CN1324048C - Method of eliminating endotoxin from interferon preparation - Google Patents

Abstract

The present invention relates to the production of recombinant protein medicine, specifically a method for removing endotoxin from interferon preparations, using chitosan, or sodium deoxycholate, or hexamethylenediamine as an affinity ligand, covalently Combined with porous media, it is operated by dynamic filtration or static adsorption to remove endotoxin in recombinant interferon preparations; the amount of affinity ligand on porous media is 0.02% to 3% nitrogen content. The method of the invention has high removal rate of endotoxin and high recovery rate of interferon activity; good specificity, suitable for production requirements; wide practical range and good prospect.

Description

A method for removing endotoxin from interferon preparations

technical field

The invention relates to the production of recombinant protein medicine, in particular to a method for removing endotoxin from interferon preparations.

Background technique

With the increasing maturity of genetic engineering technology, more and more recombinant protein drugs have been developed and utilized. Since the production process involves Gram-negative bacteria and the separation and purification process is complicated, it will cause endotoxin pollution.

Affinity technology is an effective means of separating and purifying biomacromolecules, which mainly utilizes the principle of affinity between ligands and biomacromolecules; the ligands are covalently bonded to porous media to obtain affinity media, which can be used To selectively adsorb endotoxins and achieve the goal of removing endotoxins from recombinant protein solutions; existing affinity ligands and affinity media include: monoclonal antibodies, polyclonal antibodies, antibiotics, polymyxin B, polyethylene Imine, histidine, gel filtration columns, affinity membranes, particle adsorbents, etc.

Since there are many types of recombinant proteins, their separation and purification steps are different, and the requirements for the endotoxin content in the production process are also different, so the method and effect of removing endotoxin cannot be completely universal. Korean patent (KR8902068) adopts gel filtration column Sephacryl S-200, uses the principle of molecular sieve to separate endotoxin and interferon with different molecular sizes, to remove endotoxin in interferon; but the flow rate is low, 4ml/hr, during the process Need to add other substances such as Triton X-100. The European patent (EP0800862) uses styrene-divinylbenzene copolymers containing sulfonic acid groups and uses the principle of ion exchange to remove endotoxins in recombinant protein tumor necrosis factor or interleukin, mainly to adsorb recombinant proteins. The European patent (EP0337243) uses two reversed-phase liquid chromatography columns in series to purify recombinant human IL-2. While purifying recombinant human interleukin-2, endotoxin impurities are removed, the flow rate is low, and the medium is expensive. U.S. Patent (US4885168) adopts low molecular weight chitosan, and utilizes positively charged amino groups on chitosan to interact with negatively charged nucleic acid and endotoxin to achieve the purpose of removing nucleic acid and endotoxin, but the utilization rate of chitosan Low. Chinese patent (00123290.6) Wei Guilin et al. describes a hydrophobic and positively charged affinity membrane for endotoxin removal. The amine hydrophobic and positively charged affinity ligand is bonded to the cellulose membrane to obtain the corresponding affinity membrane. ; Amines hydrophobic and positively charged affinity ligands include 2-12 carbon hydrocarbon diamines, unsaturated heterocyclic compounds with vinyl groups, quaternary ammonium salts with epoxy groups, and multiple hydrophobic groups Quaternary ammonium salt; it is mainly used in the removal of endotoxins in water. D.Petsch, T.C.Beeskow et al. (1997, Journal of Chromatography B.693, 79-91) used sodium deoxycholate or hexamethylenediamine as the affinity ligand, and used the microporous nylon membrane as the medium to make an affinity Membrane; a single affinity membrane can remove endotoxin in buffer or bovine serum albumin; but the flow rate is low, the resistance is high, the processing volume is small, and it is not easy to scale up.

For the endotoxin in the production process of interferon preparations, it is necessary to judge and select the type of affinity ligand and affinity medium according to its molecular weight, the range of isoelectric point, and the content and type of other substances in the solution to achieve the removal of endotoxin , and the purpose of reducing the loss of interferon activity as much as possible. The methods in the prior art cannot meet the requirements for the production of interferon preparations.

