CN104610436A - Preparation method of glatiramer acetate - Google Patents
Preparation method of glatiramer acetate Download PDFInfo
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- CN104610436A CN104610436A CN201510053303.4A CN201510053303A CN104610436A CN 104610436 A CN104610436 A CN 104610436A CN 201510053303 A CN201510053303 A CN 201510053303A CN 104610436 A CN104610436 A CN 104610436A
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- acetic acid
- carboxylic acid
- acid anhydride
- preparation
- copaxone
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
Abstract
The invention belongs to the technical field of preparation methods of polypeptide drugs, particularly relates to a preparation method of glatiramer acetate. Raw materials including L-alanine-N-carboxylic acid anhydride, L-tyrosine-N-carboxylic acid anhydride, protected L-glutamic acid-N-carboxylic acid anhydride and protected L-lysine-N-carboxylic acid anhydride are subjected to a ring opening polymerization reaction in the presence of dioxane medium-alkali to obtain protected glatiramer. The preparation method disclosed by the invention is simple in process operation, high in yield and low in cost and has bright application prospect and high economic value.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical field, be specifically related to a kind of preparation method of acetic acid copaxone.
Background technology
Acetic acid copaxone is the acetate of the random polymerization mixture of ALANINE, Pidolidone, 1B and TYR, and their molar average is than being 0.427:0.141:0.095:0.338, and molecular-weight average is 5000-9000Da, and structural formula is: (C
5h
9nO
4c
3h
7nO
2c
6h
14n
2o
2c
9h
11nO
3)
xxC
2h
4o
2, by Israel's pharmaceutical factory manufacture that TEVA researches and develops, commodity are called Copaxone, and check and approve in 1996 or U.S. FDA and be used for the treatment of multiple sclerosis, medicine freezes crystalline flour end for white to linen sterilizing, pack another attached water for injection and are modulated into injection liquid use.
Obstruction and rejection is had to draw the report prepared for thunder a lot of both at home and abroad; as far back as Euro.J.Immune.1242(1971; Tietelbaum et al) and US 3849550 in plaid matching draw the preparation method for thunder to be described; the method wherein described is all draw for thunder for raw material obtains shielded lattice by ring-opening polymerization with ALANINE, TYR, γ-benzyl-Pidolidone and N-TFA base-1B-N-carboxylic acid anhydride, then obtains acetic acid copaxone by acidolysis, alkaline lysis and purifying.Follow-up patent US 5800808, US 6048898, US 6362161, US 6620847, US 7199098, CN 102844325, CN 102718963, CN 102112485, CN 103641897 etc. are all prepared lattice according to the method and draw for thunder.
In patent WO 2004/043995 with ALANINE, TYR, Z or with substituting group Z 1B and benzyl ester or prepare shielded lattice with the Pidolidone-N-carboxylic acid anhydride of substituting group benzyl ester for raw material and draw for thunder; and in glacial acetic acid by directly obtaining acetic acid copaxone crude product after catalytic hydrogenolysis; purifiedly again obtain acetic acid copaxone; the advantage of the method is that synthesis step is few; and going can not cut off peptide chain in protection process compared with acidolysis, but the method need carry out improving production cost and operation easier in autoclave.
In patent CN 103265624 with ALANINE, Z or with the TYR of substituting group Z, Z or with substituting group Z 1B and benzyl ester or prepare shielded lattice with the Pidolidone-N-carboxylic acid anhydride of substituting group benzyl ester for raw material and draw for thunder, again in glacial acetic acid by obtaining acetic acid copaxone crude product after acidolysis/or hydrogenolysis, purifiedly again obtain acetic acid copaxone, the advantage of the method makes phenolic hydroxyl group not oxidizable by Z or with the protection of substituting group Z group to tyrosine, thus decrease the generation of by product; But go protection to cut off peptide chain by acidolysis thus reduce productive rate, in addition, adopting catalytic hydrogenolysis method to go protection to carry out in autoclave thus to improve production cost and operation easier.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art and provide one to enhance productivity, reduce the preparation method of the acetic acid copaxone of production cost.
