Method for preparing horse-derived immunoglobulin F (ab ') 2 by sodium caprylate precipitation process

Abstract

The invention discloses a method for preparing horse-derived immune globulin F (ab ') 2 by a sodium caprylate precipitation process, and belongs to the technical field of biological pharmacy and plasma immune globulin separation and purification. According to the method, horse source immune plasma is used as a raw material, enzymatic hydrolysate containing immune globulin F (ab ') 2 is obtained through protease enzymolysis, sodium caprylate is added to serve as a selective precipitator, non-target impure protein is selectively precipitated, and target protein F (ab') 2 is kept in the solution. After a clear feed liquid is obtained through solid-liquid separation, membrane separation or chromatographic purification treatment can be further carried out according to needs, and a horse-derived immunoglobulin F (ab ') 2 product is obtained. The precipitate formed by the invention is suitable for industrial filtration operation, solves the problems of low recovery rate of the existing ammonium sulfate precipitate and difficulty in filtration of the free octanoic acid precipitate, improves the feasibility and stability of the process, and improves the product recovery rate.

Description

A method for preparing equine immunoglobulin F(ab')2 by sodium caprylate precipitation. Technical Field

[0001] This invention relates to the fields of biopharmaceutical and plasma immunoglobulin separation and purification technology, specifically to a method for preparing equine immunoglobulin F(ab')2 using sodium caprylate precipitation. Background Technology

[0002] Equine immunoglobulin products, especially immunoglobulin F(ab')2 obtained through enzymatic hydrolysis, are widely used in antitoxin and immunotherapy. Compared with intact IgG, the F(ab')2 fragment has a smaller molecular weight and reduced complement activation capacity, resulting in better safety in clinical use.

[0003] The industrial preparation of equine immunoglobulin F(ab')2 typically involves plasma collection, enzymatic hydrolysis, and separation and purification steps. Among these, the separation and purification process after enzymatic hydrolysis directly affects the handleability, recovery rate, and product quality of the intermediates.

[0004] Currently used protein separation methods include ammonium sulfate fractionation and free octanoic acid precipitation. Ammonium sulfate fractionation is a multi-step, non-selective salting-out process that easily causes co-precipitation of non-target proteins and immunoglobulin F(ab')2, reducing the recovery rate of the active ingredient and increasing the burden on subsequent desalting and purification. Free octanoic acid precipitation can remove impurities under certain conditions, but it affects the pH of the system during precipitation. Under the same engineered processing conditions as in this invention, free octanoic acid precipitation is difficult to form a precipitation structure suitable for industrial filtration, leading to difficulties in clarification and filtration, and turbid filtrate, thus limiting its application in large-scale production.

[0005] Therefore, it is necessary to develop a separation method that can form a filterable and engineerable precipitate under mild conditions to improve the process feasibility and stability of the preparation of equine immunoglobulin F(ab')2. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing equine immunoglobulin F(ab')2 using a sodium caprylate precipitation process.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Raw material pretreatment: Using equine immune plasma as raw material, enzymatic hydrolysis is performed under acidic conditions to convert immunoglobulin IgG into immunoglobulin F(ab')2, thereby obtaining an enzymatic hydrolysate containing the target product.

[0008] Selective precipitation: Sodium caprylate is added to the enzymatic hydrolysate as the sole precipitant without introducing free fatty acids, allowing sodium caprylate to participate in the precipitation process as an anion. The precipitation process is carried out under weakly acidic conditions at the isoelectric point of non-immunoglobulin F(ab')2, avoiding non-specific precipitation of the target protein induced by acidity, while achieving selective precipitation of albumin, complement proteins, and non-target proteins generated by enzymatic hydrolysis, ensuring that immunoglobulin F(ab')2 remains in a dissolved state. Sodium caprylate can be added in one go or continuously, and the precipitation effect does not depend on precise control of the feeding rate, making the operation convenient.

[0009] Solid-liquid separation and purification: The enzymatic hydrolysate after precipitation is subjected to solid-liquid separation to obtain a clear feed solution rich in immunoglobulin F(ab')2. This feed solution has good intermediate industrial filtration adaptability and can be smoothly carried out in subsequent purification operations. The purification process includes at least one of membrane separation or chromatography to further remove residual impurities and improve product purity.

[0010] Formulation: The purified immunoglobulin F(ab')2 was subjected to formulation to obtain equine immunoglobulin F(ab')2 products.

[0011] The beneficial effects of this invention are as follows: This invention achieves selective precipitation of impurities under non-isoelectric point conditions, reducing non-specific co-precipitation; the formed precipitation structure is suitable for industrial filtration operations, and a clarified intermediate can be obtained; compared with ammonium sulfate multi-step fractionation precipitation, it reduces the co-precipitation of impurities and equine immunoglobulin F(ab')2, improving process efficiency; compared with free caprylic acid precipitation, it avoids colloidal precipitation and filtration difficulties under the same conditions, and has good process stability and scale-up feasibility.

