A method for preparing high-purity canine serum albumin

Abstract

The invention discloses a method for preparing high-purity canine serum albumin, which relates to the technical field of blood products. The specific preparation method is as follows: S1, using healthy canine serum or plasma as raw material, centrifuging, and collecting supernatant; S2, using an anion exchange chromatography column to perform chromatographic separation on the supernatant, and then using the S / D method in contact with an S / D reagent to perform virus inactivation treatment on the supernatant; S3, ultrafiltration and liquid exchange on the inactivated supernatant, and then cation exchange chromatography. The method for preparing high-purity canine serum albumin uses the supernatant after centrifugal separation of canine plasma as raw material for chromatographic treatment. Compared with the traditional low-temperature ethanol precipitation process, the separation steps of low-temperature ethanol precipitation, deep filtration, and precipitation dissolution of subsequent components are reduced, the process time is shortened, and the loss of canine serum albumin is reduced, and the purity, activity, and yield of canine serum albumin are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of blood products, in particular to a method for preparing high-purity canine blood albumin. Background Art

[0002] As pet dogs have entered thousands of households, the health care of pet dogs has attracted widespread attention. The main physiological function of canine serum albumin is to regulate osmotic pressure and act as an important substance transport carrier in metabolism. It has a wide range of indications and is often used as a hemolytic expander instead of blood transfusion. It can increase plasma colloidal osmotic pressure and increase blood volume. It is used to treat shock caused by blood loss, trauma and burns, cerebral edema and increased intracranial pressure caused by brain damage. It has a good effect on the prevention and treatment of hypoproteinemia and edema or ascites caused by cirrhosis or kidney disease. At present, the method for preparing canine serum albumin from canine plasma is mainly a low-temperature ethanol process based on the Cohn-Oncley method or the Kistler-Nitschmann method.

[0003] The Chinese patent with publication number CN1648134A discloses a low-temperature ethanol process for preparing canine blood albumin, and the process steps include: combining and melting plasma, treating the supernatant after four centrifugations to remove impurities to obtain precipitated albumin, ultrafiltration and pasteurization, and then packaging to obtain canine blood albumin. Most blood product manufacturers now prepare canine blood albumin according to this method. However, the above method has many separation steps, a long separation cycle, a complex process, and requires a low-temperature environment throughout the process, but the purity of the product is not high. The canine blood albumin products sold in China were tested by the method specified in the Chinese Pharmacopoeia, and the purity was about 90%, and the polymer content was ≥6%; however, in the above-mentioned prior art scheme, the purity of the prepared canine blood albumin was low, which would have an adverse effect on the stability of the product and the safety of clinical application; for this reason, the present invention provides a method for preparing high-purity canine blood albumin to solve the above problems. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing high-purity canine serum albumin, which solves the problems mentioned in the above background technology.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing high-purity canine blood albumin, the specific preparation method is:

[0006] S1. Using healthy dog ​​serum or plasma as raw material, centrifuging and collecting the supernatant;

[0007] S2. After the supernatant is chromatographed and separated using an anion exchange chromatography column, the supernatant is subjected to virus inactivation treatment using an S / D method in contact with an S / D reagent;

[0008] S3, performing cation exchange chromatography on the inactivated supernatant;

[0009] S4, then use 10-50 kDa ultrafiltration membrane or hollow fiber ultrafiltration and then change to gel chromatography;

[0010] S5. The supernatant after chromatography is passed through a DV20 nano-membrane to remove viruses, and the supernatant is ultrafiltrated and concentrated to a canine serum albumin concentration of 10.0±0.2 g / dL, thereby obtaining high-purity canine serum albumin.

[0011] In S1, the centrifugal temperature is 2-8°C, the centrifugal speed is 8000-12000 r / min, and the centrifugal time is 20-40 min.

[0012] Preferably, in S2, the chromatographic separation method is:

[0013] (1) Equilibrate the anion exchange chromatography column with saline until the pH and ionic strength of the effluent are the same as those of the supernatant;

[0014] (2) Dilute the supernatant with buffer and load on the sample;

[0015] (3) After loading, rinse the anion exchange chromatography column with buffer;

[0016] (4) After flushing, elute the column with buffer and collect the eluate.

[0017] Preferably, in (3), the flushing flow rate is 0.5 to 1 mL / min.

[0018] Preferably, in S2, virus inactivation is carried out at a temperature of 25 to 40° C. and is completed within 4 to 6 hours.

[0019] Preferably, in S3, the method of cation exchange chromatography specifically comprises: pumping the inactivated eluate into the balanced cation exchange chromatography column, then eluting with an elution buffer, and collecting the eluate.

[0020] Preferably, the flow rate of the eluent is 1 to 3 mL / min.

[0021] Preferably, in S4, the gel chromatography uses a Sephadex G-25 chromatography column.

