Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2-356 micrograms ml-1 with recovery of 125I-labeled C2B8 at the target sample concentration of 125 micrograms ml-1 equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9-4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.