Dog serum immune globulin and albumin as well as preparation method and application thereof

Abstract

The invention discloses dog serum immune globulin and albumin as well as a preparation method and application thereof, and relates to the technical field of biological products. The preparation method comprises the following steps: caprylic acid precipitation, one-step anion exchange chromatography, ultrafiltration concentration and virus inactivation: diluting dog serum to adjust the pH value, adding caprylic acid to precipitate impure protein, carrying out Capto Q anion exchange chromatography on the supernatant, collecting the flow-through liquid to obtain an immune globulin component, eluting with a buffer solution containing 0.2 M NaCl to obtain an albumin component, and carrying out one-step anion exchange chromatography on the albumin component. And respectively carrying out targeted ultrafiltration concentration and virus inactivation treatment to synchronously obtain two products. The process is simple and efficient, the defects of multi-step chromatography or a complex precipitation method are avoided, and the production cost is low; the purity of the prepared immunoglobulin is greater than or equal to 96%, the purity of albumin is greater than or equal to 96%, the recovery rate is in a reasonable range, the product quality is stable, and the safety requirements of biological products are met. The immune globulin and albumin can be applied to preparation of veterinary biological products, and efficient utilization of dog serum resources is realized.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to canine serum immunoglobulin and albumin, their preparation methods, and applications. Background Technology

[0002] Immunoglobulins and albumins in canine serum are proteins with important biological activities and have wide applications in veterinary medicine and biopharmaceutical research. To meet the practical demand for high-purity, high-yield canine serum immunoglobulins and albumins, efficient, safe, and economical preparation methods are urgently needed in related fields.

[0003] Currently, existing methods for separating and purifying animal serum immunoglobulins and albumin mainly include the low-temperature ethanol method, the rivanol method, the PEG method, the ammonium sulfate precipitation method, and chromatography. Among these: The low-temperature ethanol method is a classic protein separation method, but it has high operating requirements, is time-consuming, and usually requires multiple subsequent chromatography processes to obtain high-purity target products, resulting in a complicated process and significant loss of target products (e.g., patent CN1654072A). The Rivanol method, PEG method, and ammonium sulfate method have been gradually phased out due to safety issues (e.g., patents CN1648134A and CN103012581A). Chromatography is a key method for improving protein purity, but existing technologies have several shortcomings: affinity chromatography has drawbacks such as the risk of ligand shedding, low loading capacity, high cost, and poor economic efficiency, making it unsuitable for large-scale production of albumin and immunoglobulins; other patents employ two-step chromatography processes (such as patents CN112480246A, CN112521485A, CN118598979B, etc.), and the additional chromatography step increases the loss of the target product, prolongs the process time, and raises production costs; In addition, most existing patented technologies only target the separation and purification of a single target product (either immunoglobulin or albumin) in canine serum. For example, some patents only involve the preparation of canine albumin, and some patents only focus on the separation and purification of canine immunoglobulin. There is a lack of technical solutions that can simultaneously achieve efficient purification of two target products through the same simple process, resulting in low serum resource utilization and limited production efficiency.

[0004] In summary, existing methods for preparing canine serum immunoglobulins and albumins suffer from problems such as complex processes, long processing times, significant loss of target products, low utilization of serum resources, high production costs, or insufficient safety, making it difficult to meet the needs of large-scale production and practical applications. Therefore, developing a simple, efficient, safe, and economical method that can simultaneously purify canine serum immunoglobulins and albumins is of great practical significance. Summary of the Invention

[0005] The purpose of this invention is to provide canine serum immunoglobulins and albumins, their preparation methods and applications. This method is simple, efficient, safe and economical, and can simultaneously purify canine serum immunoglobulins and albumins.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing canine serum immunoglobulins and albumin, comprising the following steps: (1) Caprylic acid precipitation: After diluting and adjusting the pH of canine serum, caprylic acid was added to precipitate impurities, remove the precipitate, collect the supernatant and filter it; (2) One-step anion exchange chromatography: The supernatant filtered in step (1) is loaded onto an anion exchange chromatography column, the flow-through is collected to obtain the immunoglobulin component, and the albumin component is collected by elution.

