CN113080961B - Endotoxin-free hirudin anticoagulation vacuum blood collection tube and preparation method thereof - Google Patents
Endotoxin-free hirudin anticoagulation vacuum blood collection tube and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medical instruments, in particular to an endotoxin-free hirudin anticoagulation vacuum blood collection tube and a preparation method thereof, recombinant hirudin is coated on the inner wall of the vacuum blood collection tube, and the endotoxin content in every 100 mu g of the recombinant hirudin is less than 0.25EU/ml, wherein the recombinant hirudin is obtained by separating recombinant hirudin protein liquid by adopting anion exchange chromatography, and the method comprises the following steps: sample introduction: sampling the recombinant hirudin protein solution, wherein the sampling amount is 25-40% of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to balance the column; and (3) elution: isocratic elution is carried out by eluent, and an outflow part is collected to obtain recombinant hirudin eluent; column regeneration: isocratic elution is carried out by using the regeneration liquid, and an outflow part is collected to obtain endotoxin eluent. The hirudin for removing endotoxin by spraying on the inner wall of the vacuum blood collection tube can improve the accuracy of blood detection, is particularly suitable for the detection requirement of special items containing pyrogen in blood samples, and is safe and reliable.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to an endotoxin-free hirudin anticoagulation vacuum blood collection tube and a preparation method thereof.
Background
The vacuum blood collection tube is a medical apparatus product generally used in clinic for blood detection and collection, utilizes the repression to automatically and quantitatively collect venous blood samples, and can be divided into a common blood collection tube, a blood collection tube containing a coagulant, a blood collection tube containing an anticoagulant and the like according to different detection purposes. The blood collecting tube for the rapid plasma biochemical hemorheology test is usually added with anticoagulants such as heparin, sodium citrate and EDTA (ethylene diamine tetraacetic acid), however, the conventional anticoagulation blood collecting tube has limited anticoagulation time, has great interference on blood, is easy to cause hemolytic reaction, and the chemical anticoagulant contains more impurities and heavy metals. Hirudin is also a common anticoagulant, and for example, chinese patent with publication number CN101779959A provides a hirudin vacuum blood collection tube, which is suitable for multiple examinations.
In nature, the content of natural hirudin in leeches is limited, and a large amount of hirudin is difficult to extract from leeches and is not easy to be extractedCan meet the clinical use requirements, so that the recombination of hirudin through genetic engineering becomes the research focus of the medical community at home and abroad. The SO of the recombinant hirudin is removed from the amino acid Tyr residue at the 63 th position 3- The rest structure and property are basically the same as natural hirudin. The first recombinant hirudin cDNA was successfully cloned in 80 years of the 20 th century, and recombinant hirudin has been successfully expressed in Escherichia coli, bacillus subtilis, yeast and eukaryotic cells.
However, the focus of the present research is mainly on the production and expression of recombinant hirudins, and relatively few further studies on isolation and purification. The recombinant hirudin prepared by means of expression and fermentation of microorganisms such as bacteria or fungi has impurity components such as host protein, pigment, culture medium and the like, and also has endotoxin generated by thallus lysis, and the accumulation of the substances acts on a human body to generate a series of toxic reactions, such as fever, inflammation, even shock and the like. Therefore, recombinant hirudin prepared by fermentation culture must be subjected to reasonable purification process to remove endotoxin in protein solution, so that the recombinant hirudin can be used for preparing an anticoagulant vacuum blood collection tube, and the research on removing endotoxin in recombinant hirudin is still less at present.
Disclosure of Invention
Aiming at the technical problems, the invention provides the endotoxin-free hirudin anticoagulation vacuum blood collection tube which is simple in preparation method and suitable for large-scale industrial production.
The above object of the present invention is achieved by the following technical solutions:
an endotoxin-free hirudin anticoagulation vacuum blood collection tube, recombinant hirudin is coated on the inner wall of the vacuum blood collection tube, and the endotoxin content in 100 μ g of recombinant hirudin is less than 0.25EU/ml.
