monisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines to capillary isoelectric focusing analytical methods (2–5). Many of the publications about capillary isoelectric focusing validation focus on assay or iden-

he number of biotechnology products on the market and, particularly, in development has increased enormously during the past decade. Biological products generally are more complex than their small-molecule counterparts. Some product variants — such as deamidated, clipped, aggregated, or misfolded species — could be present in the process stream and even in the final products. Regulatory agencies expect manufacturers to demonstrate their ability to produce an identical product from batch to batch. With the structural complexities of various types of biological products, confirming batch-to-batch reproducibility requires the use of a wide range of analytical tools based upon different separation mechanisms to obtain acceptable characterization of the product. Fortunately, the advent of modern analytical techniques makes this process possible. Isoelectric focusing is an example of a modern analytical technique often used for monitoring consistency and stability and for quantifying the levels of different variants present in batches of a biotechnology product. In this technique, analytes are separated across a pH gradient formed by carrier ampholytes under the influence of an electric field. Different analytes form tightly focused bands at their isoelectric point, the pH at which their net charge is zero (1). In particular, capillary isoelectric focusing combines the resolving power of isoelectric focusing with advantages of capillary electrophoresis in quantitation and automation, and it has emerged as a very effective and useful technique for assessing charge heterogeneity in protein molecules (2–5). Santora and co-workers (3) used capillary isoelectric focusing, with a high level of precision, to separate and quantitatively measure monoclonal antibody variants that differed by a single amino acid. Other researchers have reported the validation of capillary isoelectric focusing methods for monoclonal antibodies and protein drug products (2,4,5). They found that method precision, linearity, accuracy, and robustness met target values established to demonstrate the suitability of their assays. These reports indicate capillary isoelectric focusing’s high level of reliability as a quantitative analytical method. Scientists have been successful in implementing strategies for adapting International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines to capillary isoelectric focusing analytical methods (2–5). Many of the publications about capillary isoelectric focusing validation focus on assay or identity methods, whereas the validation of capillary isoelectric focusing impurity methods has not been reported widely. In this “Validation Viewpoint” column, we look at various requirements that must be met for validating a capillary isoelectric focusing method intended for the quantitative measurement of product-related impurities found in production lots of a protein drug substance. The ICH guidelines describe product-related impurities for protein biotechnology products as molecular variants that arise from processing or that appear during storage. Examples of product-related impurities include truncated and aggregated forms and impurities resulting from the chemical modification of amino acids. One amino-acid modification is the deamidation of an asparagine or glutamine residue to aspartate or glutamate, respectively. As such, these impurities are difficult to measure quantitatively by high performance liquid chromatography (HPLC) methods, such as ion exchange, which do not always offer sufficient resolving power to discriminate between these variants. On the other hand, capillary isoelectric focusing is an ideal technique to measure deamidated impurities, because this method offers both the necessary resoGuest Authors A.S. Rathore, R.R. Kurumbail, and A.M. Lasdun