CN101298468B - Method for removing endotoxin from protein medicine in one step - Google Patents
Method for removing endotoxin from protein medicine in one step Download PDFInfo
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- CN101298468B CN101298468B CN2007100154546A CN200710015454A CN101298468B CN 101298468 B CN101298468 B CN 101298468B CN 2007100154546 A CN2007100154546 A CN 2007100154546A CN 200710015454 A CN200710015454 A CN 200710015454A CN 101298468 B CN101298468 B CN 101298468B
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Abstract
The invention discloses a simple and effective one-step removal method for esotoxins of protein drugs, the step thereof is by adopting cation exchange chromatographic column to remove the esotoxins of protein drugs by one step. The invention is characterized in that the process is simple and easy to control, the recovery rate of protein drugs is up to 90 percent above, the concentration of esotoxins in original liquid of protein drugs recycled can be lowered to less than 10 EU/ml, meeting the production requirements specified by the Pharmacopoeia; the invention also has the advantages of great amount of treatment capacity once, strong process stability and can be used in the production process of any recombinant protein drug, thus providing technical foundation for large scale production of protein drugs.
Description
Technical field
The invention belongs to the purified technology of protein in the medical preparation field, relate in particular to an endotoxic step removal method in the pharmaceutical grade protein.
Background technology
Bacteriotoxin generally can be divided into two classes, and a class is extracellular toxin (Exotoxin), and it is a kind of toxic protein, is that bacterium is secreted into the toxicant outside the thalline in process of growth; Another kind of is intracellular toxin (Endotoxin), is a kind of lipopolysaccharides on the gram-negative bacteria cell wall and the mixture of trace of albumin.
Bacterial endotoxin does not discharge when the bacterium animation, have only when the dead self-dissolving of bacterium or when sticking on other cell, just show its toxicity, its chemical ingredients is distributed widely in the lipopolysaccharides of cell wall layer of Gram-negative bacteria (as intestinal bacteria, Brucella, Corynebacterium diphtheriae, Bacillus proteus, Salmonellas etc.) and other microorganism (as chlamydozoan, Rickettsiae, spirochete etc.), mainly is made up of 0-specificity chain, core polysaccharide, lipoid A three parts.
Contain intracellular toxin or entered the human recycle system and can cause multiple transformation reactions by the preparation of contaminated with endotoxins.As high fever, shiver with cold even body gram.Therefore pharmacopeia has been done strict qualification to endotoxic content in the product that directly enters blood.
Along with development of biology, increasing recombinant protein drug is exploited, because most of recombinant protein drugs are by Gram-negative bacteria such as escherichia coli expression, must be before purifying protein earlier with the bacterial classification broken wall, to discharge albumen, lipopolysaccharides in the cell walls also is discharged in the damping fluid in a large number, easily causes endotoxic pollution.In process of production because of raw and auxiliary material, factors such as production environment and individual's operation also can cause contaminated with endotoxins simultaneously.
Because endotoxic character utmost point heterogeneity (surfactant-like on the structure, molecular weight does not wait from several thousand to tens million of), all quite stable under high temperature, strong acid, highly basic, traditional various column chromatography methods can only be removed or deactivation part intracellular toxin, but are difficult to the disposable removal of intracellular toxin.And the purifies and separates step of various recombinant proteins has nothing in common with each other, and is also variant to the requirement of endotoxin content, so be difficult to find the endotoxic method of a kind of general removal.
Usually adopt following method to carry out endotoxic removal in the bio-pharmaceuticals industry, but these methods all exist certain defective:
1, anion-exchange chromatography: can from DNA and basic protein, remove intracellular toxin, but because endotoxic unhomogeneity, as high molecular, the intracellular toxin of structure proximate adipose membrane or adipose capsule, outside electric charge is not exposed to, then can't combine with the anion-exchange chromatography medium.
2, affinity chromatography: the particularly utilization of immunosorbent in affinity chromatography make the purifying of biomacromolecule become simply, but shortcoming is the expense costliness.
3, gel permeation chromatography: utilize molecular sieve can effectively remove intracellular toxin, but its treatment capacity is little, the treatment time is longer.
