Non-animal-derived LMW (low molecular weight) heparin, preparation method therefor and application of non-animal-derived LMW heparin

A low-molecular-weight heparin, animal-derived technology, used in medical preparations, drug combinations, and pharmaceutical formulations containing active ingredients, can solve the problem of inability to achieve efficient preparation of non-animal-derived products, fine structure, large difference in weight-average molecular weight, and technology. problems such as poor stability, achieving major industrialization and clinical application prospects, easy separation, and quality controllable effects

Active Publication Date: 2020-05-15
SHANDONG UNIV
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

Studies have confirmed that the simple chemical modification method has severe reaction conditions and poor selectivity, and cannot convert the GlcA residue of the sugar chain into 2-O-sulfated iduronic acid (IdoA2S) necessary for heparin
In contrast, enzymatic modification is a more feasible strategy. For example, the research group of Robert J. Linhardt (Carbohydr Polym, 2015, 122:399-407) used chemical enzymatic modification of K5CPS to obtain anti-Xa and anti-IIa activity equivalent However, this method has low efficiency and poor process stability, and it is difficult to realize large-scale preparation, and the fine structure and weight average molecular weight of the obtained product are quite different from those of heparin
So far, it has not been possible to efficiently prepare non-animal-derived products with a typical structure of heparin / low molecular weight heparin and strong anticoagulant activity using K5CPS as a raw material

Method used

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  • Non-animal-derived LMW (low molecular weight) heparin, preparation method therefor and application of non-animal-derived LMW heparin
  • Non-animal-derived LMW (low molecular weight) heparin, preparation method therefor and application of non-animal-derived LMW heparin
  • Non-animal-derived LMW (low molecular weight) heparin, preparation method therefor and application of non-animal-derived LMW heparin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Preparation of Escherichia coli K5 exopolysaccharide K5CPS

[0076] Escherichia coli K5 is cultured in a fermenter, the medium is glucose medium, and its composition is: 20g / L glucose, 10-300mg / L vitamin B1, 13.5g / L KH 2 PO 4 , 4.0g / L (NH 4 ) 2 HPO 4 , 1.4g / L MgSO 4 ·7H 2 O, 1.7g / L citric acid, 10.0mL / L trace elements, pH 7.0. Among them, the preparation of trace elements is as follows: 10.0g FeSO 4 ·7H 2 O, 2.0g CaCl 2 , 2.2g ZnSO 4 ·7H 2 O, 0.5g MnSO 4 4H 2 O, 1.0g CuSO 4 ·H 2 O, 0.1g (NH 4 ) 6 Mo 7 o 24 4H 2 O and 0.02g Na 2 B 4 o 7 10H 2 O was dissolved in 1 L of 2M HCl. Feed medium: 300~600g / L glucose, 20g / LMgSO 4 ·7H 2 O. 0.15-0.25g / L vitamin B1.

[0077] The fermentation conditions are as follows: the inoculum size is 4%, the pH is adjusted by 30% ammonia water, the temperature is 37° C., the rotation speed is 500 rpm, and the cultivation is carried out for 36 hours.

[0078] After the fermentation is completed, the fermenta...

Embodiment 2

[0081] Example 2: Chemical N-deacetylation / N-sulfation modification of exopolysaccharide K5CPS

[0082] Dissolve 100 mg of the pure exopolysaccharide K5CPS prepared in Example 1 in 25 mL of 2M sodium hydroxide solution, react in a water bath at 60°C for 7 hours, cool to room temperature after completion, adjust the pH to 7.0-8.0 with 2M hydrochloric acid, and then Add 0.3g of sodium carbonate and 0.3g of sulfur trioxide-trimethylamine mixture, and react at 47°C for 12h, then add an equal amount of sodium carbonate and sulfur trioxide-trimethylamine mixture, and continue to react at 47°C for 12h. After the reaction, cool to room temperature, adjust the pH to 7.0 with hydrochloric acid, dialyze with a dialysis bag with a molecular weight cut-off of 3500Da to remove salt, and freeze-dry the dialysate to obtain the N-deacetylated / N-sulfated exopolysaccharide K5CPS derivative. Pure intermediate 1.

[0083] The resulting intermediate product 1 was fully cut with heparanase I, II, I...

Embodiment 3

[0084] Example 3: Heparanase III partial depolymerization of intermediate 1

[0085] Escherichia coli expressing heparanase III was constructed according to literature (Carbohydr Polym, 2017, 173:276-285). The engineered strains were cultured in LB medium with a resistance of 50 μg / mL ampicillin, cultured at 37°C until OD 600 0.6 ~ 0.8, 0.2mM IPTG induced 18 ~ 20h at 22 ℃. After the cultivation, centrifuge at 8000rpm for 15min to collect the thalli, crush and filter on ice, and then use Ni Sepharose 6B affinity column chromatography to purify. The purity of the obtained recombinant heparanase III is greater than 80%.

[0086] Weigh the intermediate product 1 that 100mg embodiment 2 makes and dissolve in 50mL buffer solution (50mM Tris-HCl, 10mM CaCl 2 , the solvent is water, the pH is 7.0), add 36 μL of heparanase III solution with a total activity of 371mIU, depolymerize in a constant temperature water bath at 37°C for 30 minutes, and deactivate the enzyme in a boiling wate...

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Abstract

The invention relates to non-animal-derived LMW (low molecular weight) heparin, a preparation method therefor and application of the non-animal-derived LMW heparin. The preparation method disclosed bythe invention comprises the steps of performing a one-step chemical method and a variety of enzyme catalysis methods in order: extracting an exopolysaccharide K5CPS, subjecting the exopolysaccharideK5CPS to chemical-method N-deacetylation / N-sulfation modification, and then, carrying out partial depolymerization in a buffer solution through heparinase III; and subjecting obtained LMW products toenzyme-method C-5 epimerization / 2-O-sulfation modification, 6-O-sulfation modification and 3-O-sulfation modification sequentially, thereby obtaining a product with anti-FXa and anti-FIIa activity. The invention provides a novel method for preparing the LMW product with typical structure characteristics of the heparin and remarkable anticoagulation activity. According to the method, raw materialsare non-animal-derived, the quality is controllable, the risk of contamination is low, adopted reaction conditions are mild and efficient, the obtained product is easy to separate, and large-scale preparation of the LMW heparin with good structural homogeneity and high anticoagulation activity can be achieved.

Description

technical field [0001] The invention relates to a non-animal source low molecular weight heparin and a preparation method and application thereof, belonging to the technical field of biomedicine. Background technique [0002] Heparin is a highly sulfated, heterogeneous glycosaminoglycan, which is formed by linking glucosamine (GlcN) with iduronic acid (IdoA) or glucuronic acid (GlcA) through 1,4-glycosidic bonds. It is a natural anticoagulant substance with a weight average molecular weight between 5000Da and 25000Da. Heparin has been used clinically as an anticoagulant drug for more than 80 years, and it has played an irreplaceable and important role so far. [0003] At present, commercialized heparin mainly includes unfractionated heparin and low molecular weight heparin. Among them, unfractionated heparin (unfractionated heparin, UFH) is mainly extracted from animal organs such as pig small intestine mucosa and bovine lung; UFH is obtained by physical, chemical or Parti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/18A61K31/727A61P7/02
CPCC12P19/26C12P19/18A61K31/727A61P7/02
Inventor 刘纯慧唐凤琰王亚利马亚卿张桂姣
Owner SHANDONG UNIV
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