In Vitro Activity Of Recombinant Human Follicle Stimulating Hormone

An accurate, rapid, convenient cell-based in vitro activity assay needs to be established to support the activity study of recombinant human Follicle Stimulating Hormone（rh FSH）. In this paper, a KGN cell-based activity assay has been established. KGN cell is a kind of ovarian granulosa cell, which expresses human FSH receptor naturally. Once the receptor being binding by rh FSH, Intracellular c AMP signaling induces steroid synthesis by increased gene expression and phosphorylation triggered activation of relevant enzymes, e.g., aromatase（ARO） to induce the synthesis and secretion of steroid, i.e. progesterone and estradiol. FSH of different concentration was used to bind the receptor on the surface of KGN cell, then accordingly different concentration of progesterone was secreted in the medium, which can be determined by commercialized ELISA kits to evaluate the bioactivity of FSH. 96-well plates and the 2nd generation of international WHO standard of FSH（Recombinant; NIBSC code 08/282） was introduced in the study to show a highly FSH dose-dependent progesterone secretion. The optimized conditions of the assay were 2×104 cell/well of inoculation cell density, 0.1²00 ng/m L of rh FSH concentration and incubation for 72 hours.The method verification, including specificity, linear range, recovery, precision, and tolerance, etc. was carried out to show a good FSH dose-dependent reaction of KGN cell as well as a satisfied specificity, a linear range between 0.1 200 ng/ml, a R2≥0.99 and the recovery between 80¹20%. Three duplicated experiments were accomplished with three batches of commercialized FSH and three bathes of samples to get the activity of（13.4±0.4）^13.5±0.8and（12.9±0.7）^14.3±1.2IU/μg respectively, which shows a CV≤15%. A good tolerance of the method has also been examined by using different brands of progesterone determination kits. Three batches of commercialized rh FSH and one batch of national standard urofollitropin were assayed comparing with the results from traditional in vivo assay to show similar results. What’s more, compared with the in vivo assay, efficiency can be greatly improved as well as the dramatically reduced costs due to the introduction of 96 well-plates. Having high sensitivity, accuracy, detection efficiency and good tolerance, the in vitro assay can be used to facilitate a great research and evaluation of FSH bioactivity and replace the traditional in vivo one.A commercialized human FSH receptor（hFSHR） antibody was introduced to further confirm the specificity of rh FSH response and the affinity of surface receptor of KGN cell, which was incubated with KGN cells in advance to initialize the binding of h FSHR antibody and h FSHR on the surface of KGN cells, then progesterone concentration was determined after rh FSH adding and incubation for 72 h. Different batches of samples and commercialized FSH was examined to confirm the similarity of affinity among different samples. The results indicated that the affinity of FSH to h FSHR was considerably higher than h FSHR antibody as the rh FSH concentration of 10ng/ml（0.3n M） and 70ng/ml（2.1n M） and accordingly ND50 of h FSHR antibody of 1.84×103ng/ml and 1.52×104ng/ml respectively, which indicated h FSHR antibody can bind the h FSHR, however, it cannot activate the response of second messenger in the cell like what rh FSH has achieved. Another antibody（anti-TMP-Fc antibody） was introduced as the negative control to successfully verify the specificity of the binding between h FSHR and h FSHR antibody.Comparing the activity changing and affinity differences of different origins and batches of rh FSH samples at the presence of h FSHR antibody, we have got the result that when h FSHR antibody concentration came to 1×104ng/ml, EC50 increased for 3-4 times（from 4⁵ng/ml to 15²5ng/ml）, which was analyzed by JMP to show no notable differences between the two groups of data（P>0.05）. The results also indicated same affection of h FSHR antibody to samples and commercialized FSH, which can weaken the affinity between rh FSH and h FSHR as well as the stimulation of rh FSH to KGN cells to inhibit the h FSHR message channel.Two aspects of intrinsic rh FSH structure has been investigated in this study, which are saliva acid degree and external environment of rh FSH protein. Special attention has been paid to the in vitro activity caused by different sialic acid concentration, in terms of different half-life in vivo due to different content of sialic acid. Non-reducing and reducing SDS-PAGE, IEF, and HPAEC-PAD have been used to examine rh FSH after different sialidase digestion conditions to reveal the property changing, p I range increasing above 6.0（due to negative charging of sialic acid） and sialic acid content（drop from 10mol/mol to undetected）. In addition, to confirm the relation of sialic acid and in vitro activity, different elution parts from Ion-exchange Chromatography（IEC） had been examined by IEF and HPAEC-PAD to show that lower p I range mean higher sialic acid content, which indicated the p I range of the protein could be used to predict its sialic acid level.After in vitro activity analysis of rh FSH with different sialic acid content, the activity of it whose the sialic acid has been completely removed increased from 13.9 IU/μg to 56.1⁸3.0 IU/μg, as 4⁶ times higher, due to the increased affinity of rh FSH and FSHR and therefore the enhanced stimulation of KGN cells. The analysis also revealed a good correlation between activity and sialic acid content, which was accomplished by JMP to show the R2=0.999 and P<0.05. The activity lose from higher sialic acid content should come from the steric factor from higher sialic molecules at the end of the protein carbohydrate chain. On the contrary, higher sialic acid content in vivo would cause the extension of half-life of the protein, which mean a negative correlation.In conclusion, the in vitro bioactivity of FSH could be used to predict the sialic acid content, isoelectric point range, in vivo metabolism performance and half-life due to the good correlation, which is also a very important and valuable reference issue in the process development, quality control, quality consistency evaluation among batches, effectiveness evaluation in vivo, clinical trial and prediction of rh FSH, etc. In this study, the CHO cell expressed and buffered by PB samples were used to investigate the affection from such conditions as temperature, light, and freeze-thaw cycles. etc., whose purity, subunit dissociation level, oxidation degree were analyzed to hopefully found the intrinsic issues of activity changing. The results revealed that light and freeze-thaw cycles would not cause such problems hereinabove, as well as incubation for 15 days under 25℃. However, under 40℃, the in vitro activity decreased slightly and subunit dissociation could be detected too, in terms of a correlation between in vitro activity and purity with the R2=0.971, P<0.05 from JMP analysis. What’s more, under 60℃, the in vitro activity lost dramatically, which plummeted to be 80% after incubation for 5 days and lost completely after 10 days, as the purity decreased dramatically at day 5 with 60% subunit dissociation and to be 20% at day 15 with 5¹0% aggregation generation, so it can be inferred that affinity lost from dissociation and aggregation caused by high temperature led to completely activity lost in vitro.In conclusion, as the major reason of activity lost in vitro, high temperature could cause protein subunit dissociation, high-level structure changing, leading to the affinity of rh FSH and h FSHR dropping. However, RT, light, and freeze-thaw cycles would not cause the structure changing and the activity in vitro could be stable.