4, reversed phase chromatography: the adsorbable endotoxic lipoid A of reversed phase chromatography medium district.But the organic reagent that reversed phase chromatography is used may have influence on activity of proteins, so seldom be used to downstream purification.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides an a kind of step simple efficient, that meet big production requirement and removes endotoxic method in the pharmaceutical grade protein, this method can guarantee that endotoxin concns reaches the officinal regulation in the pharmaceutical grade protein, can reclaim pharmaceutical grade protein efficiently again.
The inventive method is to utilize endotoxic iso-electric point lower usually, at pH〉2 o'clock electronegative, the principle of debond cation-exchange chromatography medium designs.Utilize cation-exchange chromatography,, pharmaceutical grade protein is combined on the chromatography media, and the intracellular toxin major part obtains wash-out by adjusting the condition of level pad and elution buffer.After treating that intracellular toxin is by abundant wash-out, use the damping fluid of high salt, high pH or the original purifying elution requirement wash-out of pharmaceutical grade protein to reclaim pharmaceutical grade protein.
An endotoxic step removal method in the pharmaceutical grade protein of the present invention, step is as follows:
(1) be that 5mmol/L-100mmol/L, pH value are that the level pad of 4.0-7.0 carries out balance to positively charged ion chromatography post with concentration, to baseline;
(2) get on the stoste of pharmaceutical grade protein behind the sample, use this level pad balance again, to baseline;
(3) be that 10mmol/L-100mmol/L, pH value are the elution buffer of 5.0-9.0 8-9 column volume of cation-exchange chromatography post wash-out after to last sample with concentration, to intracellular toxin by abundant wash-out;
(4) with contain 0.3mol/L-0.5mol/L salt, pH is that the elution buffer of the elution buffer of 5.0-9.0 or the former purification step of the pharmaceutical grade protein cation-exchange chromatography post after to step (3) wash-out carries out wash-out, to the abundant wash-out of pharmaceutical grade protein quilt, collect elution peak, get the stoste of pharmaceutical grade protein.
Above-mentioned steps (1) or (2) described level pad can be selected according to the difference of pharmaceutical grade protein, commonly used is acetate buffer, phosphate buffered saline buffer, Tris-HCl damping fluid or citrate buffer, the preferred 10mmol/L-70mmol/L of concentration, the preferred 4.0-6.0 of pH value.
Wherein: further preferred acetate buffer of described level pad or phosphate buffered saline buffer, concentration be 20mmol/L-50mmol/L, pH value 4.0-5.0 more preferably more preferably.
In the endotoxic step removal method, the chromatography media of described cation-exchange chromatography post is sepharose (Sepharose) class or polystyrene (Source) class in the above-mentioned pharmaceutical grade protein; Aglucon is carboxymethyl (CM), sulfopropyl (SP) or sulphur methyl (S).
The chromatography media of described cation-exchange chromatography post is further preferably: SP Sepharose F.F, SP Sepharose H.P, SP Sepharose B.B, CM Sepharose F.F, CM Sepharose CL-6B, one of SOURCE30-S.
Wherein, the chromatography media of described cation-exchange chromatography post most preferably is a SP Sepharose F.F chromatography media.
The described elution buffer of above-mentioned steps (3) can be selected according to the difference of pharmaceutical grade protein, elution buffer commonly used is: acetate buffer, phosphate buffered saline buffer or citrate buffer, concentration is preferably 20mmol/L-70mmol/L, and the pH value is preferably 5.2-8.0.
Wherein: further preferred acetate buffer of described elution buffer or phosphate buffered saline buffer, concentration is 30mmol/L-50mmol/L more preferably, and the pH value is 5.5-7.5 more preferably.
Utilize the inventive method, under the effect of the described elution buffer of step (3), intracellular toxin and pharmaceutical grade protein are owing to the difference of iso-electric point is fully separated, and method is simple, and endotoxin removal is complete, and the target protein Loss Rate is low.According to the difference of pharmaceutical grade protein, select suitable elution buffer to realize reclaiming the purpose of pharmaceutical grade protein in the step (4).After the processing of pharmaceutical grade protein through step (4), the pharmaceutical grade protein rate of recovery that obtains can be up to more than 90%, and endotoxic concentration is reduced to＜10EU/ml in the stoste of the pharmaceutical grade protein of recovery, reaches (＜10EU/ml) the production requirement of pharmacopeia regulation; And disposable treatment capacity is big, can be used for the production process of any recombinant protein drug, for the scale operation of pharmaceutical grade protein provides technical foundation.