[0007] (3) Ultrafiltration concentration and virus inactivation: After ultrafiltration concentration and buffer system replacement, the immunoglobulin component was inactivated by low pH incubation to obtain canine serum immunoglobulin. The albumin component was ultrafiltration concentration, buffer system replacement and Pasteur virus inactivation to obtain canine serum albumin. Furthermore, in step (1), the canine serum is diluted with 3-10 times the volume of purified water, and the pH is adjusted to 4.5-6.5 with acetic acid or hydrochloric acid after dilution.

[0008] Furthermore, in step (1), the final concentration of the octanoic acid is 20-35 mmol / L. After adding the octanoic acid, the pH is adjusted to the pH value before adding the octanoic acid with NaOH, stirred for 0.5-2 hours and allowed to stand for 0.5-2 hours.

[0009] Furthermore, in step (1), the removal of precipitate is carried out by centrifugation at 4000-5000g for 10-30min, or by pressure filtration or filtration, and the supernatant is filtered using a 0.22-0.45μm filter membrane.

[0010] Furthermore, in step (2), the packing material for the anion exchange chromatography is Capto Q, and the equilibration buffer and elution buffer are both acetate-sodium acetate buffer with a pH of 4.5-6.5.

[0011] Furthermore, in step (2), the albumin fraction eluted and collected is a protein fraction collected by elution with a buffer containing 0.2M NaCl.

[0012] The present invention also provides a canine serum immunoglobulin, which is prepared by the preparation method described above. After treatment with a reducing agent, the immunoglobulin exhibits two protein bands on SDS-polyacrylamide gel electrophoresis: a heavy chain with a molecular weight of approximately 50 kDa and a light chain with a molecular weight of approximately 25 kDa, with a purity of ≥96%.

[0013] The present invention also provides a canine serum albumin, which is prepared by the preparation method described above. The albumin has a molecular weight of about 66 kDa and a purity of ≥96%.

[0014] The present invention also provides the application of the canine serum immunoglobulin and / or the canine serum albumin described herein in the preparation of veterinary biological products.

[0015] The present invention also provides a veterinary biological product comprising the canine serum immunoglobulin and / or the canine serum albumin.

[0016] Beneficial effects: This invention employs a process route of "octanoic acid precipitation + one-step anion exchange chromatography + ultrafiltration concentration and virus inactivation," which offers significant advantages over existing technologies. The octanoic acid precipitation method is safe and effective, with simple operating conditions and no need for complex equipment. Compared to the low-temperature ethanol method, it significantly shortens process time, reduces operating steps, and minimizes target product loss. The one-step anion exchange chromatography, compared to two-step or affinity chromatography, avoids product loss caused by multiple steps and circumvents the drawbacks of affinity chromatography, such as ligand detachment, low loading capacity, and high cost. Furthermore, the chromatographic packing material is economical and practical, significantly reducing production costs. Simultaneously, this process can simultaneously separate and purify two target products, immunoglobulin and albumin, from canine serum, solving the problems of existing technologies that often target only a single product and have low serum resource utilization, thus achieving efficient resource utilization.

