Single-chain human follicle-stimulating hormone with a di-N-glycosylated linker.

The classic way to produce single-chain (sc) glycoprotein hormones is to fuse their two subunits through the carboxy-terminal peptide (CTP) from human Choriogonadotropin (hCG). The CTP confers a longer half-life to single-chain hormones thanks to its four O-glycosyl side chains. However, unlike syncytiotrophoblastic cells, most cells used for recombinant protein production do not transfer O-glycosyl chains efficiently. We thus choose to fuse the hFSH subunits with a linker comprising two N-glycosyl side chains (sc-hFSH LNN) or none (sc-hFSH L0N), that were generated using two expression systems, HEK293 and CHO K1 cells. Their production levels and biological activities were tested and compared. Both expression systems successfully produced biologically active sc-hFSH, but, in our hands, CHO K1 cells yielded about 30-fold higher amounts of recombinant protein than HEK293 cells. Moreover, sc-hFSH L0N was considerably less expressed than sc-hFSH LNN in both cell types. Our data show that sc-hFSH L0N and sc-hFSH LNN produced from both cell lines stimulate cAMP and progesterone production in mLTC cells expressing hFSH receptors and exhibit similar B/I (in vitro Bioactivity/Immuno activity) ratios. Finally, the ratio of in vivo/in vitro bioactivities for sc-hFSH LNN relative to natural pituitary heterodimeric hFSH increased 8-fold, most likely because of a longer half-life in the blood.

Human FSH Glycoform α-Subunit Asparagine52 Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance.

Follicle-stimulating hormone (FSH), an α/ß heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHß subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHß band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks ßAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHß glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.

Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells.

Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and ß-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography.

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C-termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

Understanding the interactions of human follicle stimulating hormone with single-walled carbon nanotubes by molecular dynamics simulation and free energy analysis.

Interactions of carbon nanotubes (CNTs) and blood proteins are of interest for nanotoxicology and nanomedicine. It is believed that the interactions of blood proteins and glycoproteins with CNTs may have important biological effects. In spite of many experimental studies of single-walled carbon nanotubes (SWCNT) and glycoproteins with different methods, little is known about the atomistic details of their association process or of structural alterations occurring in adsorbed glycoproteins. In this study, we have applied molecular dynamics simulation to investigate the interaction of follicle stimulating hormone (hFSH) with SWCNT. The aim of this work is to investigate possible mechanisms of nanotoxicity at a molecular level. We present details of the molecular dynamics, structure, and free energy of binding of hFSH on the surface of SWCNT. We find that hFSH in aqueous solution strongly adsorbs onto SWCNT via their concave surface as evidenced by high binding free energies for residues in both protein subunits. It was found that hydrophobic, π-cation, and π-π stacking interactions are the main driving forces for the adsorption of the protein at the nanotube surface.

Manufacturing of Recombinant Human Follicle-Stimulating Hormone Ovaleap® (XM17), Comparability with Gonal-f®, and Performance/Consistency.

Ovaleap® (XM17) is a recombinant human follicle-stimulating hormone to treat infertility by inducing ovulation or controlled ovarian stimulation for assisted reproductive technology (ART) procedures. Ovaleap® (follitropin-α) was approved by the European Medicines Agency in 2013 as a biosimilar medicinal product to the reference medicine, Gonal-f®. Information is often not easily accessible and/or publicly available regarding the rigorous manufacturing procedures for biosimilars. Objectives of the current analysis were to report on validation procedures for the Ovaleap® manufacturing process, to compare the characteristics of Ovaleap® versus Gonal-f®, and to describe the performance and consistency of Ovaleap®. Formal validation of the Ovaleap® manufacturing process was performed at full commercial scale, consisting of several consecutive fermentation and purification runs. Comparison with Gonal-f® involved numerous techniques to determine molecular structure, isoform distribution, biological activity, and product-related impurities. The stability of the multidose application system, targeted for long-term stability at ambient temperature, was assessed and demonstrated. All analyses showed the manufacturing process of Ovaleap® to be robust and consistent. Ovaleap® was found to have similar characteristics when compared with Gonal-f®. This analysis supports the role of Ovaleap® as a biosimilar to Gonal-f®, thus providing patients and clinicians with another therapeutic option during ART procedures.

Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice.

