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Process for the purification of human growth hormone polypeptides using affinity resins comprising specific ligands

A technology for human growth hormone and growth hormone, applied in the field of purified growth hormone polypeptide, can solve the problems of high buffer consumption, biological substance pollution, low overall yield and so on

Inactive Publication Date: 2011-10-12
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Conventional chromatography involving multiple steps suffers from one or more of the following disadvantages: low overall yield, high buffer consumption, long process time and investment in process equipment, increased labor
[0021] However, affinity resins with protein ligands are very expensive to manufacture, as well as chemically and conformationally less stable than small synthetic ligands, and inherently have purified rhGH be degraded by protein fragments from protein ligands or Risk of contamination with other biological substances from affinity resin production with protein ligands

Method used

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  • Process for the purification of human growth hormone polypeptides using affinity resins comprising specific ligands
  • Process for the purification of human growth hormone polypeptides using affinity resins comprising specific ligands
  • Process for the purification of human growth hormone polypeptides using affinity resins comprising specific ligands

Examples

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preparation example Construction

[0217] Preparation of affinity resin

[0218] Affinity resins can in principle be prepared in 2 fundamentally different ways, namely (i) by synthesis of ligands in free form and subsequent immobilization of ligands to solid phase materials either directly or via linkers (see above), or (ii ) by functionalizing the solid phase material and subsequently synthesizing one or more ligands sequentially. As far as the first variant is concerned, immobilization techniques are readily available in the art, eg in Hermanson et al. (see above). As far as the second variant is concerned, techniques are also readily available, such as solid-phase peptide synthesis techniques and derivative techniques known in the art [Fields, G. B. et al. (1992) Principles and practice of solid-phase peptide synthesis. In Synthetic Peptides: A User's Guide (Grant, G.A., ed.), pp. 77-183, W. H. Freeman] and [Fields, G. B., ed. (1997) Solid-phase peptide synthesis. Methods in Enzymology 289. 3) Dorwald, F....

Embodiment 1

[0331] Development of a small molecule affinity resin for the purification of human growth hormone (hGH) using a solid-phase combinatorial approach together with encoded bead technology.

[0332] hGH is a protein hormone that stimulates growth and cell reproduction in humans. It binds its receptor hGHbp by forming an active 1:2 (hGH:hGHbp) complex. Although the hormone binds at the same site on its receptor, the 2 binding sites on hGH are structurally different from site 1 with the highest affinity. Site 1 is a large protein surface, comprising more than 30 amino acids on each protein. Affinity was focused on a few residues of the receptor, notably Trp104 and Trp169.

[0333] In silico screening and library design.

[0334] To construct small-molecule mimics of natural ligands, a branched structure with 3 diversity points was chosen (IV)

[0335] ,

[0336] wherein AA2 and AA1 are amino acid residues, and CA is a carboxyl residue. To increase the likelihood of discov...

Embodiment 2

[0370] A ligand with the following structure was synthesized on Fractogel Amino,

[0371]

[0372] And the test was carried out by the same operation as described in Example 1. The resulting resin has a binding capacity of <0.5 mg / mL. Selectivity not tested.

[0373] The carboxylic acid residue of L19 is a substituted napthoyl and is structurally similar to CA3, CA4, CA5, CA9 and CA17 in Example 1, according to figure 2 All of these result in very low to moderate mean fluorescence values. On this basis, L19 would be expected to have a low affinity for hGH. On the other hand, according to Table 1, combining the amino acid residues of L19 (AA1, AA2) = (Tyr, Arg) is expected to result in high affinity for hGH. However, it appears that the expected positive effect of the combination of amino acid residues on the affinity of the ligand for hGH is offset by the expected negative effect of the carboxylic acid residues, resulting in a net low hGH affinity of L19.

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Abstract

The present invention relates to a novel process for the purification of growth hormone polypeptides, e.g. recombinant human Growth Hormone. The process utilizes an affinity resin comprising a solid phase material having immobilized thereto one or more low-molecular weight synthetic ligands. The affinity resins enable the separation of Growth Hormone from closely related proteins.

Description

technical field [0001] The present invention relates to novel methods for purifying growth hormone polypeptides, such as recombinant human growth hormone. The method utilizes an affinity resin comprising a solid phase material having one or more low molecular weight synthetic ligands immobilized thereto. Affinity resins enable separation of growth hormone from closely associated proteins. Background technique [0002] Therapeutic proteins are produced in living cells and must be purified from complex mixtures of proteins and other biological species before use in patients. This separation can be very laborious and expensive. Numerous chromatographic materials have been described for use in such separations. [0003] Affinity chromatography enables the selective and reversible adsorption of biological substances such as proteins to complementary binding substances, such as affinity ligands immobilized on a solid phase material, usually packed in an affinity Porous, polyme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/61C07K5/02C07K7/02
CPCC07K5/0808C07K5/0812C07K5/0815C07K5/0817C07K5/0823C07K7/02C07K14/61
Inventor J.E.拉斯姆森P.M.圣希莱雷M.罗伊斯C.B.席奥特K.J.詹森
Owner NOVO NORDISK AS
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