[0017] The canine serum immunoglobulins and albumins prepared by this invention have a purity of not less than 96%, and the recovery rate is within the reasonable range of the natural proportion of the corresponding proteins in healthy canine serum. The product quality is stable and reliable. The process is highly adaptable, and can stably produce high-purity products under different serum storage conditions and different process parameter ranges. Furthermore, through targeted virus inactivation treatment, it meets the safety requirements of biological products. The overall process is simple, economical, and efficient, suitable for large-scale production, and provides a high-quality raw material preparation solution for the field of veterinary biological products. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a reduced SDS-polyacrylamide gel electrophoresis image of serum samples and purified protein components from Example 1 of this invention; lane 1 is canine serum, lane 2 is purified immunoglobulin sample, lane 3 is purified albumin sample, lane 4 is the molecular weight standard for protein electrophoresis (the molecular weight corresponding to each protein band is shown on the right side of the figure), lane 5 is human immunoglobulin product (control), and lane 6 is human albumin product (control); this figure is used to show the protein band characteristics and purity of serum samples and purified immunoglobulin and albumin samples. Figure 2 This is an HPLC size exclusion chromatogram of the purified immunoglobulin component in Example 1 of the present invention; this chromatogram is used to show the chromatographic separation characteristics of the immunoglobulin component, wherein the main protein absorption peak that appears at about 14.9 minutes after injection is the immunoglobulin component; Figure 3 This is an HPLC size exclusion chromatogram of the albumin component purified in Example 1 of the present invention; this chromatogram is used to show the chromatographic separation characteristics of the albumin component, wherein the main protein absorption peak that appears about 16.7 minutes after injection is the albumin component; Figure 4 This is a reduced SDS-polyacrylamide gel electrophoresis image of serum samples and purified protein components in Example 2 of the present invention; wherein, lane 1 is the molecular weight standard for protein electrophoresis, lane 2 is canine serum, lane 3 is the purified immunoglobulin sample, and lane 4 is the purified albumin sample; this figure is used to show the protein band characteristics and purity of serum samples and purified immunoglobulin and albumin samples. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 (a) Experimental materials Canine serum, stored at 4°C.

[0026] (II) Experimental Procedure 1. Caprylic acid precipitation (1) Serum preparation: Take 6 mL of serum stored at low temperature, equilibrate at 18-20℃ for 30 min, dilute with 3 volumes of purified water to 24 mL, and adjust the pH value to 5.3 with acetic acid; (2) Add precipitant: At 18-20℃, slowly add octanoic acid to a final concentration of 35 mmol / L; (3) Precipitation of impurities: At 18-20℃, add NaOH to adjust the pH to 5.3, stir for 1 hour, and let stand for 1 hour; (4) Precipitation removal: Centrifuge at 4000g for 30min, collect the supernatant and discard the precipitate; (5) Filtration of supernatant: The remaining supernatant was filtered through a 0.22 μm filter membrane and then used for subsequent experiments.

[0027] 2. Anion exchange chromatography (1) Packing the column: The anion exchange chromatography packing material Capto Q (cytiva) was packed into the chromatography column (xk16 / 20, cytiva) with a height of 10cm and a packing volume of 20mL; (2) Solution preparation: Equilibration solution ①: 0.05M acetate-sodium acetate (HAc-NaAc) buffer, pH 5.3; Elution buffer ②: 0.03-0.1M HAc-NaAc buffer containing 0.2M NaCl, pH 5.3; Elution buffer ③: HAc-NaAc buffer containing 1M NaCl, pH 5.3; (3) Sample preparation: Adjust the conductivity and pH of the sample collected in the octanoic acid precipitation step to be consistent with the equilibrium solution ①; (4) Equilibrium: Equilibrium solution ① Equilibrium chromatography packing material, equilibrium volume is 120mL, flow rate is 5mL / min; (5) Sample loading and rinsing: Load the prepared sample onto the equilibrated chromatography packing material at a flow rate of 5 mL / min; collect the flow-through liquid, and after loading, continue rinsing with equilibration solution ① until the flow-through liquid no longer contains protein, and continue to collect the flow-through liquid during the process; all flow-through liquids are immunoglobulin components, stored at 4℃, and used in subsequent preparation processes; (6) Elution 1: Wash the chromatography packing material with elution buffer ② at a flow rate of 5 mL / min, collect the eluted protein fraction as albumin fraction, store at 4℃ for subsequent preparation process; (7) Elution 2: Wash the chromatography packing material with elution buffer ③ at a flow rate of 5 mL / min. The eluted protein components are impurities and should be discarded. (8) Column cleaning: Clean the chromatography packing material with 0.5M NaOH and water for injection in sequence, and preserve the chromatography packing material with 20% ethanol.

[0028] 3. Ultrafiltration concentration and virus inactivation (1) The immunoglobulin components are concentrated by ultrafiltration and replaced by buffer system to obtain the final buffer system and the product that meets the protein concentration requirements. The virus is inactivated by low pH incubation. (2) The albumin component is inactivated by pasteurization, and then concentrated by ultrafiltration and buffer system replacement to obtain the final buffer system and the product that meets the protein concentration requirements.

