CN1093428C

CN1093428C - Chemical modification of microporous nylon film and preparation of affinity film with histidine ligand

Info

Publication number: CN1093428C
Application number: CN97103649A
Authority: CN
Inventors: 商振华; 周冬梅; 郭为; 于亿年
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 1997-03-26
Filing date: 1997-03-26
Publication date: 2002-10-30
Anticipated expiration: 2017-03-26
Also published as: CN1194179A

Abstract

The present invention relates to a preparation method for nylon compatible films with histidine petunidin, which is characterized in that a nylon microporous film of which the molecular weight distribution is from 2 to 80 myriadaltons is used as raw materials; the method comprises the following steps: (1) a nylon microporous film is hydrolyzed in a hydrochloric acid solution to change an amido link on the film into an amino group; (2) an epoxy method is used to cause amino group on the nylon film after hydrolyzation to be reacted with chlorine on epoxy chloropropane, and a nylon film with an epoxy group is produced; (3) the nylon film combined with the epoxy group is used to form a spacer arm together with hexanediamine through covalent connection; (4) and a glutaraldehyde method is adopted to cause the nylon film connected with the spacerarm to form a compatible film through covalent combination with histidine petunidin. The compatible film can be used for effectively removing endotoxin from solutions, and provides an effective way for removing endotoxin.

Description

The preparation of the chemical modification of micropore nylon membrane and band histidine aglucon affinity membrane thereof

Technical field

The invention provides a kind of chemical modification method of micropore nylon membrane and utilize the crosslinked histidine of this modified micropore nylon membrane to make affinity membrane.This affinity membrane can be used for endotoxin in the solution is removed.

Background technology

In the manufacturing process of biological medicine preparation, more or less can bring some thermal source materials into, bacterial endotoxin is wherein important a kind of, it is that the Gram-negative bacteria growing discharges or produced by the cracking of okioplast film when dead.Its existence has great harm to human body, and the denier endotoxin enters human body can cause fever, blood vessel dilatation, and symptoms such as diarrhea, even cause faintness and dead.Endotoxemia is the very high very dangerous disease of a kind of death rate clinically.The endotoxin molecule is complex structure not only, and is of a great variety, and molecular weight is big, and distribution is wide, therefore removes various medical water, and medicine, the endotoxin in biochemical preparation and the blood are very difficult tasks.The physical absorption of Cai Yonging in the past, acid, alkali or oxidising agent decompose, and it is all undesirable that technology such as ion-exchange or ultrafiltration are carried out removal effect to it.Adopt in recent years high specific and optionally affinity chromatography obtained very big success, succeeded in developing several posts system and chromatographic media and can be used for removing endotoxin in water and some pharmaceutical preparations, but after chemical modification, made the affinity membrane of being with the histidine aglucon and still be not reported with the technology that this film is used for endotoxic removal with nylon 66 films.

Summary of the invention

The chemical modification method that the purpose of this invention is to provide a kind of micropore nylon membrane, and this crosslinked histidine of micropore nylon membrane through chemical modification is made affinity membrane.This affinity membrane can be used for the endotoxin in the solution is effectively removed, for endotoxic removal provides a kind of effective new way again.

The preparation method of the chemical modification of micropore Buddhist nun Buddhist nun film of the present invention and band histidine aglucon affinity membrane thereof presses step:

1. with hydrochloric acid nylon 66 miillpore filters are hydrolyzed, even the amido link on the film changes amino into, its course of reaction is as (1):

2. the activation of hydrolysis nylon membrane promptly makes amino and the reaction of the chlorine on the epoxychloropropane on nylon 66 films after the hydrolysis with epoxy method, generates nylon 66 films of band epoxy radicals, and its course of reaction is as (II):

3. being connected of activation nylon 66 films and spacerarm, promptly in conjunction with nylon 66 films and the covalently bound arm at interval of hexamethylene diamine of epoxy radicals, its course of reaction is as (III):

4. immobilized the make affinity membrane of affinity ligand on film, nylon 66 films and the histidine aglucon covalent bond that promptly adopt glutaraldehyde method to make to connect spacerarm are made affinity membrane, its course of reaction as (IV) and (V):