Generally between 70%-90%, protein recovery of the present invention is up to more than 90% for protein recovery in the prior art.In fact intracellular toxin of the present invention can be removed fully, and prior art endotoxin removal rate is generally between 50%-90%.Therefore compared with prior art, excellent results of the present invention is: technological design of the present invention is reasonable, and technological process is simple, is easy to control, is convenient to operation; The rate of recovery height of pharmaceutical grade protein, disposable processing protein content is big, and technology stability is strong, is fit to large-scale industrial production.
Embodiment
By the following examples explanation the present invention is further set forth, these embodiment should be as restriction of the present invention.
Embodiment 1: Pegylation recombined human granulocyte stimulating factors (PEG-rhG-CSF) the endotoxic removal test of stoste
1, use pH4.0, concentration is that 10mmol/L acetate-sodium acetate buffer carries out balance to sepharose (SP Sepharose F.F) positively charged ion chromatography post, to baseline;
2, get PEG-rhG-CSF stoste (concentration〉3mg/ml, buffer system: 10mmol/L acetate-sodium acetate buffer, pH4.0) doubly go up sample in the back with the described level pad of step (1) dilution 3-5, use this level pad balance to baseline again;
3, use pH5.2, concentration is that the positively charged ion chromatography post of 10mmol/L acetate-sodium acetate buffer after to last sample carries out a wash-out 8-9 column volume, makes intracellular toxin by abundant wash-out;
4, with the pH that contains 0.5mol/L NaCl be 5.0, concentration is 10mmol/L acetate-sodium acetate buffer wash-out positively charged ion chromatography post, collects elution peak, reclaims PEG-rhG-CSF stoste.
In the PEG-rhG-CSF stoste after the recovery endotoxic concentration by 10EU/3mg reduces to＜10EU/6mg, reached the intracellular toxin control criterion of PEG-rhG-CSF stoste, the rate of recovery of PEG-rhG-CSF stoste 90%.
Embodiment 2: Pegylation recombined human granulocyte stimulating factors (PEG-rhG-CSF) the endotoxic removal test of stoste
1, use pH5.0, concentration is that 10mmol/L acetate-sodium acetate buffer carries out balance to sepharose (CM Sepharose F.F) positively charged ion chromatography post, to baseline;
2, get PEG-rhG-CSF stoste (concentration〉3mg/ml, buffer system: 10mmol/L acetate-sodium acetate buffer, pH4.0) doubly go up sample in the back with the described level pad of step (1) dilution 3-5, use this 1 level pad balance to baseline again;
3, use pH5.4, concentration is that the positively charged ion chromatography post of 10mmol/L acetate-sodium acetate buffer after to last sample carries out a wash-out 8-9 column volume, makes intracellular toxin by abundant wash-out;
4, with the pH that contains 0.5mol/L NaCl be 5.0, concentration is 10mmol/L acetate-sodium acetate buffer wash-out positively charged ion chromatography post, collects elution peak, reclaims PEG-rhG-CSF stoste.
In the PEG-rhG-CSF stoste after the recovery endotoxic concentration by 10EU/3mg reduces to＜10EU/6mg, reached the intracellular toxin control criterion of PEG-rhG-CSF stoste, the rate of recovery of PEG-rhG-CSF stoste 90%.
Embodiment 3: the endotoxic removal test of recombined human granulocyte stimulating factors (rhG-CSF) stoste
1, use pH4.0, concentration is 50mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid carries out balance to sepharose (CM Sepharose F.F) positively charged ion chromatography post, to baseline;
2, get rhG-CSF stoste (buffer system: 50mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid pH5.0) is gone up sample, uses the described level pad balance of step (1) to baseline again;
3, use pH5.5, concentration is 50mmol/L Na
2HPO
4-NaH
2PO
4The positively charged ion chromatography post of damping fluid after to last sample carries out a wash-out 8-9 column volume, makes intracellular toxin by abundant wash-out;
4, use pH6.86, concentration is 50mmol/L Na
2HPO
4-NaH
2PO
4Buffer solution elution is collected elution peak, reclaims rhG-CSF stoste.
