Analysis of recombinant human erythropoietin in drug formulations by high-performance capillary electrophoresis.

A high-performance capillary electrophoresis (HPCE) method was developed for the analysis of recombinant human erythropoietin (rhEPO) in final drug preparations. All products examined were formulated with large amounts of human serum albumin (HSA) which is used as a protein excipient. Due to their similar physical characteristics in solution, rhEPO and HSA could not be resolved under HPCE conditions previously developed for the separation of bulk rhEPO. Addition of 1 mM nickel chloride to the electrophoretic buffer allowed complete separation of the two proteins as well as separation of rhEPO into several glycoform populations. The method was linear over the concentration range of 0.03-1.92 mg/ml, with limits of detection and of quantitation of 0.01 and 0.03 mg/ml, respectively. The precision of the method was evaluated from intra- and inter-day replicate injections of both rhEPO standard solution and formulation. Components of within- and between-batch variances were consistently below 5%, which constituted an acceptable level of variation. Products from two manufacturers were analyzed and showed little qualitative but appreciable quantitative lot-to-lot variations for rhEPO content when expressed in terms of units of biological activity. The method also revealed qualitative differences between the two products.