Immunochromatographic removal of albumin in erythropoietin biopharmaceutical formulations for its analysis by capillary electrophoresis.

Human serum albumin (HSA) is added to some pharmaceutical preparations as an excipient. This is the case for some of the commercial preparations of recombinant erythropoietin (rEPO). Differences in the number of the sialic acid moieties in the different rEPO glycoforms confer to these forms different net charges and different bioactivity. Knowledge of the isoforms present in each pharmaceutical product is then of interest. Differences in net charge of the rEPO forms make possible their separation by electrophoretical methods. However it has been observed in our laboratory that the amount of HSA usually present in these drug formulations interferes or even precludes separation of rEPO bands by capillary zone electrophoresis (CZE). In this work, an immunochromatographic method to remove HSA from rEPO biopharmaceutical formulations and a procedure to concentrate the sample that is needed to be performed prior to the analysis by CZE are developed. A home-made computer program to compare the percentage of correct assignments of electrophoretical bands provided by different migration parameters is used to study the effect of HSA remaining in samples on the accuracy of assignment of rEPO bands. When there exists a residual concentration of HSA in the sample (<2mg/ml) only the effective electrophoretic mobility is a reliable migration parameter to assign rEPO bands with a 100% of correct assignment. This parameter allows the correct assignment of bands of rEPO from pharmaceutical products formulated with HSA after immunochromatographic removal of HSA. Electrophoretical bands found in epoetin alpha, one of the commercial formulations of rEPO, are independent of the molecular mass of the excipients. The methodology used in this work for the analysis by CZE and the assignment of rEPO isoforms, as well as for the immunochromatographic HSA removal in the pharmaceutical products could be of high interest for the health authorities to control the quality of the product in marketing surveillance studies and for the quality control laboratories of the manufacturers.

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis.

Capillary zone electrophoresis of samples of recombinant human erythropoietin is performed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of alpha- and beta-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin alpha, the novel erythropoiesis-stimulating protein.