The recombinant hirudin can be specifically and specifically combined with thrombin in blood, has a remarkable anticoagulation effect, and has little interference on a detection result when being used as an anticoagulant coating on the inner wall of a blood collection tube. Moreover, the hirudin anticoagulant used by the vacuum blood collection tube provided by the invention hardly contains endotoxin, has high purity, single anticoagulation component, sterility, no toxicity and good safety, is suitable for detection of special items such as pyrogen in blood and the like, and improves the accuracy of blood detection.
Preferably, the recombinant hirudin is obtained by separating recombinant hirudin protein liquid by high performance liquid anion exchange chromatography.
Anion exchange chromatography uses an ion exchanger as a separation stationary phase, charged ions of the same type can freely exchange with each other and compete for binding, for example, between proteins or between a protein and other molecules, ions of the same charge type can competitively bind to a stationary phase medium with opposite charge. In the invention, the recombinant hirudin has weaker adsorption acting force with the surface of the ion exchanger, the recombinant hirudin preferentially flows out of a chromatographic column during elution, and endotoxin components with strong adsorption acting force with a stationary phase need to be washed by a regeneration solution with stronger elution capability.
The invention also aims to provide a preparation method of the endotoxin-free hirudin anticoagulation vacuum blood collection tube, which comprises the following steps:
s1, cleaning and drying a blood collection tube by using ultrasonic waves;
s2, coating a siliconizing agent on the inner wall surface of the blood collection tube, and curing the siliconizing agent at a high temperature;
s3, sterilizing the blood collection tube by using ethylene oxide, and removing residual ethylene oxide;
s4, removing endotoxin in the recombinant hirudin by adopting a high performance liquid anion exchange chromatography, diluting the recombinant hirudin by pyrogen-free water for injection, and then sterilizing and filtering to obtain a sterile and endotoxin-free hirudin anticoagulant;
and S5, spraying a hirudin anticoagulant on the inner wall of the blood collection tube from top to bottom at a position of 2cm in the sterile environment, drying, vacuumizing and sealing the blood collection tube.
Preferably, the silicifying agent in step S2 is dimethyldichlorosilane or dimethyldisilane.
Preferably, the temperature for curing the siliconizing agent in step S2 is 130 to 180 ℃.
Preferably, the high performance liquid anion exchange chromatography in step S4 specifically comprises the steps of:
sample introduction: sampling the recombinant hirudin protein solution, wherein the sampling amount is 25-40% of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to balance the column;
and (3) elution: isocratic elution is carried out by the eluent, and the outflow part is collected to obtain recombinant hirudin eluent;
column regeneration: the endotoxin is eluted isocratically with the regeneration liquid, and the effluent fraction is discarded.
The sample amount in the anion exchange chromatography separation also has a remarkable influence on the separation effect. Because the maximum sample loading amount of the chromatographic column is influenced by factors such as the size of filler particles, the bonding density of the filler surface, the size of the chromatographic column and the like, the sample loading amount is often determined according to the saturated adsorption amounts of different chromatographic columns. However, the inventors have found in practice that the total efficiency of the purification recovery and the removal of impurities of recombinant hirudin protein is not high even if the sample is introduced at the maximum loading. The sample amount of the present invention is far less than the maximum sample loading amount of the chromatographic column, because the sample amount is continuously increased, the endotoxin removing effect is greatly reduced although the recovery rate of the recombinant hirudin protein is not reduced. If the sample loading amount is less than 25% of the maximum sample loading amount, the recovery rate of the recombinant hirudin protein can be obviously reduced, but the endotoxin removal effect is obviously improved. Therefore, the invention realizes the effect of removing the endotoxin in the recombinant hirudin protein solution and the optimal hirudin recovery rate by reasonably regulating and controlling the sample amount.
More preferably, the anion exchange chromatography of the present invention is carried out in an amount of 30% of the maximum loading of the column.
Preferably, the equilibrium solution of the present invention is Tris-HCl buffer solution having a concentration of 8 to 20mmol/L and a pH of 6.5 to 7.5.
