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Gonadotrophins for folliculogenesis

A follicle and superovulation technology, applied in the field of gonadotropins, can solve problems such as limiting the number of embryos

Inactive Publication Date: 2005-04-20
MERCK SERONO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a limiting factor in the success of assisted reproductive techniques as it limits the number of embryos that can be transferred and / or cryopreserved

Method used

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  • Gonadotrophins for folliculogenesis
  • Gonadotrophins for folliculogenesis
  • Gonadotrophins for folliculogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] The mensuration of embodiment 1 Z numerical value

[0102] Based on the glycan profile, the Z value of the glycoprotein can be determined.

[0103] Recombinant human FSH was reacted with hydrazine at 100°C for 5 hours, and glycan moieties were released using an Oxford GlycoSciences GlycoPrep(R) 1000 automatic analyzer or equivalent.

[0104] Glycans are separated from unreacted hydrazine and amino acid hydrazides using a glass bead-coated column. Glycans were eluted with sodium acetate reagent.

[0105] Glycans are acetylated with acetic anhydride. Excess reagents were removed using a mixed bed ion exchange column. Any unreduced glycans were collected in dilute acetate buffer.

[0106] Glycans were collected on 0.5 meter filters (Oxford GlycoSciences) and lyophilized. Under acidic conditions, the dry polysaccharides are reacted with a reducing agent with a fluorophore (such as 2-aminobenzamide) at 65° C. for 120 minutes to label the dry polysaccharides.

[0107] T...

Embodiment 2

[0124] Embodiment 2 Measurement of Antenna Index (AI)

[0125] Glycans were released from the peptide backbone by hydrazinolysis as described in Example 1, and these carbohydrates were then fluorescently labeled with 2-aminobenzamide.

[0126] 2-Aminobenzamide-labeled glycans were enzymatically desialylated with sialidase (Vibrio cholerae) in 250 mM ammonium acetate pH 5.5 containing 20 mM calcium chloride for 18 hours at 37°C. A starting amount of glycans of 100 μg rhFSH uses about 0.05 U sialidase.

[0127] The desialylated glycans were vacuum-dried and stored at -20°C until separation by preparative reverse-phase HPLC under the following conditions:

[0128] The column is a GlycoSep® R column;

[0129] The flow rate of the mobile phase is 0.7 ml / min;

[0130] Eluent A: Ammonium acetate 50mM pH6.0

[0131] Eluent B: Ammonium acetate 50mM pH6.0 with 8% acetonitrile

[0132] Detect with a fluorometer and set λ 激发 : 330nm, λ 发射 : 420nm;

[0133] • Column temperature: 30...

Embodiment 3

[0145] Example 3 FSH is separated into different components based on the degree of sialylation

[0146] Recombinant FSH was separated into acidic and basic components by anion exchange chromatography on DEAE Sepharose FF.

[0147] The column used was packed with DEAE Sepharose FF resin: Φ 1.6 x 20 cm (XK Pharmacia or equivalent) for experimental grade purity (about 60 mg whole protein), Φ 3 for larger scale purity .4 x 40 cm (Vantadge Amicon or whatever);

[0148] The flow rate of the mobile phase is 150-250 cm / hour;

[0149] Equilibrium buffer 1: 2M Tris-HCl pH7.0±0.1;

[0150] Equilibrium buffer 2: 25mM Tris-HCl pH7.0±0.1, conductivity is 2.15±1.5mS / cm;

[0151] Elution buffer 1: 25mM Tris pH 7.0±0.1, 35mM NaCl, conductivity 5.8±0.4mS / cm (this buffer elutes the more basic isoform);

[0152] Elution buffer 2: 25mM Tris pH 7.0±0.1, 150mM NaCl, conductivity 18.3±0.5mS / cm (this buffer elutes the more acidic isoform);

[0153] Regeneration solution: 0.5M sodium hydroxide, 1M...

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Abstract

The invention provides a follicle stimulating hormone (FSH) preparation with high sialylation and high efficacy.

Description

technical field [0001] The present invention relates to the field of gonadotropins, in particular their use in assisted reproductive techniques, ovulation induction, intrauterine insemination (IUI) and infertile male patients. Background of the invention [0002] Gonadotropins are a group of heterodimeric glycoproteins that include follicle stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotropin (CG). These hormones regulate male and female reproductive function. [0003] Each of these hormones consists of two non-covalently linked subunits: one is an α-subunit common to FSH, LH, and hCG, and the other is unique to each hormone and confers biological Specific β-subunits. [0004] In all gonadotropins, each subunit has an asparagine-linked (N-linked) oligosaccharide side chain. They are linked at positions 52 and 78 in the common α-subunit of the human hormone. In human FSH and CG, two N-linked oligosaccharide side chains are attached to the β-subu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02A61K38/24A61P5/24A61P15/08C07K14/59
CPCA61K38/24C07K14/59A61P15/08A61P5/24
Inventor E·罗梅耶C·E·吉亚托西欧
Owner MERCK SERONO SA
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