Biological characterization of recombinant human follicle stimulating hormone isoforms

It has been established that follicle stimulating hormone (FSH) circulates in the bloodstream as a heterogeneous population of molecules, Individual F SH isoforms, while displaying identical amino acid sequences, differ in the ir extent of post-translational modification. As a result of these variatio ns, the FSH isoforms exhibit differences in overall charge, degree of siali c acid or sulphate incorporation, receptor binding affinity and plasma half -life, Taking advantage of the fact that these forms can be separated from each other on the basis of their charge, we have evaluated in rats the meta bolic clearance rates of the acidic [with an isoelectric point (pI) less th an or equal to 4.8] and the less acidic (pI > 4.8) isoforms of recombinant human FSH (rhFSH) obtained after chromatofocusing. The less acidic isoform group was found to have a faster clearance from the circulation in rats as compared with the acidic isoform group, This finding is in agreement with t he lower bioactivity in vivo (as determined by the Steelman-Pohley assay) o f the less acidic isoform group, compared with the acidic one. The mass spe ctra of the two groups of isoforms showed a difference in the sialic acid c ontent thus highlighting the importance of these residues on the in-vivo ac tivity of FSH. Conversely, when the two groups of isoforms were tested in v itro by using the Y1 human FSH receptor (Y1 hFSHR) assay and a reporter gen e assay, no significant differences in:the biological activities between th ese preparations were detected when test concentrations were based on mass.