Preferably, in the present invention, the eluent and the regeneration liquid are both buffer solutions having a pH value of 6.5 to 7.5.
Preferably, the eluent and the regeneration liquid of the invention are both Tris-HCl buffer solution containing NaCl, and the concentration of chloride ions in the eluent is less than that in the regeneration liquid.
Endotoxin is a negatively charged lipopolysaccharide substance, and has strong binding force with anion exchanger at pH > 2. Under the separation condition of the invention close to neutrality, the adsorption strength of the endotoxin on the ion exchange chromatographic packing is far greater than that of the recombinant hirudin. Therefore, the recombinant hirudin preferentially flows out of the chromatographic column under the action of the eluent with lower salt concentration, and then is eluted by the regeneration liquid with high salt concentration for desorption, so that the residual endotoxin component in the chromatographic column flows out along with the regeneration liquid, and the effective separation of the recombinant hirudin and the endotoxin impurities is realized.
More preferably, in the eluent of the present invention, the concentration of NaCl is 100 to 150mmol/L, and the concentration of Tris-HCl is 8 to 20mmol/L.
More preferably, in the regeneration liquid of the present invention, the concentration of NaCl is 1.0 to 2.0mol/L, and the concentration of Tris-HCl is 8 to 20mmol/L.
Preferably, the recombinant hirudin protein solution used in the invention is prepared by purifying recombinant hirudin fermentation liquor, and the purity is more than or equal to 95%.
Preferably, the sample injection flow rate of the invention is 70-100 ml/min.
Preferably, the invention can be loaded and separated again by eluting with the regeneration solution and then washing with the equilibrium solution.
The invention uses Diethylaminoethyl (DEAE) bonded silica gel as anion exchange chromatographic packing for separation, increases the salt concentration of a mobile phase step by step for isocratic elution, preferentially flows out recombinant hirudin with weak binding force, dissociates endotoxin with high salt concentration, realizes effective separation and completes the regeneration of a chromatographic column. The recovery rate of the recombinant hirudin protein prepared by the method can reach more than 95 percent, and the endotoxin content in 100 mu g of recombinant hirudin is less than 0.25EU/ml.
Preferably, the amount of the hirudin anticoagulant sprayed in step S5 is 20 to 50 μ g of hirudin per ml of the blood collection tube.
Preferably, the degree of vacuum in step S5 is ≦ 100pa.
Compared with the prior art, the invention has the following beneficial effects:
(1) The hirudin anticoagulant for removing endotoxin is sprayed on the inner wall of the vacuum blood collection tube, so that the accuracy of blood detection can be improved, the hirudin anticoagulant is particularly suitable for the detection requirement of special items containing pyrogen in blood samples, and the recombinant hirudin has high purity, good safety and small interference on detection results;
(2) According to the invention, the anion exchange chromatography is adopted to remove endotoxin in the recombinant hirudin protein solution, a good removal effect is realized by regulating and controlling the sample amount, and meanwhile, a higher protein recovery rate is ensured;
(3) According to the strength of the adsorption effect of recombinant hirudin and endotoxin with an anion exchanger under a neutral condition, isocratic elution is carried out by increasing the salt concentration in a mobile phase, the recombinant hirudin with weak adsorption force preferentially flows out, and the endotoxin component with strong action force is dissociated under the action of a regeneration liquid with high salt concentration;
(4) The invention effectively removes the endotoxin in the recombinant hirudin by a simple and quick method, and simultaneously ensures that the prepared anticoagulation vacuum blood collection tube has wide detection range, long service time, small pollution, safety and reliability, and is suitable for industrial production.
Detailed Description
The technical solution of the present invention is further described and illustrated by the following specific examples. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified. It should be understood that the specific embodiments described herein are only for the purpose of facilitating understanding of the present invention, and are not intended to limit the present invention specifically.
