Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography.

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Ultrasensitive biosensor based on magnetic microspheres enhanced microfiber interferometer.

A critical barrier for the successful development of fiber sensors for bio-chemical processes is their limitedly improved sensitivity, restricted by the sensor structural design. To solve this, in this paper, a novel concept was proposed using functionalised modified magnetic microspheres (MMSs) to "amplify" the effect of target bio-chemical analytes to significantly improve the fiber sensor's sensitivity, which has been demonstrated using human chorionic gonadotropin (hCG) as an example. Two types of antibody hCG, (ß and α, both can specifically bind with hCG), were adhered on the surface of fibre sensor and MMSs respectively. Both hCG and MMSs will be specifically captured by the fibre sensor, where MMSs act as an "amplifier" to improve the sensor sensitivity. Experimentally immunomagnetic detection limit of 0.0001 mIU/mL has been achieved, which is the highest reported so far. This newly developed methodology opens a new direction for sensitivity improvement and could be further explored to applications require ultrahigh sensitivity detections such as earlier medical diagnostics.

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction-tandem mass spectrometry to detect hCG use.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free ß-subunit (hCGß) and ß-subunit core fragment (hCGßcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGß and hCGßcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGß and hCGßcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

The relationship between fat and progesterone, estradiol, and chorionic gonadotropin levels in Quebec cow's milk.

PURPOSE: The majority of milk in industrialized countries is obtained from pregnant cows, which contains increased levels of estrogen and progesterone compared to non-pregnant cows. The aim of this study was to quantify the amount of hormones present in milk with different fat content because previous studies on humans have shown potential effects of increased milk consumption on serum and urine hormone levels as well as on sperm parameters. However, it is unclear whether consumption of milk at the currently recommended levels would lead to systemic effects. METHODS: Samples of cow's milk of varying fat concentrations (0, 1, 2, 3.25, 10, and 35%) were analyzed via competitive ELISA assays. RESULTS: Progesterone concentrations were significantly correlated to increasing fat content of milk (r = 0.8251, p = 0.04). CONCLUSIONS: Research on conditions in which additional progesterone may have an effect on human health should consider inclusion of limitation of milk intake and its effects. Further studies are needed to determine the concentration of progesterone in milk of different fat content in other regions and countries and to quantify the potential pathophysiologic role.

A Label-Free and Ultrasensitive Immunosensor for Detection of Human Chorionic Gonadotrophin Based on Graphene FETs.

We report on a label-free immunosensor based on graphene field effect transistors (G-FETs) for the ultrasensitive detection of Human Chorionic Gonadotrophin (hCG), as an indicator of pregnancy and related disorders, such as actopic pregnancy, choriocarcinoma and orchic teratoma. Pyrene based bioactive ester was non-covalently anchored onto the graphene channel in order to retain the sp² lattice. The G-FET transfer characteristics showed repeatable and reliable responses in all surface modifying steps using a direct current (DC) readout system. The hCG concentration gradient showed a detection limit of ~1 pg·mL-1. The proposed method facilitates the cost-effective and viable production of graphene point-of-care devices for clinical diagnosis.

Ultra-high sensitivity of the non-immunological affinity of graphene oxide-peptide-based surface plasmon resonance biosensors to detect human chorionic gonadotropin.

