antibodies with high isoelectric point are frequent since most of them are constructed from the same framework. Classically, cation exchange chromatography is used as a standard method for the determination of antibody charge heterogeneity. In contrast, in this study highly linear pH gradients were achieved by keeping the buffering capacity over the length of the gradient constant. The buffering compounds were selected to be unretained on the column and their respective concentration was adjusted in the start and end buffer of the pH gradient to achieve constant buffering capacity. This helps conserve linearity and stability of the gradient. The method allows quantification of charge variant distribution and the determination of chromatographic isoelectric point. To demonstrate the effectiveness of this novel method, a ProPac WCX-10 column was used to separate isoforms of trastuzumab biosimilar antibodies. Effects of pH gradient linearity and of varying the analytical amount of sample on the separation are shown.