[0029] 4. Testing The purity of the prepared samples was determined using a high-performance liquid chromatography system and a size exclusion column; the purity of the prepared samples was determined using protein gel electrophoresis; and the protein concentration was determined using the BCA method, and the protein recovery rate was calculated.

[0030] (III) Results of purity and recovery rate tests 1. Purity testing Canine serum immunoglobulin has a typical four-chain monomer structure, consisting of two heavy chains (H chains) and two light chains (L chains) linked by disulfide bonds, with a molecular weight of approximately 150 kDa. Upon treatment with a reducing agent, it cleaves into a heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa. Canine serum albumin is a single-chain protein with a molecular weight of approximately 66 kDa.

[0031] Serum samples, purified immunoglobulin and albumin samples were treated with reducing agent and then loaded onto SDS-PAGE gels. As a control, intravenous immunoglobulin and albumin products from a domestic blood products manufacturer were treated with reducing agent and then loaded onto SDS-polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue and images were acquired (e.g., ...). Figure 1 (As shown): Lane 4 is the protein molecular weight standard, and the molecular weight represented by each protein band is as follows: Figure 1 As shown on the right: Lane 1 is a serum sample, showing the presence of various protein components; Lane 2 is a purified immunoglobulin sample, showing two protein bands with molecular weights of 50 kDa and 25 kDa, indicating high purity and molecular weight characteristics consistent with canine immunoglobulin; Lane 3 is a purified albumin sample, showing a protein band with a molecular weight of approximately 66 kDa, indicating high purity and molecular weight characteristics consistent with canine serum albumin; the electrophoretic patterns of Lanes 2 and 3 are consistent with those of the human immunoglobulin product (Lane 5) and albumin product (Lane 6) used as controls.

[0032] The prepared immunoglobulin sample was loaded onto an HPLC size-exclusion column, and the results are as follows: Figure 2 As shown, by Figure 2 It can be seen that a protein uptake peak appeared at about 14.9 minutes after sample injection, which was an immunoglobulin component, accounting for about 96.30% of the total protein content.

[0033] The prepared albumin sample was loaded onto an HPLC size-exclusion column, and the results are as follows: Figure 3 As shown, by Figure 3 It can be seen that a protein absorption peak appeared about 16.7 minutes after sample injection, which was albumin, accounting for about 98.29% of the total protein content.

[0034] 2. Protein recovery rate detection In healthy dogs, immunoglobulins account for approximately 12%–18% of total serum protein, and albumin accounts for approximately 55%–60% of total serum protein. The immunoglobulins and albumin purified in this example contain 12.62% and 51.76% of the initial total serum protein, respectively, demonstrating high recovery efficiency. Specific data are shown in Table 1 below. Table 1. Total protein content and recovery rate in Example 1

[0035] Example 2 (a) Experimental materials Canine serum, stored at -20°C.

[0036] (II) Experimental Procedure 1. Caprylic acid precipitation (1) Serum preparation: Take 10 mL of frozen serum, thaw it at 18-20℃ and equilibrate for 30 min, dilute it with 4 volumes of purified water to 50 mL, and adjust the pH value to 6.0 with acetic acid; (2) Add precipitant: At 18-20℃, slowly add octanoic acid to a final concentration of 25 mmol / L; (3) Precipitation of impurities: At 18-20℃, add NaOH to adjust the pH to 6.0, stir for 2 hours, and let stand for 2 hours; (4) Precipitation removal: Centrifuge at 5000g for 20min, collect the supernatant and discard the precipitate; (5) Filtration of supernatant: The remaining supernatant was filtered through a 0.45 μm filter membrane and then used for subsequent experiments.