[0022] Preferably, in S5, the ultrafiltration concentration and liquid replacement is performed by using a pH 8.6±0.1 phosphate buffer solution.

[0023] Beneficial Effects

[0024] The present invention provides a method for preparing high-purity canine serum albumin. Compared with the prior art, the method has the following beneficial effects:

[0025] (1) The method for preparing high-purity canine serum albumin uses the supernatant after centrifugal separation of canine plasma as the raw material for chromatography. Compared with the traditional low-temperature ethanol precipitation process, it reduces the separation steps of low-temperature ethanol precipitation, deep filtration and precipitation dissolution of subsequent components, shortens the process time, reduces the loss of canine serum albumin, and improves the activity and yield of canine serum albumin.

[0026] (2) The method for preparing high-purity canine serum albumin can effectively remove various macromolecular and small molecular impurity proteins in the product through anion exchange chromatography, cation exchange chromatography, ultrafiltration concentration and ultrafiltration overnight concentration, thereby improving the purity of the product. The purity of the canine serum albumin obtained by the method is at least 99.9%, which greatly improves the safety of application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention discloses a canine serum albumin HPLC detection spectrum provided for a method for preparing high-purity canine serum albumin. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the technical scheme, during the elution process, a 20% sulfosalicylic acid solution is used to detect the activity of the effluent. When no precipitation occurs after the effluent is mixed with the sulfosalicylic acid solution, it indicates that the active substance has been completely collected.

[0030] Example 1

[0031] A method for preparing high-purity canine serum albumin, the specific preparation method is:

[0032] S1. Using healthy dog ​​serum or plasma as raw material, centrifuging at a temperature of 2°C, a speed of 8000 r / min, and a time of 20 min, and collecting the supernatant;

[0033] S2. The supernatant is chromatographed and separated using an anion exchange chromatography column. The anion exchange chromatography elution buffer includes 1 mmol / L sodium acetate and 0.01 mol / L sodium chloride. The pH of the elution buffer is 4.8. The specific method is as follows: the anion exchange chromatography is balanced with physiological saline until the pH and ionic strength of the effluent are the same as those of the supernatant; the supernatant is diluted with the buffer and then loaded; after loading, the anion exchange chromatography column is rinsed with the elution buffer at a flow rate of 0.5 mL / min; after rinsing, the chromatography column is eluted with the buffer and the eluate is collected; and the supernatant is subjected to a virus inactivation treatment at 25°C for 4 hours using the S / D method in contact with the S / D reagent;

[0034] S3, the supernatant after inactivation is subjected to cation exchange chromatography, wherein a phosphate buffer solution of pH 6.1 is used for ultrafiltration and liquid exchange, and the cation exchange chromatography elution buffer comprises 1 mmol / L sodium citrate and 0.5 mol / L sodium chloride, and the specific method is: the inactivated eluate is pumped into the balanced cation exchange chromatography column, and then the elution buffer is used for elution at a flow rate of 1 mL / min, and the eluate is collected;

[0035] S4, ultrafiltration using a 10 kDa ultrafiltration membrane and then changing to a Sephadex G-25 chromatography column for chromatography;

[0036] S5. The supernatant after chromatography is passed through a DV20 nano-membrane to remove viruses, and ultrafiltration concentration is exchanged for a phosphate buffer solution with a pH of 8.5 to ultrafiltration and liquid exchange until the canine serum albumin concentration is 9.8 g / dL, thereby obtaining high-purity canine serum albumin.

[0037] Example 2

[0038] A method for preparing high-purity canine serum albumin, the specific preparation method is:

[0039] S1. Using healthy dog ​​serum or plasma as raw material, centrifuging at a temperature of 5°C, a speed of 10,000 r / min, and a time of 30 min, and collecting the supernatant;

[0040] S2. The supernatant is chromatographed and separated using an anion exchange chromatography column. The anion exchange chromatography elution buffer includes 25 mmol / L sodium acetate and 0.25 mol / L sodium chloride. The pH of the elution buffer is 5.4. The specific method is as follows: the anion exchange chromatography is balanced with physiological saline until the pH and ionic strength of the effluent are the same as those of the supernatant; the supernatant is diluted with the buffer and then loaded; after loading, the anion exchange chromatography column is rinsed with the elution buffer at a flow rate of 0.8 mL / min; after rinsing, the chromatography column is eluted with the buffer and the eluate is collected; and the supernatant is subjected to a virus inactivation treatment at 35°C for 5 hours using the S / D method in contact with the S / D reagent;

[0041] S3, the supernatant after inactivation is subjected to cation exchange chromatography, wherein a phosphate buffer solution of pH 6.2 is used for ultrafiltration and liquid exchange, and the cation exchange chromatography elution buffer comprises 25 mmol / L sodium citrate and 1.5 mol / L sodium chloride, and the specific method is: the inactivated eluate is pumped into the balanced cation exchange chromatography column, and then the elution buffer is used for elution at a flow rate of 2 mL / min, and the eluate is collected;

[0042] S4, ultrafiltration using a 30 kDa ultrafiltration membrane and then changing to a Sephadex G-25 chromatography column for chromatography;

[0043] S5. The supernatant after chromatography is passed through a DV20 nano-membrane to remove viruses, and ultrafiltration concentration is exchanged for a phosphate buffer solution with a pH of 8.6 to ultrafiltration and liquid exchange until the canine serum albumin concentration is 10.0 g / dL, thereby obtaining high-purity canine serum albumin.