The hormone - specific FSHß subunit of the human FSH heterodimer consists of N-linked glycans at Asn7 and Asn24 residues that are co-translationally attached early during subunit biosynthesis. Differences in the number of N-glycans (none, one or two) on the human FSHß subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH21/18, missing either an Asn24 or Asn7 N-glycan chain on the ß - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7 and Asn24 N-linked glycans on the ß - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH21/18 and FSH24) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH21/18 exhibits faster FSH- receptor binding kinetics and is much more active than FSH24 in every assay tested to date. However, the in vivo bioactivity of the hypo-glycosylated FSH glycoform has never been tested. Here, we evaluated the in vivo bioactivities of FSH glycoforms in Fshb null mice using a pharmacological rescue approach. In Fshb null female mice, both hypo- and fully-glycosylated FSH elicited an ovarian weight gain response by 48 h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH24. Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH21/18 elicited a response similar to that by fully-glycosylated FSH24 in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH21/18 was more active than the fully-glycosylated FSH24 in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH21/18 glycoform elicits bioactivity in vivo similar to the fully-glycosylated FSH. Our studies may have clinical implications particularly in formulating FSH-based ovarian follicle induction protocols using a combination of different human FSH glycoforms.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

Follicle-Stimulating Hormone: A Review of Form and Function in the Treatment of Infertility.

The use of gonadotropin therapy, including follicle-stimulating hormone (FSH), represents an indispensable part of fertility treatment. There are a number of FSH preparations commercially available or in development, including both urinary-derived products (urinary-derived FSH [uFSH]) and FSH produced through recombinant techniques (recombinant FSH [rFSH]). Differences in the glycosylation patterns of FSH give rise to a number of naturally occurring isoforms that may differ functionally. The relative concentrations of these isoforms vary over the course of the menstrual cycle and the lifetime, indicating that these differences in glycosylation may have physiologic relevance. Although both uFSH and rFSH contain human FSH, there are differences in the glycosylation patterns, which may give rise to differences in biologic activity between products. Current FSH products have been shown to have high purity and to exhibit consistent, favorable efficacy and safety profiles for the treatment of infertility, regardless of urinary or recombinant origin.

A large, multicentre, observational, post-marketing surveillance study of the 2:1 formulation of follitropin alfa and lutropin alfa in routine clinical practice for assisted reproductive technology.

BACKGROUND: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) both have a role to play in follicular development during the natural menstrual cycle. LH supplementation during controlled ovarian stimulation (COS) for assisted reproductive technology (ART) is used for patients with hypogonadotropic hypogonadism. However, the use of exogenous LH in COS in normogonadotropic women undergoing ART is the subject of debate. The aim of this study was to investigate characteristics of infertile women who received the 2:1 formulation of follitropin alfa and lutropin alfa (indicated for stimulation of follicular development in women with severe LH and FSH deficiency) in German clinical practice. METHODS: A 3-year, multicentre, open-label, observational/non-interventional, post-marketing surveillance study of women (21-45 years) undergoing ART. Primary endpoint: reason for prescribing the 2:1 formulation of follitropin alfa and lutropin alfa. Secondary variables included: COS duration/dose; oocytes retrieved; fertilization; clinical pregnancy; ovarian hyperstimulation syndrome (OHSS). RESULTS: In total, 2220 cycles were assessed; at least one reason for prescribing the 2:1 formulation was given in 1834/2220 (82.6%) cycles. Most common reasons were: poor ovarian response (POR) (39.4%), low baseline LH (17.8%), and age (13.8%). COS: mean dose of the 2:1 formulation on first day, 183.1/91.5 IU; mean duration, 10.8 days. In 2173/2220 (97.9%) cycles, human chorionic gonadotrophin was administered. Oocyte pick-up (OPU) was attempted in 2108/2220 (95.0%) cycles; mean (standard deviation) 8.0 (5.4) oocytes retrieved/OPU cycle. Fertilization (≥1 oocyte fertilized) rates: in vitro fertilization (IVF), 391/439 (89.1%) cycles; intracytoplasmic sperm injection (ICSI)/IVF + ICSI, 1524/1613 (94.5%) cycles. Clinical pregnancy rate: all cycles, 25.9%; embryo transfer cycles, 31.3%. OHSS: hospitalization for OHSS, 8 (0.36%) cycles, Grade 2, 60 (2.7%), and Grade 3, 1 (0.05%). CONCLUSIONS: In German routine clinical practice, the most common reasons for using the 2:1 formulation of follitropin alfa and lutropin alfa for women undergoing ART were POR, low baseline LH, and age. Severe OHSS incidence was low and similar to that reported previously.

Hypo-glycosylated human follicle-stimulating hormone (hFSH(21/18)) is much more active in vitro than fully-glycosylated hFSH (hFSH(24)).

Hypo-glycosylated hFSH(21/18) (possesses FSHß(21) and FSHß(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHß(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation.

Dynamic changes in glycosylation and glycan composition of serum FSH and LH during natural ovarian stimulation.