The object of the present invention is achieved like this:
A preparation method for acetic acid copaxone, step is as follows:
1), with ALANINE-N-carboxylic acid anhydride, TYR-N-carboxylic acid anhydride, shielded Pidolidone-N-carboxylic acid anhydride and shielded 1B-N-carboxylic acid anhydride for raw material, in dioxane, carry out ring-opening polymerization under base catalysis, prepare shielded lattice and draw for thunder;
2), the shielded lattice obtained in step 1) are drawn carry out scission reaction for thunder and alkaline hydrolysis agent after obtain lattice and draw for thunder crude product;
3), by step 2) in the lattice that obtain draw and carry out purifying for thunder crude product and obtain acetic acid copaxone.
In the preparation method of above-mentioned acetic acid copaxone, the structure of described ALANINE-N-carboxylic acid anhydride is:
In the preparation method of above-mentioned acetic acid copaxone, the structure of described TYR-N-carboxylic acid anhydride is:
In the preparation method of above-mentioned acetic acid copaxone, the structure of described shielded Pidolidone-N-carboxylic acid anhydride is:
Wherein R
1for methyl or ethyl.
In the preparation method of above-mentioned acetic acid copaxone, the structure of described 1B-N-carboxylic acid anhydride is:
Wherein R
2for trifluoroacetyl group.
Based on the above, the reaction consumption mol ratio of the ALANINE-N-carboxylic acid anhydride in described step 1), TYR-N-carboxylic acid anhydride, shielded Pidolidone-N-carboxylic acid anhydride and shielded 1B-N-carboxylic acid anhydride is: 0.90-1.05:0.20-0.25:0.25-0.30:0.75-0.85.
Based on the above, the solvent in described step 1) is Isosorbide-5-Nitrae-dioxane, and its consumption is 16-20 times of protected amino acid total mass used.
Based on the above, the polymerization temperature in described step 1) is 25-35 DEG C.
Based on the above, the alkaline catalysts in described step 1) is diethylamine or triethylamine, and its consumption is the 0.6-1.0% of protected amino acid total mass used.
Based on the above, described step 2) in the concentration of alkaline hydrolysis agent be 5-10%wt, its consumption is that every gram of shielded lattice draw and need the agent of 10-20 mL alkaline hydrolysis for thunder, and described alkaline lysis temperature of reaction is 25-35 DEG C, and the reaction times is 5-10 hour.
Based on the above, the purifying in described step 3) is dialysis purifying, ultrafiltration;
Described dialysis purifying is dialysis tubing acetic acid copaxone crude product solution being put into 2000 Da, and be placed in pH be 5.5 ± 0.3 ammonium acetate buffer solution dialyse to pH and reach 6.5-7.0, again by retentate with 1.0% acetic acid reclaim until pH reaches 4.0-4.5 again, and with water diafiltration until pH reaches 5.0-5.5, carry out concentrated also freeze-drying finally by 3000 Da molecular weight mwco membranes;
Described ultrafiltration is that acetic acid copaxone crude product solution Glacial acetic acid is adjusted pH to 5.0-5.5, adopts 3000Da filter membrane to carry out ultrafiltration.
Described step 2) in alkaline hydrolysis agent be sodium hydroxide with the one of the aqueous solution of the aqueous solution of the aqueous solution of methyl alcohol or ethanol, hydrated barta and methyl alcohol or ethanol, lithium hydroxide and methyl alcohol or ethanol or two kinds and mix above.
Shielded Pidolidone-N-carboxylic acid anhydride in described step 1) adopts ester group to protect Pidolidone-N-carboxylic acid anhydride, and described ester group is ethyl ester or methyl esters.
The preparation method of acetic acid copaxone provided by the invention; adopt the Pidolidone-N-carboxylic acid anhydride of alcohol carboxylic esterification protection and the N-carboxylic acid anhydride of other three seed amino acids to be that raw material is prepared shielded lattice and drawn for thunder, only go protection and purifying to obtain acetic acid copaxone (reaction process is shown in Fig. 1) through ring-opening polymerization, alkaline lysis.Preparation method's technological operation provided by the invention is simple, yield is high and cost is low, and is with a wide range of applications and economic worth.Adopt ester group to protect to Pidolidone-N-carboxylic acid anhydride, after repolymerization reaction terminates, by basic hydrolysis deprotection base, avoid using autoclave shortening deprotection base, reduce operation easier, be beneficial to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is reacting flow chart of the present invention.
Embodiment
Embodiment 1
The preparation of acetic acid copaxone
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 10.35 g, TYR-N-carboxylic acid anhydride 4.14 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 4.38 g and N-TFA base-1B-N-carboxylic acid anhydride 20.10 g is added in there-necked flask; add 624 mL 1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.4 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 2000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 30.82 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 79.1%.