[0044] Example 3

[0045] A method for preparing high-purity canine serum albumin, the specific preparation method is:

[0046] S1. Using healthy dog ​​serum or plasma as raw material, centrifuging at a temperature of 8°C, a speed of 12000 r / min, and a time of 40 min, and collecting the supernatant;

[0047] S2. The supernatant is chromatographed and separated using an anion exchange chromatography column. The anion exchange chromatography elution buffer includes 50 mmol / L sodium acetate and 0.5 mol / L sodium chloride. The pH of the elution buffer is 6.0. The specific method is as follows: the anion exchange chromatography is balanced with physiological saline until the pH and ionic strength of the effluent are the same as those of the supernatant; the supernatant is diluted with the buffer and then loaded; after loading, the anion exchange chromatography column is rinsed with the elution buffer at a flow rate of 1 mL / min; after rinsing, the chromatography column is eluted with the buffer and the eluate is collected; and the supernatant is subjected to a virus inactivation treatment at 40°C for 6 hours using the S / D method in contact with the S / D reagent;

[0048] S3, the supernatant after inactivation is subjected to cation exchange chromatography, wherein a phosphate buffer solution of pH 6.3 is used for ultrafiltration and liquid exchange, and the cation exchange chromatography elution buffer comprises 50 mmol / L sodium citrate and 2.0 mol / L sodium chloride, and the specific method is: the inactivated eluate is pumped into the balanced cation exchange chromatography column, and then the elution buffer is used for elution at a flow rate of 3 mL / min, and the eluate is collected;

[0049] S4, ultrafiltration using a 50 kDa ultrafiltration membrane and then changing to a Sephadex G-25 chromatography column for chromatography;

[0050] S5. The supernatant after chromatography is passed through a DV20 nano-membrane to remove viruses, and ultrafiltration concentration is exchanged for a phosphate buffer solution with a pH of 8.7 to ultrafiltration liquid exchange until the canine serum albumin concentration is 10.2 g / dL, thereby obtaining high-purity canine serum albumin.

[0051] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0052] It should be noted that the present invention only uses canine plasma as an example to obtain high-purity canine serum albumin through a three-step chromatography method. High-purity albumin obtained from the plasma of other animals according to the method of the present invention is also within the protection scope of the present invention. In addition, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0053] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing high-purity canine serum albumin, characterized in that: The specific preparation method is: S1. Using healthy dog ​​serum or plasma as raw material, centrifuging and collecting the supernatant; S2. After the supernatant is chromatographed and separated using an anion exchange chromatography column, the supernatant is subjected to virus inactivation treatment using an S / D method in contact with an S / D reagent; S3, performing cation exchange chromatography on the inactivated supernatant; S4, then use 10-50 kDa ultrafiltration membrane or hollow fiber ultrafiltration and then change to gel chromatography; S5. The supernatant after chromatography is passed through a DV20 nano-membrane to remove viruses, and ultrafiltration and concentration are performed to replace the liquid until the canine serum albumin concentration is 10.0±0.2 g / dL, thereby obtaining high-purity canine serum albumin; In S1, the centrifugal temperature is 2-8°C, the centrifugal speed is 8000-12000 r / min, and the centrifugal time is 20-40 min; In said S2, the chromatographic separation method is: (1) Equilibrate the anion exchange chromatography column with saline until the pH and ionic strength of the effluent are the same as those of the supernatant; (2) Dilute the supernatant with buffer and load on the sample; (3) After loading, rinse the anion exchange chromatography column with buffer; (4) After washing, the column is eluted with a buffer solution and the eluate is collected; In (3), the flushing flow rate is 0.5 to 1 mL / min; In S2, the virus inactivation is carried out at a temperature of 25 to 40° C. and is completed within 4 to 6 hours; In S3, the method of cation exchange chromatography specifically comprises: pumping the inactivated eluate into the balanced cation exchange chromatography column, then eluting with an elution buffer, and collecting the eluate; The flow rate of the eluent is 1 to 3 mL / min; In S4, the gel chromatography uses a Sephadex G-25 chromatography column; In S5, the ultrafiltration concentration and liquid replacement is performed by using a pH 8.6±0.1 phosphate buffer solution.

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