Specific peptide aptamers can be used in place of expensive antibody proteins, and they are gaining increasing importance as sensing probes due to their potential in the development of non-immunological assays with high sensitivity, affinity and specificity for human chorionic gonadotropin (hCG) protein. We combined graphene oxide (GO) sheets with a specific peptide aptamer to create a novel, simple and label-free tool to detect abnormalities at an early stage of pregnancy, a GO-peptide-based surface plasmon resonance (SPR) biosensor. This is the first binding interface experiment to successfully demonstrate binding specificity in kinetic analysis biomechanics in peptide aptamers and GO sheets. In addition to the improved affinity offered by the high compatibility with the target hCG protein, the major advantage of GO-peptide-based SPR sensors was their reduced nonspecific adsorption and enhanced sensitivity. The calculation of total electric field intensity (ΔE) in the GO-based sensing interfaces was significantly enhanced by up to 1.2 times that of a conventional SPR chip. The GO-peptide-based chip (1mM) had a high affinity (KA) of 6.37×1012M-1, limit of detection of 0.065nM and ultra-high sensitivity of 16 times that of a conventional SPR chip. The sensitivity of the slope ratio of the low concentration hCG protein assay in linear regression analysis was GO-peptide (1mM): GO-peptide (0.1mM): conventional chip (8-mercaptooctanoic acid)-peptide (0.1mM)=8.6: 3.3: 1. In summary, the excellent binding affinity, low detection limit, high sensitivity, good stability and specificity suggest the potential of this GO-peptide-based SPR chip detection method in clinical application. The development of real-time whole blood analytic and diagnostic tools to detect abnormalities at an early stage of pregnancy is a promising technique for future clinical application.

Absence of TGF-ß Receptor Activation by Highly Purified hCG Preparations.

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-ß receptor (TGFßR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-ß1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFßR in mink lung epithelial cells transfected with a reporter gene for TGFßR activation. No such activation was found with highly purified hCG or its free ß-subunit (hCGß), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFßR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Sequential Versus Continual Purified Urinary FSH/hCG in Men With Idiopathic Hypogonadotropic Hypogonadism.

CONTEXT: Gonadotropin therapy using a human chorionic gonadotropin (hCG) and FSH preparation is an effective regimen in inducing masculinization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, the high cost of medication and frequent injections affect compliance. OBJECTIVE: The aim of this study was to determine the efficacy of sequential use of highly purified urinary FSH (uFSH)/hCG in men with IHH. DESIGN AND SETTING: A randomized, open-label, prospective, controlled noninferiority trial with an 18-month follow-up was conducted in 9 tertiary hospitals. PATIENTS AND INTERVENTION: A total of 67 Chinese men with IHH were randomly allocated into group A receiving continual uFSH (75 U, 3 times a week) and hCG (2000 U, twice a week) injection and group B receiving sequential uFSH (75 U, 3 times a week every other 3 months) and hCG (2000 U, twice a week) injection. MAIN OUTCOME MEASURE: The primary outcome was the proportion of subjects with a sperm concentration of ≥ 1.0 × 10(6)/mL during the 18 months. The efficacy between groups A and B was compared for noninferiority. RESULTS: Of the patients, 17/33 (51.5%) receiving continual uFSH/hCG and 19/34 (55.9%) receiving sequential uFSH/hCG achieved sperm concentrations of ≥ 1.0 × 10(6)/mL. The efficacy in the sequential uFSH/hCG group was not inferior to that in the continual uFSH/hCG group (noninferiority, P = .008) by intention-to-treat analysis. CONCLUSIONS: The efficacy of the sequential uFSH/hCG regimen is not inferior to that of the continual uFSH/hCG regimen in inducing spermatogenesis and masculinization of patients with IHH.

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation.

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type High Gradient Magnetic Fishing (HGMF) system for large-scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single-cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively.

Gold-linked electrochemical immunoassay on single-walled carbon nanotube for highly sensitive detection of human chorionic gonadotropin hormone.