The invention provides an endotoxin-free hirudin anticoagulation vacuum blood collection tube, which is prepared by the following steps:
s1, cleaning and drying a blood collection tube by using ultrasonic waves;
s2, coating a siliconizing agent on the inner wall surface of the blood collection tube, and curing the siliconizing agent at the temperature of 130-180 ℃;
s3, sterilizing the blood collection tube by using ethylene oxide, and removing residual ethylene oxide;
s4, removing endotoxin in the recombinant hirudin by adopting an anion exchange chromatography, diluting the recombinant hirudin with pyrogen-free water for injection, and sterilizing and filtering to obtain a sterile hirudin anticoagulant without endotoxin;
s5, spraying a hirudin anticoagulant on the inner wall of the blood collection tube from top to bottom at a position of 2cm in the sterile environment, spraying 20-50 mu g of hirudin in each ml of blood collection tube, drying, vacuumizing the blood collection tube to be less than or equal to-100 pa, and sealing to obtain the hirudin anticoagulant;
wherein, the high performance liquid anion exchange chromatography comprises the following steps:
sample introduction: taking a recombinant hirudin protein solution with the purity of more than or equal to 95%, injecting the sample at the flow rate of 70-100 ml/min, wherein the sample injection amount is 25-40% of the maximum sample loading amount of the chromatographic column, and washing with a Tris-HCl buffer solution balance solution with the concentration of 8-20 mmol/L and the pH value of 6.5-7.5 to perform column balance, wherein the washing volume is 3-6 times of the column volume;
and (3) elution: eluting with Tris-HCl buffer solution containing NaCl and with the pH value of 6.5-7.5 as a mobile phase, wherein the concentration of NaCl is 100-150 mmol/L and the concentration of Tris-HCl is 8-20 mmol/L, and collecting eluent of an outflow part to obtain recombinant hirudin;
regeneration: eluting with 3-6 times of column volume of regeneration liquid, wherein the regeneration liquid is Tris-HCl buffer solution with pH value of 6.5-7.5 and containing NaCl, the concentration of NaCl is 1.0-2.0 mol/L, the concentration of Tris-HCl is 8-20 mmol/L, and removing residual endotoxin in the chromatographic column; finally, the column is washed by equilibrium liquid with 3 to 6 times of column volume, and the column can be used for sample loading and separation again.
The recombinant hirudin protein solution is obtained by purifying recombinant hirudin fermentation liquor which is fermented and secreted and expressed by escherichia coli or saccharomycetes, and the purification process is a conventional chromatographic separation method and is not described again.
After the endotoxin is removed by the method, the protein recovery rate of the recombinant hirudin is more than or equal to 95 percent, and the endotoxin content in 100 mu g of the recombinant hirudin is less than 0.25EU/ml by the detection of a limulus reagent gel method.
Example 1
(1) Firstly, removing endotoxin in a recombinant hirudin protein solution by adopting a high performance liquid anion exchange chromatography, and filling a DEAE bonded silica gel chromatographic column (the particle size is 5 mu m, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500 mm) with a filler, and the method comprises the following specific steps:
sample introduction: sampling recombinant hirudin protein solution with the purity of 95% at the flow rate of 80ml/min, wherein the sampling amount is 30% of the maximum sample loading amount of the chromatographic column, and washing with Tris-HCl buffer solution equilibrium liquid with the concentration of 10mmol/L and the pH value of 7.0 to perform column balance, wherein the washing volume is 5 times of the column volume;
and (3) elution: eluting with Tris-HCl buffer solution containing NaCl and with pH of 7.0 as mobile phase, wherein the concentration of NaCl is 120mmol/L and the concentration of Tris-HCl is 10mmol/L, and collecting eluate of the eluate to obtain recombinant hirudin;
regeneration: eluting with 5 times column volume of regeneration solution, wherein the regeneration solution is Tris-HCl buffer solution with pH of 7.0 and containing NaCl at concentration of 1.5mol/L and Tris-HCl at concentration of 10mmol/L, and removing residual endotoxin in the chromatographic column; finally, washing with 5 times of column volume of equilibrium solution, and then loading and separating again;
(2) Then preparing the vacuum blood collection tube by recombinant hirudin according to the following method:
s1, cleaning and drying a blood collection tube by using ultrasonic waves;
s2, coating dimethyldichlorosilane on the inner wall surface of the blood collection tube, and curing a silicification agent at the temperature of 150 ℃;
s3, sterilizing the blood collection tube by using ethylene oxide, and removing residual ethylene oxide;
s4, removing endotoxin in the recombinant hirudin by adopting an anion exchange chromatography, diluting the recombinant hirudin with pyrogen-free water for injection, sterilizing and filtering to obtain a sterile and endotoxin-free hirudin anticoagulant;
and S5, spraying hirudin anticoagulant on the inner wall of the blood collection tube from top to bottom in a sterile environment, spraying 20-50 mu g of hirudin into each ml of the blood collection tube, freeze-drying, vacuumizing the blood collection tube to-200 pa, and sealing to obtain the non-endotoxin hirudin anticoagulant vacuum blood collection tube.