Specifically, in the above-mentioned reaction (1), be that nylon 66 films with molecular weight distribution from 20,000 to 800,000 dalton's (ton) are raw material, in 1～6MHCl solution in 25～40 ℃ of following oscillating reactions 10～72 hours, make the abundant hydrolysis of amido link on nylon 66 films become amino, fully wash with distilled water after the hydrolysis to neutrality.In the said hydrolyzed process as can shorten the reaction time with dense HCl solution; Reaction (II) is carried out in alkaline solution, under 50～60 ℃, hydrolysis nylon 66 films are under agitation put into the dimethyl sulfoxide (DMSO) be the epoxychloropropane of solvent to react 3～5 hours, make amino and the reaction of the chlorine on the epoxychloropropane on the nylon membrane, generate nylon 66 films of band epoxy radicals, reaction finishes the back earlier with rare HCl solution, repeatedly film is rinsed well with distilled water again.Can replace epoxychloropropane with glutaraldehyde in the above-mentioned reaction.Reaction (III) is carried out in ethanol water, with NaOH or HCl the pH value of solution value is transferred to 11, to join while stirring in the ethanol water that contains hexamethylene diamine through the film that reaction (II) is handled, under 50～60 ℃, reacted 2～4 hours, reaction finishes the back earlier with rare HCl solution, and is with deionized water that film is fully clean again, and sops up water remaining on the film with filter paper.The also available hexamethylene diamine of hexamethylene diamine in the above-mentioned reaction, octamethylenediamine, the certain herbaceous plants with big flowers diamines replaces.Reaction (IV) is at NaH ₂PO ₄Make glutaraldehyde and nylon 66 film reactions that connect spacerarm in the buffer solution (pH7.2～7.5), reaction temperature is 20～30 ℃, reaction carries out making in 2～4 hours the amino on the spacerarm to change aldehyde radical into, and reaction finishes the back and uses the buffer solution flushing membrane, uses the distilled water washes clean again.Above-mentioned reaction also can replace glutaraldehyde to carry out with epoxychloropropane.Reaction (V) is also at NaH ₂PO ₄Finish in the buffer solution, the pH value is 7.2～7.5, under 25～30 ℃, make histidine and nylon 66 film reactions that activated with glutaraldehyde 4～6 hours, histidine is immobilized to film, after reaction finishes, film is clean with distilled water flushing, and promptly making of the present inventionly has interactional nylon 66 affinity membranes of specificity to endotoxin.

The specific embodiment

Below by embodiment technology of the present invention is given to illustrate further.

The preparation of embodiment 1 nylon 66 affinity membranes

With average pore size is 0.45 μ m, thickness is 0.12mm, molecular weight distribution is a raw material from 2～800,000 dalton's's (ton) nylon 66 films, in 2MHCl solution in 35 ℃ of following oscillating reactions 50 hours, finish the hydrolysis (1) of nylon 66 films, with distilled water fully wash until neutrality, be cut into the film that diameter is 50mm thereafter.

In 50ml water, add 12gNaOH and stir even all backs adding 100ml dimethyl sulfoxide (DMSO), 20ml epoxychloropropane, 0.5gKBH ₄, after stirring, saying good-bye adds 50 nylon 66 films after the hydrolysis while stirring, 60 ℃ of down reactions 3 hours, finishes reaction (II), thereafter earlier with the rare HCl of 0.01m ol/L, again with the distilled water flushing membrane repeatedly of saying good-bye.

Preparation 80ml contains 50% ethanol water, add the 1g hexamethylene diamine, with 0.1mol/LNaOH or 0.1mol/L HCl solution the pH value of solution value is transferred to 11 after the stirring and dissolving, say good-bye and add nylon 66 films that obtain after above-mentioned (II) reaction while stirring, in 50 ℃ of following oscillating reactions 2 hours, reaction (III) finishes the back and use earlier 0.01NHCl, and is with deionization that film is thoroughly clean and sop up water remaining on the film with filter paper again.

At 80ml 0.05mol/L NaH ₂PO ₄Add the 75g25% glutaraldehyde solution in the buffer solution (pH7.2～7.5), after stirring, saying good-bye adds nylon 66 films that above-mentioned reaction (III) is handled, and in 25 ℃ of following oscillating reactions 3 hours, finishes reaction (IV), uses earlier 0.1mol/L NaH ₂PO ₄Buffer solution is said good-bye and is washed 3 times, washs film with distilled water again.

At 80ml 0.1mol/NaH ₂PO ₄Add the 0.5g histidine, 0.4gKBH in (pH7.2～7.4) buffering ₄Stir, in nylon 66 films that add while stirring under 30 ℃ after above-mentioned reaction (IV) is handled, reaction can be finished reaction (V) in 5 hours under vibration, and film is taken out, and saying good-bye to rinse well with distilled water just to make nylon 66 affinity membranes of the present invention.The content of the immobilized affinity ligand of this film is 50～100 μ mol/g, and the desorption constant is 3.0 * 10 ^-8Mol/L, this medium is suitable for doing affinity purification.

Endotoxic removal in the embodiment 2 medical water

Be assembled into membrane separator with embodiment 1 50 prepared nylon 66 affinity membranes, allow 1 liter of endotoxin concns be 1.3eu/ml, at flow is by this separator under 0.5～2ml/ divides, collecting and measuring by endotoxin concns in the water behind the membrane separator with the quantitative chromogenic assay method of tripeptides UV absorption is 0.03eu/ml, and endotoxic clearance is more than 95%.

3 couples of embodiment contain endotoxic removal in the amino acid whose medical injection

Utilize the membrane separator of example 2, the medical injection 500ml that forms by essential amino acids such as Gly, Thr, Val, Mat, Leu, Phe, Lys, make it contain endotoxin 220eu, under 0.5～1.5ml/ shunt volume, pass through example 2 described affinity membrane separators, collect endotoxin and various amino acid whose content in the liquid after measuring film respectively, the result shows endotoxic clearance more than 75%, to the amino acid whose rate of recovery more than 90%.