BACKGROUND: Glycosylation and glycan composition are of fundamental importance for the biological properties of FSH and LH. The aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum FSH and LH throughout the normal menstrual cycle. METHODS: Serum samples were collected from 79 healthy women with regular menstrual cycles. The mean numbers of anionic monosaccharide (AMS), sialic acid (SA), and sulfonated N-acetylgalactosamine (SU) residues per FSH and LH molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins. RESULTS: Di-glycosylated glycoforms (FSHdi, LHdi) were detected in serum in addition to tetra-glycosylated FSH (FSHtetra) and tri-glycosylated LH (LHtri). FSHdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. FSHtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. There were lower concentrations of LHdi than LHtri, except at midcycle when the opposite occurred. The mean numbers of SA and SU residues per molecule of FSH and LH in serum showed four different patterns during the cycle, all with highly significant (P < 0.0001) differences between levels at different phases of the cycle. The pattern of SA residues on FSH was 'M'-shaped, and that of SU on LH 'V'-shaped. CONCLUSION: Serum FSH and LH governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. This new information on the FSH and LH molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women.

The glycan structure in recombinant human FSH affects endocrine activity and global gene expression in human granulosa cells.

The aim of this study was to analyse the biological response to different recombinant human FSH (rhFSH) glycosylation variants on the endocrine activity and gene expression at whole-genome scale in human granulosa-like tumor cell line, KGN. The effects of differences in rhFSH sialylation and oligosaccharide complexity were determined on steroid hormone and inhibin production. A microarray approach was used to explore gene expression patterns induced by rhFSH glycosylation variants. Set enrichment analysis revealed that hormone sialylation and oligosaccharide complexity in rhFSH differentially affected the expression of genes involved in essential biological processes and molecular functions of KGN cells. The relevance of rhFSH oligosaccharide structure on steroidogenesis was confirmed assessing gene expression by real time-PCR. The results demonstrate that FSH oligosaccharide structure affects expression of genes encoding proteins, growth factors and hormones essential for granulosa cells function.

[The molecular design and drug development of recombinant long-acting follicle stimulating hormone].

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.

Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product.

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies.

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone.

BACKGROUND: Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. METHODS: To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. RESULTS: Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED(50) values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. CONCLUSION: High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin.

Toward fully synthetic homogeneous beta-human follicle-stimulating hormone (beta-hFSH) with a biantennary N-linked dodecasaccharide. synthesis of beta-hFSH with chitobiose units at the natural linkage sites.

A highly convergent synthesis of the sialic acid-rich biantennary N-linked glycan found in human glycoprotein hormones and its use in the synthesis of a fragment derived from the beta-domain of human Follicle-Stimulating Hormone (hFSH) are described. The synthesis highlights the use of the Sinay radical glycosidation protocol for the simultaneous installation of both biantennary side-chains of the dodecasaccharide as well as the use of glycal chemistry to construct the tetrasaccharide core in an efficient manner. The synthetic glycan was used to prepare the glycosylated 20-27aa domain of the beta-subunit of hFSH under a Lansbury aspartylation protocol. The proposed strategy for incorporating the prepared N-linked dodecasaccharide-containing 20-27aa domain into beta-hFSH subunit was validated in the context of a model system, providing protected beta-hFSH subunit functionalized with chitobiose at positions 7 and 24.

Single-chain human gonadotropin analogs induce follicle development in sheep.

The biopotency of single-chain analogs of human hFSH, human chorionic gonadotropin (hCG), and a dually active gonadotropin construct (FcCGbetaalpha) was examined. Sheep (bwt=61.4+/-1.1 kg; n=6 ewes/treatment) received a single injection (5 IU/kg, i.v.) of the hFSH analog (Fcalpha), the hCG analog (CGbetaalpha), FcCGbetaalpha, or Fcalpha and CGbetaalpha. Control animals received conditioned media. Ovulation was induced 3 days after analog administration using hCG (1000 IU, i.v.). Basal serum concentrations of estradiol (E(2)) were maintained in control animals. Neither Fcalpha nor CGbetaalpha alone induced significant E(2) production during the pre-hCG period. Conversely, serum concentrations of E(2) were increased (P<0.05) 2 days after administration of FcCGbetaalpha or Fcalpha+ CGbetaalpha. Although P(4) concentrations were maintained at basal levels in control animals, significant increase was noted in all other treatment groups during the post-hCG period. Final ovarian weight was significantly increased (P<0.05) in animals receiving Fcalpha, Fcalpha+ CGbetaalpha, or FcCGbetaalpha, but not CGbetaalpha alone. Most of the ovarian enlargement was attributed to the formation of corpora lutea. Collectively, these observations demonstrate that the single-chain analogs of the human gonadotropins are active in sheep. The construct with singular FSH activity supports follicle development but not E(2) production. Conversely, the construct that incorporates beta-domains from both CG and FSH has dual activity. The long-lived nature of the single-chain constructs suggests that these recombinant gonadotropins may be effective alternatives to pituitary- or placenta-derived gonadotropins in out-of-season breeding and/or superovulation protocols.

Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology.

This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.