A new sensitive gold-linked electrochemical immunoassay (GLEIA) for the detection of the pregnancy marker human chorionic gonadotropin (hCG) has been developed using the direct electrochemical detection of Au nanoparticles. We utilized single-walled carbon nanotube (SWCNT) microelectrodes; 24 SWCNT microelectrodes were arrayed on a single Si substrate 25×30 mm² in size, for the development of a new GLEIA (SWCNT-GLEIA). This SWCNT-GLEIA provided convenient and cost-effective tests with the required antibody and antigen sample volumes as small as 2.0 µL for a group of 4 SWCNT microelectrodes. In addition, this assay also exhibited properties of high sensitivity and selectivity benefitting from the intrinsic extraordinary features of SWCNTs. Using scanning electron microscopy, we also observed Au nanoparticle-labeled antigen-antibody complexes immobilized on the surface of the SWCNT microelectrodes. The concentration of the pregnancy marker (hCG) showed a linear relationship with the current intensity obtained from differential pulse voltammetry measurements with a limit of detection (LOD) of 2.4 pg/mL (0.024 mIU/mL) hCG. This LOD is 15 times more sensitive than a previous GLEIA, which used screen-printed carbon electrodes.

Gonadotropin purification from horse serum applying magnetic beads.

The glycoprotein hormone equine chorionic gonadotropin (eCG) is a commercial product used in animal breeding as well as in veterinary medicine. The current state of the art for the purification of eCG from serum is pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. Two simplified processes, including the use of magnetic microsorbents for the purification of eCG have been developed. The processes reduce or even omit the use of organic solvents and the required solid-liquid separation steps, thus making them potential candidates for a first commercial application of magnetic beads in bioprocessing.

Evaluation of process efficiency and bioequivalence of biosimilar recombinant human chorionic gonadotropin (rhCG).

BACKGROUND: Human chorionic gonadotropin (hCG) is a therapeutic protein used for ovulation induction in women with infertility. Dong-A Pharm. Co. has developed recombinant hCG (rhCG) [product code DA-3803] produced in Chinese hamster ovary cells and evaluated its biologic properties, such as biologic potency, efficacy, and pharmacokinetic profile, compared with a reference product, Ovidrel®. OBJECTIVE: The purpose of this study was to evaluate the efficiency of the purification process of Dong-A rhCG (DA-3803) and its bioequivalence from a biosimilar perspective. METHODS: The efficiency of the purification process was estimated through scale-down clearance studies for viruses, endotoxins, host cell DNAs (HCDs) and host cell proteins (HCPs). To confirm bioequivalence, the in vivo/in vitro biologic potency, ovulation induction rate, and pharmacokinetic profile of DA-3803 were compared with those of Ovidrel®. RESULTS: In the clearance studies, the lowest log reduction value (LRV) for model viruses was 8.43. LRVs for endotoxins, HCDs, and HCPs were greater than 5.27, 16.36, and 3.37, respectively. DA-3803 showed equivalent potency with Ovidrel®, and similarity between DA-3803 and Ovidrel® was observed in an efficacy evaluation that measured ovulation induction. The bioequivalence was also confirmed in a rat pharmacokinetic study, which compared pharmacokinetic parameters such as maximum serum concentration, area under the concentration-time curve, time to reach maximum serum concentration, and half-life. In a comparison of different isoform groups of DA-3803, it was shown that the potency and pharmacokinetic profile depend on the sialic acid content. CONCLUSION: The purification process of DA-3803 was effective in removing the major process impurities, and DA-3803 showed similar biologic properties to the reference drug, Ovidrel®.

Simplified purification of equine chorionic gonadotropin (eCG)--an example of the use of magnetic microsorbents for the isolation of glycoproteins from serum.

Classical purification of the glycoprotein equine chorionic gonadotropin (eCG) from serum includes pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. A simplified process requiring only 1/3 of the solvent and improving the yield from 53 to 65% has been developed. The process comprises an ultra-/diafiltration step after the first ethanol precipitation, directly followed by an adsorption/desorption procedure based on magnetic microadsorbents with N,N-diethyl-ammonium functionalization. The process reaches an overall purification factor of eCG of more than 1800 and an average product activity of 1300 IU(ELISA)/mg. After adapting the parameters of the fractionation and the type of magnetic microadsorbents, the new concept is likely to be transferable to other serum proteins.

