药物分析学报

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Ibio-Number Assay: A Physicochemical Assay that Predicts the Bioactivity of Erythropoietin with High Precision and Accuracy and May Replace the Mouse Bioassay in the Quality Control of EPO Batch Release

Hermentin P

The Ibio-number assay, based on capillary zone electrophoresis data of EPO samples, is evaluated as a physicochemical assay that enables to calculate the bioactivity of EPO medicinal products. In part 1, the CZE data of the candidate biological reference preparation EPO cBRP3 of a collaborative study of 10 laboratories (published in 2007) are used to calculate the bioactivity of EPO cBRP3 and to determine the interlaboratory precision of the assay and its accuracy against the stated bioactivity of EPO BRP3. This retrospective analysis by the author revealed an inter-laboratory precision of CV=0.8% (n=9 labs, 1 outlier lab excluded). The bioactivity calculated for cBRP3 (139.6 IU/μg), compared with the stated bioactivity of BRP3 (141.1 IU/μg=100%), provided an accuracy of 98.9%. In part 2, the CZE data of an epoetin alfa drug substance secondary standard and two epoetin alfa drug product samples (40,000 IU/mL and 2,000 IU/mL) from Centocor and two artificial epoetin beta concentrated solution samples from Roche (the mean of 40 epoetin beta ‘training batches’ and the mean of 17 epoetin beta ‘validation batches’) were retrospectively analyzed by the author using the Ibio-number assay. Moreover, the two hypothetical epoetin alfa/beta 1:1 mixtures of the Centocor and the Roche concentrated solution materials were prepared and the Ibionumber compared with the stated bioactivity of EPO BRP3. The results are summarized as follows: The Ibio-number assay applied to Centocor’s epoetin alfa secondary standard provided a potency of 142.3 IU/μg. The same assay applied to the mean of the 40 epoetin beta ‘training batches’ respectively the mean of the 17 epoetin beta ‘validation batches’ from Roche provided potencies of 133.8 IU/μg and 139.9 IU/μg, respectively. The two hypothetical alfa/beta 1:1 mixtures yielded bioactivities of 138.0 IU/μg and 141.1 IU/μg, respectively, which matched the stated bioactivity of BRP3 (141.1 IU/μg) with accuracies of 97.8% and 100.0%, respectively. The Ibio-number assay applied to Centocor’s formulated 40,000 IU/mL and 2,000 IU/mL epoetin alfa drug product samples revealed that the polysorbate 80 removal from the formulated samples decreased the bioactivity of the products by -1.7% (40 k sample) respectively -2.9% (2 k sample). This data demonstrates the suitability of the Ibio-number assay to calculate and compare the bioactivity of EPO samples with up till now unmet precision and accuracy. It is suggested that a prospective collaborative validation study will show that the Ibio-number assay is suitable to replace the mouse bioassay of EPO drug substance batch release, which will provide an increase in bioactivity precision and accuracy without any loss of product safety.