In the rhG-CSF stoste after the recovery endotoxic concentration by 10EU/300 μ g reduces to＜10EU/6mg, and the rate of recovery of rhG-CSF stoste 90%.
Embodiment 4: the endotoxic removal test of recombined human granulocyte stimulating factors (rhG-CSF) stoste
1, use pH4.0, concentration is 100mmol/L Na
2HPO
4-citrate buffer solution carries out balance to sepharose (CM SepharoseCL.6B) positively charged ion chromatography post, to baseline;
2, get rhG-CSF stoste (buffer system: 50mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid pH4.0) is gone up sample, uses the described level pad balance of step (1) to baseline again;
3, use pH5.2, concentration is 100mmol/L Na
2HPO
4The positively charged ion chromatography post of-citrate buffer solution after to last sample carries out a wash-out 8-9 column volume, makes intracellular toxin by abundant wash-out;
4, use pH6.86, concentration is 50mmol/L Na
2HPO
4-NaH
2PO
4Buffer solution elution is collected elution peak, reclaims rhG-CSF stoste.
In the rhG-CSF stoste after the recovery endotoxic concentration by 10EU/300 μ g reduces to＜10EU/6mg, and the rate of recovery of rhG-CSF stoste 90%.
Embodiment 5: the endotoxic removal test of regrouping human interleukin-11 (rhIL-11) stoste
1, use pH7.0, concentration is 10mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid carries out balance to sepharose (SP Sepharose B.B) positively charged ion chromatography post, to baseline;
2, get rhIL-11 stoste (buffer system: 10mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid pH7.0) is gone up sample, uses the described level pad balance of step (1) to baseline again;
3, use pH9.0, concentration is 10mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid 8-9 column volume of positively charged ion chromatography post wash-out after to last sample makes intracellular toxin by abundant wash-out;
4, with the pH that contains 0.3mol/LNaCl be 7.0, concentration is 10mmol/L Na
2HPO
4-NaH
2PO
4Buffer solution elution is collected elution peak, reclaims rhIL-11 stoste.
In the rhIL-11 stoste after the recovery endotoxic concentration by 10EU/1.5mg reduces to＜10EU/6mg the rate of recovery of rhIL-11 stoste 90%.
Embodiment 6: the endotoxic removal test of regrouping human interleukin-11 (rhIL-11) stoste
1, use pH7.0, concentration is that 20mmol/L Tris-HCl damping fluid p-poly-phenyl ethene (SOURCE30-S) positively charged ion chromatography post carries out balance, to baseline;
2, get rhIL-11 stoste (buffer system: 10mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid pH7.0) is gone up sample, uses the described level pad balance of step (1) to baseline again;
3, use pH9.0, concentration is 20mmol/L Tris-HCl damping fluid 8-9 column volume of positively charged ion chromatography post wash-out after to last sample, makes intracellular toxin by abundant wash-out;
4, with the pH that contains 0.3mol/L NaCl be 7.0, concentration is 10mmol/L Na
2HPO
4-NaH
2PO
4Buffer solution elution is collected elution peak, reclaims rhIL-11 stoste.
In the rhIL-11 stoste after the recovery endotoxic concentration by 10EU/1.5mg reduces to＜10EU/6mg the rate of recovery of rhIL-11 stoste 90%.
Embodiment 7: the endotoxic removal test of rhMGF (rhSCF) stoste
1, use pH4.0, concentration is 20mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid carries out balance to sepharose (SP Sepharose H.P) positively charged ion chromatography post, to baseline;
2, get rhIL-11 stoste (buffer system: 20mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid pH4.0) is gone up sample, uses the described level pad balance of step (1) to baseline again;
3, use pH5.6, concentration is 20mmol/L Na
2HPO
4-NaH
2PO
4Damping fluid 8-9 column volume of positively charged ion chromatography post wash-out after to last sample makes intracellular toxin by abundant wash-out;
4, with pH be 7.0, concentration is 20mmol/L Na
2HPO
4-NaH
2PO
4Buffer solution elution is collected elution peak, reclaims rhSCF stoste.
In the rhSCF stoste after the recovery endotoxic concentration by 10EU/600 μ g reduces to＜10EU/6mg the rate of recovery of rhSCF stoste 90%.