Endotoxic removal in the solution such as 4 pairs of albumin of embodiment, lysozyme

Allow the concentration be that bovine serum albumin(BSA) (BSA) and the lysozyme of 5mg/ml, 1mg/ml absorbs (in them contained in plain concentration be 100eu/ml) by by diameter 50mm respectively, the membrane separator of 30 nylon 66 histidine affinity membranes assemblings, in solution salt concentration is 0.1～0.2mol/L, the pH value is 3～8, flow is to pass through membrane separator under the experiment condition that divides of 0.5～2ml/, measure to collect the concentration of endotoxin and BSA in the liquid, lysozyme respectively, endotoxic clearance is respectively 82% and 85%, and the rate of recovery of BSA and lysozyme can reach 95% and 90% respectively.

Nylon 66 affinity membranes that utilize the inventive method preparation are at solution salt concentration 0.1～0.2mol/L, in the solution of pH value 3～8, interior plain in the multiple pharmaceutical preparation are had preferable removal effect.

The regeneration of embodiment 5 nylon 66 affinity membranes

Used nylon 66 affinity membranes of example 2～4 can be with the NaOH that contains 0.2mol/L and 0.5%, the NaTDC of W/W does not have heat source water solution and washs, make affinity media obtain regeneration, nylon affinity membrane after the regeneration carries out the endotoxic separation process of example 2～4 removals equally can reach same effect, and regenerative process can be carried out repeatedly.

Comparative example

The agarose Sepharose4B that biotechnology and applied biology magazine (volume was 134 pages in 1988 the 10th) have been reported the production of human Sweden Pharmacia companies such as Japanese H.Matsumae is a raw material, the post affinity chromatograph filling of the band histidine affinity ligand of after chemical modification, making, aglucon content is 0.2～0.5mg/g, the affinity chromatographic column of loading with this affinity media is about 90% to endotoxic clearance in the water, to bovine serum albumin(BSA), endotoxic clearance is about 80% in the biologic products such as lysozyme, and their rate of recovery is respectively 90% and 86%.Compare with the film affinity chromatography, the major defect of post affinity chromatography is to be difficult to realize scale, serialization, automation mechanized operation, and might make the large biological molecule inactivation.

Claims

1. preparation method with histidine aglucon nylon affinity membrane is characterized in that with molecular weight distribution being that 2～800,000 dalton's's (ton) nylon-microporous membrane is a raw material, presses step:

1), make the amido link on the film change amino into in the hydrochloric acid solution nylon-microporous membrane being hydrolyzed;

2) make amino and the reaction of the chlorine on the epoxychloropropane on the nylon membrane after the hydrolysis with epoxy method, generate the nylon membrane of band epoxy radicals;

3) with in conjunction with the nylon membrane and the covalently bound arm at interval of hexamethylene diamine of epoxy radicals;

4) adopt glutaraldehyde method to make the nylon membrane that connects spacerarm become affinity membrane with histidine aglucon covalent bond.

2. according to the described preparation method of claim 1, it is characterized in that step:

1) be in 1～6MHCl solution in 25～40 ℃ of following oscillating reactions 10～72 hours, make the amido link hydrolysis on the nylon membrane become amino, fully wash to neutrality with distilled water after the hydrolysis;

2) be in alkaline solution, to carry out, under 50～60 ℃, the hydrolysis nylon membrane is under agitation put into the dimethyl sulfoxide (DMSO) be the epoxychloropropane of solvent to react 3～5 hours, make amino and the reaction of the chlorine on the epoxychloropropane on the nylon membrane, generate the nylon membrane of band epoxy radicals, reaction finishes the back earlier with rare HCl solution, repeatedly film is rinsed well with distilled water again;

3) in ethanol water, carry out, with NaOH or HCl the pH value of solution value is transferred to 11, will be through reacting 2) film handled joins in the ethanol water that contains ethylenediamine while stirring, under 50～60 ℃, reacted 2～4 hours, reaction finishes the back earlier with rare HCl solution, and is with deionized water that film is fully clean again, and sops up water remaining on the film with filter paper;

4) be at NaH ₂PO ₄Make glutaraldehyde and second connect the nylon membrane reaction of spacerarm in the buffer solution (pH7.2～7.5), reaction temperature is 20～30 ℃, reaction carries out using in 2～4 hours the amino on arm to change aldehyde radical into, and reaction finishes the back and uses the buffer solution flushing membrane, uses the distilled water washes clean again; Then at NaH ₂PO ₄In the buffer solution, the pH value is 7.2～7.5, makes histidine and the nylon membrane reaction that activated with glutaraldehyde 4～6 hours under 25～30 ℃, and histidine is immobilized to film, and is after reaction finishes that film is clean with distilled water flushing, makes nylon affinity membrane.

3. according to the described preparation method of claim 1, it is characterized in that step 2) replace epoxychloropropane to react with glutaraldehyde.

4. according to the described preparation method of claim 1, it is characterized in that the step 3) ethylenediamine, octamethylenediamine or certain herbaceous plants with big flowers diamines replace hexamethylene diamine to react.