The medium packing and the recombinant hirudin protein solution of the chromatographic column used in this example are both produced by Ningbo Rui Va Biotech Limited.
Example 2
This example differs from example 1 only in that the sample loading was 25% of the maximum loading of the column, and the remaining operating method and process conditions were the same as in example 1.
Example 3
This example differs from example 1 only in that the sample loading was 40% of the maximum loading of the column, and the remaining operating method and process conditions were the same as in example 1.
Comparative example 1
(1) Firstly, removing endotoxin in a recombinant hirudin protein solution by adopting an anion exchange chromatography, and filling a DEAE bonded silica gel chromatographic column (the particle size is 5 mu m, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500 mm) with the following specific steps:
sample introduction: sampling recombinant hirudin protein solution with the purity of 95% at the flow rate of 80ml/min, wherein the sampling amount is 30% of the maximum sample loading amount of the chromatographic column, and washing with Tris-HCl buffer solution equilibrium solution with the concentration of 10mmol/L and the pH value of 7.0 to perform column equilibrium, wherein the washing volume is 5 times of the column volume;
and (3) elution: performing gradient elution by using a mobile phase A-a mobile phase B, wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 7.0 and containing NaCl, the concentration of the NaCl is 120mmol/L, and the concentration of the Tris-HCl is 10mmol/L; the mobile phase B is a Tris-HCl buffer solution with the pH value of 7.0 and containing NaCl, the concentration of the NaCl is 1.5mol/L, and the concentration of the Tris-HCl buffer solution is 10mmol/L; the elution procedure was: 0-60min:100% → 0% mobile phase a,0% → 100% mobile phase B; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV 254nm, and collecting the main elution peak part when the base line rises to obtain recombinant hirudin for later use; the remaining effluent fraction is an eluent containing endotoxin components;
regeneration: eluting with mobile phase B2 times of column volume, and washing with balance solution 5 times of column volume to obtain sample for separation;
(2) And preparing the vacuum blood collection tube by recombinant hirudin, wherein the specific operation steps are the same as those in example 1 and are not described again.
Comparative example 2
Comparative example 2 differs from example 1 only in that the sample loading is 50% of the maximum loading of the column, and the remaining operating method and process conditions are the same as in example 1.
Comparative example 3
Comparative example 3 is different from example 1 only in that the inner wall of the blood collection tube is directly sprayed with the recombinant hirudin protein solution, endotoxin is not removed by anion exchange chromatography, and the remaining operation method and process conditions are the same as those of example 1.
Comparative example 4
Comparative example 4 is a general commercial heparin anticoagulation vacuum blood collection tube (manufacturer is American BD company).
The endotoxin content in the hirudin anticoagulants of examples 1 to 3 and comparative examples 1 to 4 was measured by reference to the thrombin titration method specified in section 1 of the "Chinese pharmacopoeia" 2010 edition, and 1,3-D glucan and galactomannan antigens were detected by the G test and the GM test, respectively, and measured in parallel 3 times. The results are shown in Table 1.