[0037] 2. Anion exchange chromatography (1) Packing the column: The anion exchange chromatography packing material Capto Q (cytiva) was packed into the chromatography column (xk16 / 20, cytiva) with a height of 10cm and a packing volume of 20mL; (2) Solution preparation: Equilibration solution ①: 0.1M acetate-sodium acetate (HAc-NaAc) buffer, pH 6.0; Elution buffer ②: 0.03-0.1M HAc-NaAc buffer containing 0.2M NaCl, pH 6.0; Elution buffer ③: HAc-NaAc buffer containing 1M NaCl, pH 6.0; (3) Sample preparation: Adjust the conductivity and pH of the sample collected in the octanoic acid precipitation step to be consistent with the equilibrium solution ①; (4) Equilibrium: Equilibrium solution ① Equilibrium chromatography packing material, equilibrium volume is 120mL, flow rate is 3.3mL / min; (5) Sample loading and rinsing: Load the prepared sample onto the equilibrated chromatography packing material at a flow rate of 3.3 mL / min; collect the flow-through liquid, and after loading, continue rinsing with equilibration solution ① until the flow-through liquid no longer contains protein, and continue to collect the flow-through liquid during the process; all flow-through liquids are immunoglobulin components, stored at 4℃, and used in subsequent preparation processes; (6) Elution 1: Wash the chromatography packing material with elution buffer ② at a flow rate of 3.3 mL / min, collect the eluted protein fraction as albumin fraction, store at 4℃ for subsequent preparation process; (7) Elution 2: Wash the chromatography packing material with elution buffer ③ at a flow rate of 3.3 mL / min. The eluted protein fraction is a contaminant protein, which should be discarded. (8) Column cleaning: Clean the chromatography packing material with 0.5M NaOH and water for injection in sequence, and preserve the chromatography packing material with 20% ethanol.

[0038] 3. Ultrafiltration concentration and virus inactivation (1) The immunoglobulin components are concentrated by ultrafiltration and replaced by buffer system to obtain the final buffer system and the product that meets the protein concentration requirements. The virus is inactivated by low pH incubation. (2) The albumin component is inactivated by pasteurization, and then concentrated by ultrafiltration and buffer system replacement to obtain the final buffer system and the product that meets the protein concentration requirements.

[0039] 4. Testing The purity of the prepared samples was determined by protein gel electrophoresis; the protein concentration was determined by the BCA method, and the protein recovery rate was calculated.

[0040] (III) Results of purity and recovery rate tests 1. Purity testing Serum samples, purified immunoglobulin and albumin samples were treated with a reducing agent and then loaded onto an SDS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and images were acquired, as detailed below. Figure 4 As shown: Lane 1 is the molecular weight standard for protein electrophoresis; Lane 2 is a serum sample, showing the presence of various protein components; Lane 3 is a purified immunoglobulin sample, showing two main protein bands with molecular weights of 50 kDa and 25 kDa, indicating high purity and molecular weight characteristics consistent with canine immunoglobulins; Lane 4 is a purified albumin sample, showing a main protein band with a molecular weight of approximately 66 kDa, indicating high purity and molecular weight characteristics consistent with canine serum albumin.

[0041] 2. Protein recovery rate detection The immunoglobulins and albumins purified in this example contained 11.67% and 49.44% of the initial serum total protein, respectively, demonstrating high recovery efficiency. Specific data are shown in Table 2 below. Table 2. Total protein content and recovery rate in Example 2

[0042] In summary, this invention employs a process route of "octanoic acid precipitation + one-step anion exchange chromatography + ultrafiltration concentration and virus inactivation," which enables the simultaneous separation and purification of two target products, canine serum immunoglobulin and albumin, from canine serum under different storage conditions (short-term storage at 4°C and long-term cryopreservation below -20°C), thus achieving efficient utilization of canine serum resources.

[0043] Regarding purity, in Example 1, SDS-polyacrylamide gel electrophoresis showed that the purified immunoglobulin sample mainly exhibited two protein bands with molecular weights of 50 kDa (heavy chain) and 25 kDa (light chain), while the albumin sample mainly exhibited a protein band with a molecular weight of 66 kDa, both consistent with the molecular weight characteristics of their respective proteins. HPLC size exclusion chromatography showed that the immunoglobulin purity reached 96.30%, and the albumin purity reached 98.29%, consistent with the electrophoretic patterns of the control human immunoglobulin and albumin products. In Example 2, SDS-polyacrylamide gel electrophoresis also showed that the purified immunoglobulin sample mainly exhibited two protein bands with molecular weights of 50 kDa and 25 kDa, while the albumin sample mainly exhibited a protein band with a molecular weight of 66 kDa, both consistent with the molecular weight characteristics of their respective proteins, indicating high purity.