[0012] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0013] Figure 1 is a schematic diagram of the recovery rate of immunoglobulin F(ab')2 according to an embodiment of the present invention; Figure 2 is a schematic diagram of the purity of immunoglobulin F(ab')2 according to an embodiment of the present invention; Figure 3 is a schematic diagram of the protein activity recovery rate according to an embodiment of the present invention. Detailed Implementation

[0014] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0015] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the field; unless otherwise specified, the materials described are all from commercial channels.

[0016] Referring to Figures 1-3, this invention provides a method for preparing equine immunoglobulin F(ab')2 using a sodium caprylate precipitation process, as detailed in the following examples.

[0017] Example 1: Preparation of equine immunoglobulin F(ab')2 by sodium caprylate precipitation. Equine immune plasma was used as the starting material. Under normal acidic conditions, pepsin was added for enzymatic hydrolysis to convert immunoglobulin IgG into immunoglobulin F(ab')2, and the hydrolysate was obtained.

[0018] Adjust the pH of the enzymatic hydrolysate to 4.5, then add sodium octanoate aqueous solution to the enzymatic hydrolysate to a final concentration of 120 mM, and stir the reaction for 30 minutes.

[0019] After the reaction, solid-liquid separation was performed, followed by clarification and filtration to obtain a filtrate rich in immunoglobulin F(ab')2. The filtrate was clear and transparent, meeting the basic engineering requirements for subsequent purification steps. The filtrate was then subjected to membrane separation or chromatographic purification as needed to obtain the equine immunoglobulin F(ab')2 product.

[0020] Specifically, the recovery rate (%) of immunoglobulin F(ab')2 = overall protein recovery rate × purity.

[0021] The recovery rate of immunoglobulin F(ab')2 in this embodiment was 20.73±0.22%, the purity of immunoglobulin F(ab')2 was 86.93±0.54%, and the protein activity recovery rate was 67.11±4.40%. The obtained product showed good process repeatability in multiple batches of preparation, and the batch-to-batch coefficient of variation of the overall protein recovery rate was 0.45%.

[0022] Comparative Example 1: Preparation of equine immunoglobulin F(ab')2 by ammonium sulfate fractionation precipitation. Equine immune plasma from the same source as in Example 1 was subjected to pepsin enzymatic hydrolysis under the same conditions to obtain an enzymatic hydrolysate containing immunoglobulin F(ab')2.

[0023] Under the same temperature and processing volume conditions as in Example 1, ammonium sulfate fractional precipitation was used for treatment. After the reaction, solid-liquid separation was performed, followed by dissolution, desalting, and subsequent purification steps to prepare immunoglobulin F(ab')2.

[0024] The recovery rate of immunoglobulin F(ab')2 prepared by ammonium sulfate fractionation precipitation was 14.25 ± 0.27%, the purity of immunoglobulin F(ab')2 was 80.92 ± 0.28%, and the protein activity recovery rate was 46.63 ± 0.08%. The batch-to-batch coefficient of variation for the overall protein recovery rate was 1.55%. Compared with Example 1, the recovery rates of immunoglobulin F(ab')2 and protein activity were significantly reduced.

[0025] Comparative Example 2: Preparation of equine immunoglobulin F(ab')2 by precipitation with free caprylic acid of the same concentration. Equine immune plasma from the same source as in Example 1 was used and subjected to pepsin enzymatic hydrolysis under the same protein concentration, processing volume and operating conditions to obtain an enzymatic hydrolysate containing immunoglobulin F(ab')2.

[0026] Under the same protein concentration, treatment volume, and pH conditions as in Example 1, the only difference was that the precipitant was replaced with free caprylic acid, and the amount added was the same as the molar amount of sodium caprylate added in Example 1, based on the caprylate ion. Precipitation treatment was then performed.

[0027] During the precipitation process, it was observed that the precipitate formed in the system was in a fine flocculent or colloidal dispersion state, making it difficult to form a dense precipitate phase with a clear phase interface. After solid-liquid separation following the reaction, turbidity persisted, and a significant increase in filtration resistance occurred during the clarification filtration process, making it impossible to complete the filtration step. These phenomena were observed in multiple repeated experiments, indicating that under the same engineering processing conditions as in the example, it is difficult to obtain a clear intermediate that meets the requirements of subsequent membrane separation and chromatography using free octanoic acid under the same conditions; the process becomes impractical at this stage. Due to the above engineering limitations, Comparative Example 2 could not continue with the subsequent purification process of Example 1.

[0028] Comparison of Experimental Results and Explanation of Effects: By comparing and analyzing the experimental results of Example 1 with those of Comparative Examples 1 and 2, it can be clearly seen that there are essential differences in the precipitation behavior and technical effects exhibited by different precipitants under the same engineering treatment conditions.

