Prediction of the In-vivo Biological Activity of Human Recombinant Follicle-stimulating Hormone Using Quantitative Isoelectric Focusing. Optimization of the Model

Follicle-stimulating hormone (FSH) is a glycoprotein consisting of two non-covalently linked subunits, α and β. FSH exists as multiple isoforms due to the attachment of oligosaccharides. The oligosaccharide moiety of these isohormones differs in branching pattern as well as in the sialic acid (N-acetyl neuraminic acid) content. The isohormone-related inherent microheterogeneity can be easily visualized using charge-based separation methods such as isoelectric focusing (IEF). Isohormones of recombinant FSH (rFSH, follitropin-β) display largely differing and isoelectric point (pI)-dependent in-vivo bioactivity. This has formed the basis for correlation between the charge distribution as determined by IEF (amount of isohormones focusing below pI 5) and the in-vivo bioactivity. Here we show that the sialic acid content is responsible for the differences in the in-vivo bioactivity. Gradual removal of sialic acid by in-vitro neuraminidase treatment of rFSH reduces the in-vivo bioactivity and results in a more basic IEF profile. The data allowed the refinement of the IEF in-vivo bioactivity model especially for samples with low in-vivo bioactivity (less than 5000 IU mg−1). In the optimized model the average pI was found to be a better parameter than the percentage of isohormones below pI 5. The average pI model for the in-vivo bioactivity was subsequently used to predict the in-vivo bioactivity of 27 production batches. The mean difference between the experimental and predicted in-vivo bioactivity was—1% (95% confidence interval—13 to 11%). These data further substantiate the use of IEF to accurately predict in-vivo bioactivity and offers an attractive alternative for the in-vivo bioassay currently in use for the potency declaration of rFSH. Similarly, since there is a strong correlation between sialic-acid content and the in-vivo bioactivity, this parameter may also be used to determine the potency of rFSH.