Contents of the invention

The purpose of the present invention is to provide a method for removing endotoxin from interferon preparations with high flow rate, large processing capacity and meeting production requirements.

In order to achieve the above object, the technical scheme adopted in the present invention is: with chitosan, or sodium deoxycholate, or hexamethylenediamine as the affinity ligand, covalently bound to the porous medium, with dynamic filtration or static adsorption ( or static soaking) to remove the endotoxin in the recombinant interferon preparation; wherein the amount of the affinity ligand on the porous medium is: the nitrogen content is 0.02% to 3%.

The porous medium is a polysaccharide medium containing multiple hydroxyl groups, such as cellulose, dextran or agarose; or a synthetic medium containing multiple hydroxyl groups, such as polyvinyl alcohol; the pH of the recombinant interferon solution is pH3.5-6.0; Chitosan is used as the affinity ligand, and the molecular weight of chitosan ranges from 10,000 to 1 million; the type of the porous medium is flat membrane, hollow fiber membrane, pellet or particle; the dynamic filtration of the recombinant interferon solution The flow rate is 1-20ml/min; the operating condition of static adsorption is from 4°C to room temperature, and stand for 2-24 hours.

The present invention has the following advantages:

1. The method of the present invention has a high removal rate of endotoxin and a high recovery rate of interferon activity. The endotoxin concentration of the filtered solution is reduced from >80EU/ml to <10EU/ml, and the recovery rate of interferon activity (potency) is 90%.

2. The present invention has good specificity (different concentrations or different activities of recombinant interferons are processed), and is suitable for production requirements. The endotoxin concentration (<10EU/ml) of the filtered solution meets the production requirements of the Pharmacopoeia.

3. The present invention has a wide practical range and good prospects. It can remove endotoxin pollutants contained in the production of interferon preparations, solve its product quality problems, and meet the production requirements stipulated in the Pharmacopoeia; it is also expected to be applied to the production process of other recombinant protein drugs to remove harmful substances such as endotoxins in biological drugs, Improve the level of drug production in our country; create higher economic benefits.

All in all, the affinity membrane method not only combines the advantages of high flow rate, large surface area, uniform pore size, and small diffusion radius between the liquid and the ligand, but also combines the advantages of high selectivity and high efficiency of the affinity method. The membrane is prepared as a corresponding remover, which can be easily scaled up to meet the needs of drug production. Therefore, the use of affinity membrane to remove endotoxin in interferon preparations has high flow rate, good removal effect, and high recovery rate of active ingredients, which is better than column affinity chromatography.

Detailed ways

Example 1.

Preparation of affinity membrane with chitosan as ligand:

Take 50 pieces of cellulose membranes with a diameter of 47 mm, rinse them with a large amount of water, place them in a 200ml beaker, add 40ml of epichlorohydrin, 120ml of 1M NaOH and 0.19g of NaBH , mix well, and let stand overnight at room temperature. Activation; take the activated membrane the next day, wash it with a large amount of water until neutral, drain, then add 150ml of 1% chitosan (molecular weight: 350,000) solution (prepared by 1% acetic acid solution), overnight at 60°C (16 Hours or more); the next day, take the activated membrane, wash it with a large amount of water until it is neutral, drain it, then add 100m10.25% sodium borohydride solution (prepared with 0.1M pH8.5 phosphate buffer), and perform a reduction reaction at room temperature for 4 hours , take the reduced film, wash it with a large amount of water until neutral, drain and dry. The nitrogen content of the affinity membrane is 0.02%.

Application effect of affinity membrane with chitosan as ligand:

10 pieces of affinity membranes were made into corresponding separators, and 200ml of interferon preparations were filtered at a flow rate of 9ml/min (interferon titer 91406IU/ml, solution pH3.5), and the endotoxin concentration of the solution after filtration was from> 80EU/ ml is reduced to <10 EU/ml, and the recovery rate of interferon activity (titer) is 90%.

Example 2.