Higher birth rate after recombinant hCG triggering compared with urinary-derived hCG in single-blastocyst IVF antagonist cycles: a randomized controlled trial.

In a prospective randomized controlled trial, 119 patients were randomized to receive either recombinant hCG (250 µg) or urinary-derived hCG (10,000 IU) for final oocyte maturation in an antagonist protocol with a fixed dose of recombinant FSH (187.5 IU) and predefined single blastocyst transfer. The delivery rate was improved in the recombinant hCG group compared with the urinary-derived hCG group (44.1 vs. 25.7, respectively); however, adequately powered randomized controlled trials are justified to ascertain whether this difference is true.

Divergent immunomodulatory effects of recombinant and urinary-derived FSH, LH, and hCG on human CD4+ T cells.

This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4(+) T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19-30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4(+) T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4(+) T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-gamma (IFN-gamma) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4(+) T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy.

Gonadotropin preparations: past, present, and future perspectives.

This Educational Bulletin offers past, present and future perspectives on gonadotropin preparations.

Purified human chorionic gonadotropin induces apoptosis in breast cancer.

Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study, the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breast cancer xenografts grown in nude mice were examined. It was shown that intratumoral injection of purified hCG increased the apoptotic index in breast cancer xenografts. These results were supported by the findings that exposure of breast cancer cells to purified hCG decreased cell viability in five different breast cancer cell lines. In some of these cell lines, the effects of hCG in cell viability appear to correlate with activation/expression of the hCG/luteinizing hormone receptor. Preoperative apoptotic induction by factors such as purified hCG may improve local control or work synergistically with neoadjuvant chemotherapy to improve complete pathologic response of locally advanced breast cancer.

Carbon-protein covalent conjugates in non-instrumental immunodiagnostic systems.

An original technique for obtaining stable conjugates using colloid carbon particles as indicator labels has been developed. The reliability and stability of the diagnostic reagents obtained is provided by covalent binding of various affine compounds on the surface of carbon particles. The stability of the reagents has been studied under various storage conditions for 3-10 years. It was shown that even when storage conditions were outside the optimal range, some conjugates preserved their analytical characteristics for 10 years. A number of systems for analytical detection of various ligands have been designed based on the carbon-protein conjugates synthesized. The major characteristics of these systems are their high sensitivity and specificity, reliability, and reproducibility, simplicity in operation, and quick results.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains--selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG.

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose-glycerol-methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8-15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Effect of highly purified human chorionic gonadotropin preparations on the gene expression signature of stromal cells derived from endometriotic lesions: potential mechanisms for the therapeutic effect of human chorionic gonadotropin in vivo.

OBJECTIVE: To investigate alterations in the overall gene expression profile of endometriosis-derived stroma with increasing concentrations of hCG by using the Affymetrix GeneChip U133 Set. DESIGN: In vitro study. SETTING: Academic research institution. PATIENT(S): Women undergoing diagnostic laparoscopic surgery for endometriosis. INTERVENTION(S): Increasing concentrations of hCG, added to fibroblast monocultures from endometriotic lesions. RESULT(S): We have found that hCG concentrations of 0.1 U/mL and higher lead to a dose-dependent increase in the expression of 68 genes. Most of the up-regulated genes encoded proteins that are involved in cell adhesion, intercellular communication, extracellular matrix (ECM) remodeling, apoptosis, and inflammation. We then incubated stromal monocultures from nine patients treated with and without 50 U/mL of hCG and performed reverse transcriptase-polymerase chain reaction (RT-PCR) for selected, highly up-regulated genes to validate our DNA array findings and to confirm that the alterations in the gene expression signature are exemplary of patients with endometriosis. CONCLUSION(S): We have shown that hCG induces dose-dependent alterations in the gene expression profile of stromal cells obtained from endometriotic lesions and have, for the first time, identified potential mechanisms by which hCG might exert its therapeutic effect on endometriotic lesions.