Prediction of the in vivo biological activity of human recombinant follicle stimulating hormone using quantitative isoelectric focusing.

Currently, the in vivo biopotency of commercial preparations containing the glycoprotein follicle-stimulating hormone (FSH) is declared on the basis of the ovarian weight augmentation assay as described in the British, European and United States Pharmacopoeias. Human FSH contains approximately 35% (W/W) carbohydrate which introduces considerable microheterogeneity (isohormones). Analysis of isohormones of recombinant FSH has revealed a relation between the isoelectric point (pl) and the in vivo bioactivity. Isohormones in the range of pl 3.5 are 100- to 200-fold more potent in the in vivo bioassay than isohormones with a pl of 5.5-6.0. These data suggest that quantification of the isohormone profile should enable us to predict the in vivo bioactivity. Thus, isohormones of recFSH samples were separated by isoelectric focusing (IEF), visualized by Coomassie brilliant blue G250 staining and quantified using densitometry. The data from 21 samples were compared with the in vivo bioassay data using partial least square (PLS) regression. A close correlation was found using a model with 2 PLS factors (correlation coefficient (r)=0.95, standard error of estimation SD 1. 02 lU/microg protein). In addition, ordinary least square (OLS) regression revealed a similar correlation between the fraction of isohormones between pl 3.9 and 4.9 and the in vivo bioactivity: r=0. 95, sd=1.4 lU/microg protein. Thus, an increase in the acidic isohormone fraction results in an increase in the in vivo bioactivity. The reverse is true for the amount of isohormones focusing between pl 5.1 and 5.7. An increase of this fraction results in a decrease of the in vivo bioactivity. These data are consistent with what might be expected from the in vivo bioassay data of the isohormones. The OLS model was subsequently validated using the guidelines of the European Centre for Validation of Alternative Methods (ECVAM) using 10 samples of recFSH that had not been used for the calibration. The relative standard deviation (RSD) of the mean difference between experimental and predicted in vivo bioactivity was approximately 6%. A Student's t-test performed on the experimental and predicted bioactivity data indicated that the predicted bioactivities do not deviate significantly from the experimental in vivo bioactivity data (P<0.05). These results demonstrate that the IEF scanning data can be used to predict the in vivo bioactivity with reasonable accuracy. This may be the first step towards replacing the in vivo bioassay for highly purified FSH by a physicochemical alternative. In general, quantitative charge-based separation methods like chromatofocusing, high performance capillary electrophoresis and ion exchange chromatography may also be considered as alternatives. Finally, quantitative charge profiling may prove to be as important for the estimation of the potency of other therapeutic glycoproteins like luteinizing hormone (LH), chorionic gonadotropin (CG), thyroid-stimulating hormone (TSH) and erythropoietin (EPO). Copyright 1997 The International Association of Biological Standardization.