Claims (8)
1. an endotoxic step removal method in the pharmaceutical grade protein, step is as follows:
(1) be that 5mmol/L-100mmol/L, pH value are that the level pad of 4.0-7.0 carries out balance to positively charged ion chromatography post with concentration, to baseline;
(2) get on the stoste of pharmaceutical grade protein behind the sample, use the described level pad balance of step (1) again, to baseline;
(3) be that 10mmol/L-100mmol/L, pH value are the elution buffer of 5.0-9.0 8-9 column volume of cation-exchange chromatography post wash-out after to last sample with concentration, to intracellular toxin by abundant wash-out;
(4) with contain 0.3mol/L-0.5mol/L salt, pH is that the elution buffer of the 5.0-9.0 cation-exchange chromatography post after to step (3) wash-out carries out wash-out, to pharmaceutical grade protein by abundant wash-out, collect elution peak, the stoste of pharmaceutical grade protein;
Wherein, step (1) or (2) described level pad are acetate buffer, phosphate buffered saline buffer, Tris-HCl damping fluid or citrate buffer; The described elution buffer of step (3) is: acetate buffer, phosphate buffered saline buffer or citrate buffer.
2. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 1, it is characterized in that: step (1) or (2) described level pad are acetate buffer, phosphate buffered saline buffer, Tris-HCl damping fluid or citrate buffer, and concentration is that 10mmol/L-70mmol/L, pH value are 4.0-6.0.
3. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 2, it is characterized in that: step (1) or (2) described level pad are acetate buffer or phosphate buffered saline buffer, and concentration is that 20mmol/L-50mmol/L, pH value are 4.0-5.0.
4. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 1, it is characterized in that: the chromatography media of described cation-exchange chromatography post is sepharose class or polystyrene type; Aglucon is carboxymethyl, sulfopropyl or sulphur methyl.
5. remove an endotoxic step removal method in the pharmaceutical grade protein according to a described step of claim 4, it is characterized in that: the chromatography media of described cation-exchange chromatography post is: SP Sepharose F.F, SP Sepharose H.P, SP SepharoseB.B, CM Sepharose F.F, CM Sepharose CL-6B, one of SOURCE30-S.
6. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 5, it is characterized in that: the chromatography media of described cation-exchange chromatography post is: SP Sepharose F.F chromatography media.
7. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 1, it is characterized in that: the described elution buffer of step (3) is: acetate buffer, phosphate buffered saline buffer or citrate buffer, concentration is 20mmol/L-70mmol/L, and the pH value is 5.2-8.0.
8. according to an endotoxic step removal method in the described pharmaceutical grade protein of claim 7, it is characterized in that: the described elution buffer of step (3) is: acetate buffer or phosphate buffered saline buffer, concentration are 30mmol/L-50mmol/L, and the pH value is 5.5-7.5.
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| CN2007100154546A CN101298468B (en) | 2007-04-30 | 2007-04-30 | Method for removing endotoxin from protein medicine in one step |
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| CN2007100154546A CN101298468B (en) | 2007-04-30 | 2007-04-30 | Method for removing endotoxin from protein medicine in one step |
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| CN102850450B (en) * | 2011-07-01 | 2014-08-13 | 齐鲁制药有限公司 | Purification method of pegylated recombinant human granulocyte colony stimulating factor |
| CN107312809A (en) * | 2016-04-27 | 2017-11-03 | 辽宁师范大学 | The rLj-RGD3 protein preparation methods of endotoxin-free |
| CN114452444B (en) * | 2022-01-27 | 2022-10-21 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1123550A (en) * | 1994-02-16 | 1996-05-29 | Csl有限公司 | Process for removing endotoxins |
| CN1523037A (en) * | 2003-02-21 | 2004-08-25 | 中国科学院大连化学物理研究所 | Method of eliminating endotoxin from interferon preparation |
-
2007
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1123550A (en) * | 1994-02-16 | 1996-05-29 | Csl有限公司 | Process for removing endotoxins |
| CN1523037A (en) * | 2003-02-21 | 2004-08-25 | 中国科学院大连化学物理研究所 | Method of eliminating endotoxin from interferon preparation |
Non-Patent Citations (1)
| Title |
|---|
| Zhou et al..pH–conductivity hybrid gradient cation-exchange chromatography for process-scale monoclonal antibody purification.《Journal of Chromatography A》.2007,第1175卷第69-71页. * |
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