Preparation of affinity membrane with hexamethylenediamine as ligand:

Get 320 pieces of cellulose films with a diameter of 47 mm, rinse them with a large amount of water, put them in a 1000ml beaker, add 650ml of oxidizing solution (containing 2% NaIO ), and activate them overnight in a 30°C oven; the next day, take The activated membrane was washed with a large amount of water until it was neutral, drained, and then 800ml of 1% hexamethylenediamine (prepared in 0.1M pH=7 phosphate buffer) was added to the membrane, and the cross-linking reaction was carried out in a 30°C oven overnight; the cross-linked membrane was taken the next day. The membrane was washed with a large amount of water until it was neutral, drained, added 800ml of 0.6% NaBH4 (prepared with 0.1MpH=9 phosphate buffer) for reduction, and baked at 30°C overnight. Wash again with plenty of water until neutral, drain and tumble dry. The nitrogen content of the affinity membrane is 2.2%.

Application effect of affinity membrane with hexamethylenediamine as ligand:

10 affinity membranes were made into corresponding separators, and 225ml of interferon preparations were filtered at a flow rate of 18ml/min (interferon titer 93976IU/ml, solution pH4.2), and the endotoxin concentration of the solution after filtration was from> 80EU/ ml is reduced to <10EU/ml, and the recovery rate of interferon activity (titer) is 88%.

Example 3.

Preparation of affinity membrane with sodium deoxycholate as ligand:

Take 50 pieces of cellulose membranes with a diameter of 47mm, wash them with a large amount of water, put them in a 200ml beaker, add 40ml of epichlorohydrin, 120ml of 1M NaOH and 0.19g of NaBH4, mix well, and let stand overnight at room temperature , to activate; the next day, take the activated membrane, wash it with a large amount of water until it is neutral, drain it, then add 125ml of 1% hexamethylenediamine solution (prepared with 1% pH9 phosphate buffer), overnight at 60°C (more than 16 hours) ); Take the membrane the next day, wash it with a large amount of water until it is neutral, drain it, then add 125ml of 0.1M MES buffer solution with pH 6.0, then add 2.5 grams of sodium deoxycholate and 1.25 grams of EDC, mix well, and leave overnight at room temperature . Take the cross-linked film, wash it with plenty of water until neutral, drain and dry. That is, the affinity membrane with sodium deoxycholate as ligand.

Application effect of affinity membrane with sodium deoxycholate as ligand:

10 affinity membranes were made into corresponding separators, and 200ml of interferon preparation was filtered at a flow rate of 9ml/min (interferon titer 91406IU/ml, solution pH6), and the endotoxin concentration of the filtered solution was reduced from > 80EU/ml To <40EU/ml, the recovery rate of interferon activity (titer) is 70%.

Claims (6)

1. from interferon formulation, remove endotoxic method for one kind, it is characterized in that: with chitosan, or sodium deoxycholate, or hexanediamine is as affinity ligand, be covalently bound on the porous medium, mode with dynamic filtration or Static Adsorption is operated, in order to remove the intracellular toxin in the recombinant interferon preparation; Wherein the amount of affinity ligand on porous medium is nitrogen content 0.02%～3%.

2. remove endotoxic method according to claim 1 is described from interferon formulation, it is characterized in that: described porous medium is to contain polyhydric polysaccharide media fibers element, dextran or agarose; Or contain polyhydric synthetic medium polyvinyl alcohol.

3. remove endotoxic method according to claim 1 is described from interferon formulation, it is characterized in that: the pH of interferon formulation is pH 3.5-6.0.

4. remove endotoxic method according to claim 1 is described from interferon formulation, it is characterized in that: be affinity ligand with the chitosan, the molecular weight ranges of chitosan is 10,000-1,000,000.

5. remove endotoxic method according to claim 1 is described from interferon formulation, it is characterized in that: the pattern of porous medium is flat sheet membrane, hollow-fibre membrane, bead or particle.

6. according to the described a kind of endotoxic method of removing from interferon formulation of claim 1, it is characterized in that: the flow velocity of described dynamic filtration interferon formulation is 1-20ml/min.