[0044] Regarding recovery efficiency, in Example 1, the immunoglobulin recovery rate was 12.62%, and the albumin recovery rate was 51.76%, both within the reasonable range of the natural proportions of the corresponding proteins in the serum of healthy dogs (immunoglobulin 12%-18%, albumin 55%-60%). In Example 2, the immunoglobulin recovery rate was 11.67%, and the albumin recovery rate was 49.44%, also maintaining a high recovery efficiency.

[0045] In terms of process adaptability, under different process parameters (such as final octanoic acid concentration of 25 mmol / L-35 mmol / L, pH value of 5.3-6.0, centrifugation speed of 4000g-5000g, chromatography flow rate of 3.3mL / min-5mL / min, and filter membrane pore size of 0.22μm-0.45μm), the preparation process of the present invention can stably obtain the target product with high purity and high recovery efficiency, demonstrating good process stability and adaptability.

[0046] Regarding virus inactivation, this invention achieves virus inactivation of the product by using low-pH incubation to inactivate the immunoglobulin component and pasteurization to inactivate the albumin component, thus meeting the safety requirements of biological products.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for the preparation of canine serum immunoglobulin and albumin, characterized in that, The method comprises the following steps: (1) octanoic acid precipitation: dilute the dog serum, adjust the pH, add octanoic acid to precipitate impurities, remove the precipitate, collect the supernatant and filter; (2) one-step anion exchange chromatography: load the filtered supernatant of step (1) into an anion exchange chromatography column, collect the flow-through to obtain an immunoglobulin component, and subsequently collect an albumin component by elution; (3) ultrafiltration concentration and virus inactivation: concentrate the immunoglobulin component by ultrafiltration, replace the buffer system, and perform virus inactivation by low-pH incubation to obtain a dog serum immunoglobulin, and concentrate the albumin component by ultrafiltration, replace the buffer system, and perform pasteurization to obtain a dog serum albumin.

2. The production method according to claim 1, characterized by, In step (1), the dog serum is diluted with 3-10 times the volume of purified water, and the pH is adjusted to 4.5-6.5 with acetic acid or hydrochloric acid after dilution.

3. The production method according to claim 1, characterized by, In step (1), the final concentration of octanoic acid is 20-35 mmol / L, and the pH is adjusted to the pH before the addition of octanoic acid with NaOH after the addition of octanoic acid, stirred for 0.5-2 h and allowed to stand for 0.5-2 h.

4. The method of claim 1, wherein, In step (1), the precipitate is removed by centrifugation at 4000-5000 g for 10-30 min, or by pressure filtration or filtration, and the supernatant is filtered using a 0.22-0.45 μm filter membrane.

5. The preparation method according to claim 1, characterized in that, In step (2), the packing material for anion exchange chromatography is Capto Q, and the equilibration buffer and elution buffer are both acetic acid-sodium acetate buffers with a pH of 4.5-6.

5.

6. The method of claim 1, wherein, In step (2), the albumin component collected by elution is a protein component collected by elution with a buffer containing 0.2 M NaCl.

7. A canine serum immunoglobulin, characterized in that, The immunoglobulin prepared by the preparation method of any one of claims 1-6 has a heavy chain with a molecular weight of about 50 kDa and a light chain with a molecular weight of 25 kDa after treatment with a reducing agent, and the purity is ≥96% as shown by SDS-polyacrylamide gel electrophoresis.

8. A canine serum albumin, characterized in that, The albumin prepared by the preparation method of any one of claims 1-6 has a molecular weight of about 66 kDa, and the purity is ≥96%.

9. Use of the dog serum immunoglobulin of claim 7 and / or the dog serum albumin of claim 8 in the preparation of a veterinary biological product.

10. A veterinary biological product, characterized in that, The veterinary biological product comprises the dog serum immunoglobulin of claim 7 and / or the dog serum albumin of claim 8.

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