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B-chain modified monomer quick-acting insulin and method for preparing same

A technology of insulin and monomers, applied in the fields of medicine and pharmacy, can solve problems such as chemical side reactions, difficulty in large-scale production, and side effects

Inactive Publication Date: 2005-11-23
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process is difficult to produce on a large scale due to the following main disadvantages: (1) small peptides such as GFFY(But)Obut need to be chemically synthesized; (2) when trypsin enzymatically transpeptides, the yield is within 80%; (3) ) After enzymatic transpeptidation, the intermediate needs to be treated with strong acid, such as trifluoroacetic acid (TFA) to remove the protecting group; (4) The process is complicated and there are chemical side reactions
[0007] In addition, currently available monomeric insulins are highly immunogenic, especially when used in high doses, or when used across species (e.g., human insulin for dogs, or porcine insulin for humans). ), will cause certain side effects
Therefore, there is a lack of monomeric insulin that has a wide range of applications and has reduced immunogenicity for pets such as humans and dogs.

Method used

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  • B-chain modified monomer quick-acting insulin and method for preparing same
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  • B-chain modified monomer quick-acting insulin and method for preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Construction of expression vector

[0076] The codon usage of P. pastoris is basically similar to that of S. cerevisiae, but there are some differences in individual amino acids such as Glu [Zhao, X., Huo, K.K. and Li, Y.Y.(2000). Synonymous codon usage in Pichia pastoris Sheng Wu Gong Cheng XueBao16(3):308-11.]. Chemical synthesis of monomeric insulin B following the codon preference of P. pastoris 27 K-DTrI precursor gene fragment (Figure 1).

[0077] Chemically synthesize code B by the same method 1 A B 27 K-DTrI (Figure 2) and (DesB 1 )B 27 DNA of K-DTrI (Fig. 3). Similar to S.cerevisiae, a suitable prepro-leader is helpful for the secretion and expression of insulin precursors in P.pastoris, and the chemically synthesized DNA fragments are cloned into the EcoRI and NotI sites of the pPIC9K plasmid (Invitrogen Company) to form α- Mating factor leader-EAEAYVEFK-MIP expression framework. Among them, the EA-rich spacer peptide (such as EAEAYVEFK, SEQ ID NO: 9) ...

Embodiment 2

[0079] Plasmid transformation and screening of MIP expression strains

[0080] 3ug of pPIC9K / MIP plasmid linearized by Bgl II was electrotransformed into Pichia pastoris GS115(his4) (purchased from NRRL, deposit number NRRL Y-15851, US6,730,499), 0.4cm electric shock cup, 2.4KV, 5.3ms, continuous electric shock Twice, MD plates were coated with transformed yeast cells. After the plasmid pPIC9K / MIP is linearized by Bgl II, after entering the yeast cells, part of it will be re-circularized and integrated into the chromosome through single-point insertion to obtain Mut+ phenotype transformants, and the other part of the linear plasmid will be integrated into the chromosome by substitution to obtain Muts Phenotypic transformants, during which multicopy insertion integrations spontaneously form. Pick 200 single clones from the MD plate and inoculate them on YPD plates containing 0, 0.5, and 4 mg / ml of G418 respectively. The clones that grow colonies on all three plates enter the p...

Embodiment 3

[0082] Shake Flask Fermentation and Purification of Insulin Precursor

[0083] Inoculate high-expressing MIP yeast strains into 100 ml of YPD medium, culture in a 500 ml Erlenmeyer flask at 30°C for 48 hours, let the cells settle after standing, pour off the supernatant, and then add 100 ml of MM medium (containing 1% methanol) , continue culturing at 30°C for 96 hours, add methanol once every 24 hours, and collect the fermentation broth by centrifugation after the fermentation is completed.

[0084] The hydrophobic adsorption column XAD-7 (purchased from Sigma Company) has good adsorption capacity for MIP, and can remove inorganic salts, most polysaccharides, pigments and miscellaneous proteins in the fermentation broth. The XAD-7 column was washed with methanol at 5Vc (that is, 5 times the total column bed volume, the same below), and balanced with 10Vc water, then centrifuged at 10Vc to decellularize the fermentation broth, then washed with 10Vc water, 2Vc 15% ethanol conta...

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Abstract

The invention provides a B-chain modified monomer quick-acting insulin and method for preparation and use, wherein the insulin analog not only possesses the property of the monomer insulin (such as physiological pH, non-polymeric at high concentration), thus the overall biologos of the normal insulin is sustained, but also the immunogen property is lowered down substantially.

Description

technical field [0001] The present invention relates to the fields of medicine and pharmacy. More specifically, the present invention relates to insulin B chains with reduced immunogenicity, monomeric insulins containing the B chains, pharmaceutical compositions containing the monomeric insulins, and methods of making and uses thereof. Background technique [0002] The incidence of diabetes is increasing year by year, and it can cause death from cardiovascular disease. It is one of the major diseases threatening human health today. Hyperglycemia is the cause of various diabetic complications, and strictly controlling the blood sugar concentration within the normal range 24 hours a day is the most critical factor to prevent and delay the complications of late diabetes. [0003] Insulin has been an irreplaceable drug since it was used to treat diabetes in the 1920s. However, the secretion of insulin in a normal human body is regulated by the blood sugar concentration. Genera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62
Inventor 张友尚费俭丁金国崔大敷蔡国强石嘉豪
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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