[0029] In Example 1, sodium octanoate was used as a precipitant, which formed a precipitate with a well-defined phase interface without significantly altering the pH of the system. The resulting precipitate had a stable structure and suitable particle size, facilitating subsequent solid-liquid separation. The resulting filtrate was clear and transparent, demonstrating good industrial filtration suitability. Testing showed that immunoglobulin F(ab')2 exhibited a high effective recovery rate and low batch-to-batch variability, indicating that this precipitation method has good repeatability and scalability.

[0030] In contrast, when ammonium sulfate fractionation was used in Comparative Example 1, although a certain degree of protein separation could be achieved, the recovery rate of immunoglobulin F(ab')2 was significantly reduced during the precipitation process. This may be due to the co-precipitation of immunoglobulin F(ab')2 with other proteins. The overall process efficiency was significantly lower than that of Example 1.

[0031] In Comparative Example 2, under the same protein concentration, treatment volume, and operating conditions as Example 1, only the precipitant was replaced with free octanoic acid, and the same molar addition was performed. The purpose of this comparison was to evaluate the differences in precipitation behavior of different precipitants under the same engineering conditions, rather than to explore optimal conditions for each precipitant individually. Experimental results showed that the precipitate formed in this system was in a fine flocculent or colloidal dispersion state, making it difficult to form a dense precipitate phase suitable for industrial filtration operations. This resulted in a significant increase in filtration resistance during clarification filtration, and the resulting filtrate remained turbid. These phenomena were observed in multiple repeated experiments, indicating that free octanoic acid is difficult to meet the engineering separation requirements in this system.

[0032] The results above show that sodium octanoate, ammonium sulfate, and free octanoic acid exhibit significant differences in precipitation behavior within the system of this invention. Sodium octanoate, by participating in the precipitation process as an anion, can selectively precipitate non-immunoglobulin proteins under relatively mild conditions, forming engineerable precipitation structures. Ammonium sulfate precipitation, however, is limited by a non-selective salting-out mechanism, resulting in low recovery rates of immunoglobulin F(ab')2 and protein activity. Free octanoic acid, on the other hand, readily forms colloidal precipitates, making effective filtration difficult.

[0033] The aforementioned technical effects cannot be achieved simply by replacing the precipitant or adjusting conventional process parameters; rather, they stem from the differences in the morphology and mechanism of action of the precipitants. Therefore, the sodium caprylate precipitation method employed in this invention demonstrates superior overall technical performance in the industrial preparation of immunoglobulin F(ab')2.

[0034] The equine immunoglobulin F(ab')2 prepared by the method described in this invention can be used to prepare antitoxin serum or other equine immunotherapy products, and has good prospects for industrial application.

[0035] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0036] 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 preparing equine immunoglobulin F(ab')2 by sodium caprylate precipitation, characterized in that, The process includes the following steps: S1, providing equine immune plasma and performing enzymatic hydrolysis under acidic conditions to obtain an enzymatic hydrolysate containing immunoglobulin F(ab')2; S2, adding sodium caprylate as a precipitant without introducing free fatty acids, allowing sodium caprylate to exist in a dissociated state, thereby selectively precipitating non-immunoglobulin impurities; S3, performing solid-liquid separation on the precipitate system obtained in step S2 to obtain a clear solution rich in immunoglobulin F(ab')2; S4, performing at least one purification treatment on the clear solution to obtain immunoglobulin F(ab')2; S5, performing formulation treatment on the purified immunoglobulin F(ab')2 to obtain equine immunoglobulin F(ab')2 products; wherein the precipitate formed is a precipitate structure suitable for industrial filtration operations.

2. The method according to claim 1, characterized in that, The precipitant used in step S2 is only sodium octanoate, excluding free octanoic acid and octanoate salts formed by adding free octanoic acid and then neutralizing it.

3. The method according to claim 1, characterized in that, The precipitation process in step S2 is carried out under the isoelectric point conditions of non-immunoglobulin F(ab')2.

4. The method according to claim 1, characterized in that, In step S2, the pH of the precipitation system is in a weakly acidic range to avoid acid-induced nonspecific precipitation of immunoglobulin F(ab')2.

5. The method according to claim 1, characterized in that, The amount of sodium octanoate added in step S2 must meet the following requirements: it should be able to achieve selective precipitation of non-immunoglobulin contaminants and keep immunoglobulin F(ab')2 in a dissolved state.

6. The method according to claim 1, characterized in that, In step S2, sodium caprylate preferentially precipitates albumin, complement protein, and non-target proteins generated during enzymatic hydrolysis, while immunoglobulin F(ab')2 remains in the feed solution.

7. The method according to claim 1, characterized in that, Compared with the ammonium sulfate fractionation precipitation method, this method can improve the effective recovery rate of immunoglobulin F(ab')2 while reducing the co-precipitation of impurities with immunoglobulin F(ab')2.

8. The method according to claim 1, characterized in that, In step S2, sodium octanoate can be added either in one go or continuously, and the precipitation effect does not depend on the precise control of the feeding rate.

9. The method according to claim 1, characterized in that, The purification process in step S4 includes at least one of membrane separation or chromatography.

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