TABLE 1 test results of endotoxin, fungus and mold in blood collection tubes in examples 1 to 3 and comparative examples 1 to 4
| Examples | Endotoxin content in 100. Mu.g recombinant hirudin | G test | GM test |
| Example 1 | <0.25EU/ml | - | - |
| Example 2 | <0.25EU/ml | - | - |
| Example 3 | <0.5EU/ml | - | - |
| Comparative example 1 | <0.5EU/ml | - | - |
| Comparative example 2 | ≥0.75EU/ml | + | + |
| Comparative example 3 | >10EU/ml | + | + |
| Comparative example 4 | <0.25EU/ml | - | - |
Note: in the table, "+" indicates that the test result was positive, and "-" indicates that the test result was negative.
The technical solutions claimed in the above embodiments are not exhaustive in terms of their values within the parameters and the equivalent substitutions for a single or multiple features in the technical solutions of the embodiments are also within the scope of the invention, and all the parameters involved in the technical solutions of the invention have no mutually exclusive combinations unless otherwise specified.
The specific embodiments described herein are merely illustrative of the spirit of the invention and do not limit the scope of the invention. The technical solutions similar or similar to the present invention can be obtained by those skilled in the art through equivalent replacement or equivalent transformation, and all fall within the protection scope of the present invention.
Claims (4)
1. An endotoxin-free hirudin anticoagulation vacuum blood collection tube is characterized in that recombinant hirudin is coated on the inner wall of the vacuum blood collection tube, and the endotoxin content in 100 mu g of the recombinant hirudin is less than 0.25EU/ml;
the preparation method of the vacuum blood collection tube comprises the following steps:
s1, cleaning and drying a blood collection tube by using ultrasonic waves;
s2, coating a siliconizing agent on the inner wall surface of the blood collection tube, and curing the siliconizing agent at a high temperature;
s3, sterilizing the blood collection tube by using ethylene oxide, and removing residual ethylene oxide;
s4, removing endotoxin in the recombinant hirudin by adopting an anion exchange chromatography, diluting the recombinant hirudin with pyrogen-free water for injection, sterilizing and filtering to obtain a sterile and endotoxin-free hirudin anticoagulant;
s5, spraying a hirudin anticoagulant on the inner wall of the blood collection tube in an aseptic environment, drying, vacuumizing and sealing the blood collection tube;
wherein the temperature of the curing siliconizing agent in the step S2 is 130 to 180 ℃;
the anion exchange chromatography comprises the following steps:
sample introduction: injecting a recombinant hirudin protein solution into a sample, wherein the sample injection amount is 25 to 40 percent of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to carry out column balance;
and (3) elution: isocratic elution is carried out by eluent, and an outflow part is collected to obtain recombinant hirudin eluent;
column regeneration: isocratic elution of endotoxin with the regenerated liquid and discarding the effluent;
the eluent and the regeneration liquid are both Tris-HCl buffer solution containing NaCl with the pH value of 6.5 to 7.5, and the concentration of chloride ions in the eluent is less than that of the chloride ions in the regeneration liquid;
the anion exchange chromatography uses a diethylaminoethyl bonded silica gel matrix as a chromatographic column, and the matrix is as follows: the particle size is 5 μm, the aperture is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm;
in the eluent, the concentration of NaCl is 100 to 150mmol/L, and the concentration of Tris-HCl is 8 to 20 mmol/L;
in the regeneration liquid, the concentration of NaCl is 1.0 to 2.0mol/L, and the concentration of Tris-HCl is 8 to 20mmol/L.
2. The endotoxin-free hirudin anticoagulant vacuum blood collection tube according to claim 1, wherein the recombinant hirudin is obtained by separating recombinant hirudin protein solution by anion exchange chromatography.
3. The endotoxin-free hirudin anticoagulant vacuum blood collection tube according to claim 1, wherein the equilibrium solution is Tris-HCl buffer solution with a concentration of 8 to 20mmol/L and a pH value of 6.5 to 7.5.
4. The endotoxin-free hirudin anticoagulant vacuum blood collection tube according to claim 1, wherein the recombinant hirudin protein solution is prepared by purifying recombinant hirudin fermentation broth, and the purity is more than or equal to 90%.
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