Sodium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid solution is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.91 g acetic acid copaxone, yield is 69.1%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 8745 dalton; The average molecular fraction of L-Ala, L-glutamic acid, tyrosine, Methionin is respectively: 0.431,0.145,0.082,0.327.
Embodiment 2
The preparation of acetic acid copaxone
The shielded lattice that Example 1 obtains draw the hydrated barta/methanol-water solution for thunder 10 g, 120 mL8% to join in flask, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid solution is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.81 g acetic acid copaxone, yield is 68.1%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 7613Da.
Embodiment 3
The preparation of acetic acid copaxone
The shielded lattice that Example 1 obtains draw the lithium hydroxide/methanol-water solution for thunder 10 g, 120 mL8% to join in flask, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.93 g acetic acid copaxone, yield is 69.3%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 6897Da.
Embodiment 4
The preparation of acetic acid copaxone
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 10.35 g, TYR-N-carboxylic acid anhydride 4.14 g, Pidolidone-ethyl ester-N-carboxylic acid anhydride 4.73 g and N-TFA base-1B-N-carboxylic acid anhydride 20.10 g is added in there-necked flask; add 630 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.4 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 2000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 31.18 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 79.3%.
Lithium hydroxide/the ethanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.95 g acetic acid copaxone, yield is 69.5%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 6928 Da.
Embodiment 5
The preparation of acetic acid copaxone
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 5.18 g, TYR-N-carboxylic acid anhydride 2.07 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.19 g and N-TFA base-1B-N-carboxylic acid anhydride 10.05 g is added in there-necked flask; add 312 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.3 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 16.99 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 87.2%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.92 g acetic acid copaxone, yield is 69.2%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 6286.
Embodiment 6
The preparation of acetic acid copaxone
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 5.18 g, TYR-N-carboxylic acid anhydride 2.07 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.19 g and N-TFA base-1B-N-carboxylic acid anhydride 10.05 g is added in there-necked flask; add 312 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.4 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 16.91 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 86.8%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.92 g acetic acid copaxone, yield is 69.2%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 8546.
Embodiment 7
Protected lattice draw the preparation for thunder
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 6.04 g, TYR-N-carboxylic acid anhydride 2.59 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.63 g and N-TFA base-1B-N-carboxylic acid anhydride 11.39 g is added in there-necked flask; add 363 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.3 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 19.68 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 86.9%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.95 g acetic acid copaxone, yield is 69.5%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 6634Da.
Embodiment 8
Protected lattice draw the preparation for thunder
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 5.18 g, TYR-N-carboxylic acid anhydride 2.07 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.19 g and N-TFA base-1B-N-carboxylic acid anhydride 10.05 g is added in there-necked flask; add 390 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.3 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 16.95 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 87.0%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.91 g acetic acid copaxone, yield is 69.1%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 8146Da.
Embodiment 9
Protected lattice draw the preparation for thunder
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 5.60 g, TYR-N-carboxylic acid anhydride 2.33 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.40 g and N-TFA base-1B-N-carboxylic acid anhydride 10.72 g is added in there-necked flask; add 312 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.3 mL diethylamine, under nitrogen protection stirring reaction 24 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 18.10 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 87.9%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.97 g acetic acid copaxone, yield is 69.7%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 8592Da.
Embodiment 10
Protected lattice draw the preparation for thunder
Under nitrogen protection; ALANINE-N-carboxylic acid anhydride 5.60 g, TYR-N-carboxylic acid anhydride 2.33 g, Pidolidone-methyl esters-N-carboxylic acid anhydride 2.40 g and N-TFA base-1B-N-carboxylic acid anhydride 10.72 g is added in there-necked flask; add 312 mL1; 4-dioxane; at 25-35 DEG C stirring and dissolving completely after; add 0.3 mL diethylamine, under nitrogen protection stirring reaction 30 h.Slowly poured into by mixture in 1000 mL water, after stirring 30 min, solid collected by filtration, obtain the shielded lattice of 18.54 g after being placed in vacuum drying oven drying under reduced pressure 5 h of 30-35 DEG C and draw for thunder, yield is 88.1%.
Lithium hydroxide/the methanol-water solution for thunder, 120 mL8% is drawn to join in flask the shielded lattice of 10 g, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.96 g acetic acid copaxone, yield is 69.6%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 7298Da.
Embodiment 11
The preparation of acetic acid copaxone
The shielded lattice that Example 4 obtains draw the lithium hydroxide/methanol-water solution for thunder 10 g, 240 mL8% to join in flask, 5 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid solution is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.79 g acetic acid copaxone, yield is 67.9%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 6951Da.
Embodiment 12
The preparation of acetic acid copaxone
The shielded lattice that Example 4 obtains draw the hydrated barta/methanol-water solution for thunder 10 g, 120 mL8% to join in flask, 10 h are stirred at 25-35 DEG C, after reaction terminates reaction mixture to be poured in 2 KDa molecular weight mwco membranes and to be placed in ammonium acetate buffer (pH=5.5 ± 0.3) diafiltration, until the pH value of penetrating fluid reaches 6.5-7.0; Retentate 1.0% acetic acid solution is reclaimed again, for the unnecessary acetic acid deionized water diafiltration of removing is until pH value reaches 5-5.5 after reaching 4.0-4.5 to pH value, carry out concentrated also freeze-drying finally by 3 KDa molecular weight mwco membranes, obtain 6.72 g acetic acid copaxone, yield is 67.2%.
The peak-average molecular weight of the acetic acid copaxone that GPC measures is: 8841Da.
Claims (9)
1. a preparation method for acetic acid copaxone, is characterized in that: step is as follows:
1), with ALANINE-N-carboxylic acid anhydride, TYR-N-carboxylic acid anhydride, shielded Pidolidone-N-carboxylic acid anhydride and shielded 1B-N-carboxylic acid anhydride for raw material, in dioxane, carry out ring-opening polymerization under base catalysis, prepare shielded lattice and draw for thunder;
2), the shielded lattice obtained in step 1) are drawn carry out scission reaction for thunder and alkaline hydrolysis agent after obtain lattice and draw for thunder crude product;
3), by step 2) in the lattice that obtain draw and carry out purifying for thunder crude product and obtain acetic acid copaxone.
2. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: the reaction consumption mol ratio of the ALANINE-N-carboxylic acid anhydride in described step 1), TYR-N-carboxylic acid anhydride, shielded Pidolidone-N-carboxylic acid anhydride and shielded 1B-N-carboxylic acid anhydride is: 0.90-1.05:0.20-0.25:0.25-0.30:0.75-0.85.
3. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: the solvent in described step 1) is Isosorbide-5-Nitrae-dioxane, and its consumption is 16-20 times of protected amino acid total mass used.
4. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: the polymerization temperature in described step 1) is 25-35 DEG C.
5. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: the alkaline catalysts in described step 1) is diethylamine or triethylamine, and its consumption is the 0.6-1.0% of protected amino acid total mass used.
6. the preparation method of a kind of acetic acid copaxone according to claim 1; it is characterized in that: described step 2) in the concentration of alkaline hydrolysis agent be 5-10%wt; its consumption is that every gram of shielded lattice draw and need the agent of 10-20 mL alkaline hydrolysis for thunder; described alkaline lysis temperature of reaction is 25-35 DEG C, and the reaction times is 5-10 hour.
7. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: the purifying in described step 3) is dialysis purifying, ultrafiltration;
Described dialysis purifying is dialysis tubing acetic acid copaxone crude product solution being put into 2000 Da, and be placed in pH be 5.5 ± 0.3 ammonium acetate buffer solution dialyse to pH and reach 6.5-7.0, again by retentate with 1.0% acetic acid reclaim until pH reaches 4.0-4.5 again, and with water diafiltration until pH reaches 5.0-5.5, carry out concentrated also freeze-drying finally by 3000 Da molecular weight mwco membranes;
Described ultrafiltration is that acetic acid copaxone crude product solution Glacial acetic acid is adjusted pH to 5.0-5.5, adopts 3000Da
Filter membrane carries out ultrafiltration.
8. the preparation method of a kind of acetic acid copaxone according to claim 1, is characterized in that: described step 2) in alkaline hydrolysis agent be sodium hydroxide with the one of the aqueous solution of the aqueous solution of the aqueous solution of methyl alcohol or ethanol, hydrated barta and methyl alcohol or ethanol, lithium hydroxide and methyl alcohol or ethanol or two kinds and mix above.
9. the preparation method of a kind of acetic acid copaxone according to claim 1; it is characterized in that: the shielded Pidolidone-N-carboxylic acid anhydride in described step 1) adopts ester group to protect Pidolidone-N-carboxylic acid anhydride, and described ester group is ethyl ester or methyl esters.
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