Method for purifying recombinant canine serum albumin from pichia fermenting liquor

Abstract

This application relates to a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The method includes the following steps: performing compound anion exchange chromatography on the supernatant of the Pichia pastoris fermentation broth to prepare a first chromatographic product; performing hydrophobic chromatography on the first chromatographic product to prepare a second chromatographic product; and performing weak anion exchange chromatography on the second chromatographic product to achieve purification of recombinant canine serum albumin. This application obtains recombinant canine serum albumin by sequentially purifying it using compound anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography. This method is efficient, simple, and yields recombinant canine serum albumin with satisfactory purity, which has significant practical implications for clinical treatment.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. Background Technology

[0002] Canine serum albumin (CSA) is commonly used in the treatment of canine distemper or parvovirus. It can enhance the dog's immunity, promote development, and help the dog maintain its strength and fight off the virus.

[0003] There are many methods for preparing albumin, mainly including low-temperature ethanol precipitation, column chromatography, and ultrafiltration. Low-temperature ethanol precipitation uses low-temperature ethanol at different concentrations to fractionally precipitate plasma, extracting various plasma protein components. These components are then processed through ultrafiltration or freeze-drying for alcohol removal, concentration, and freeze-drying to obtain albumin. Column chromatography separates and purifies the target protein using different column packing materials and elution buffers under specific pH, ionic strength, and concentration conditions. Ultrafiltration uses pressure differences to separate proteins of different molecular weights through ultrafiltration membranes with different pore sizes. Currently, the main source of canine albumin reported in studies is canine blood, which limits the availability of raw materials and increases costs. Currently, methods such as ammonium sulfate precipitation, column chromatography, and ultrafiltration all suffer from low albumin purity or yield and high costs, failing to meet the needs of practical production. Therefore, the production of canine serum albumin using recombinant DNA technology has been developed and utilized.

[0004] Researchers used gene editing technology to edit the plasmid genome, inserting the canine serum albumin gene into the editable region, causing Pichia pastoris to produce recombinant canine serum albumin (rCSA). Large quantities of high-purity rCSA were obtained by purifying the Pichia pastoris fermentation broth. The rCSA isolated and purified from the Pichia pastoris fermentation broth must meet the requirements for clinical drug use and biochemical research; therefore, the isolation and purification of rCSA from the fermentation broth supernatant is a crucial step.

[0005] Finding an efficient and simple method to isolate and purify recombinant canine serum albumin from transgenic Pichia pastoris fermentation broth, and obtaining high-purity recombinant canine serum albumin, has significant practical implications for clinical treatment and biochemical research. Summary of the Invention

[0006] Based on this, one or more embodiments of this application provide a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. This includes the following technical solutions:

[0007] One or more embodiments of this application provide a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, the method comprising the following steps:

[0008] The supernatant of Pichia pastoris fermentation broth was subjected to complex anion exchange chromatography to prepare the first chromatographic product;

[0009] The first chromatographic product was subjected to hydrophobic chromatography to prepare a second chromatographic product; and,

[0010] The second chromatographic product was subjected to weak anion exchange chromatography to purify recombinant canine serum albumin.

[0011] In some embodiments of this application, the supernatant contains a protein stabilizer;

[0012] Optionally, the protein stabilizer includes one or more of caprylic acid and sodium caprylate;

[0013] Optionally, the supernatant is supplemented with 45 mM to 55 mM of the protein stabilizer.

[0014] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0015] (1) The media for composite anion exchange chromatography include NM 90Agarose HAM;

[0016] (2) The media for hydrophobic chromatography include UniHR Butyl-80L; and,

[0017] (3) The media for weak anion exchange chromatography include: UniGel 30DEAE.

[0018] In some embodiments of this application, the second chromatographic product is subjected to ultrafiltration followed by weak anion exchange chromatography;

[0019] Optionally, the ultrafiltration treatment conditions include: using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuging at 5800 rpm to 6200 rpm for 8 min to 12 min.

[0020] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0021] 1) The conductivity of the loading solution for composite anion exchange chromatography is 19 mS / cm to 23 mS / cm;

[0022] 2) The conductivity of the sample loading solution for hydrophobic chromatography is 45 mS / cm to 49 mS / cm; and,

[0023] 3) The conductivity of the loading solution for weak anion exchange chromatography is 2 mS / cm to 4 mS / cm.

[0024] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0025] (A) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in the composite anion exchange chromatography process are each independently 6.8 to 7.2; optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in the composite anion exchange chromatography process are kept consistent.

[0026] (B) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are each independently 6.9–7.1; optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are kept consistent; and,

[0027] (C) In the process of weak anion exchange chromatography, the pH values ​​of the equilibration buffer and the eluent are each independently set to 7.8–8.1, and the pH value of the elution buffer is 5.8–6.1.

[0028] In some embodiments of this application, the composite anion exchange chromatography satisfies one or more of the following conditions:

[0029] A) The equilibrium solution consists of a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.2 M–0.25 M sodium chloride;

[0030] B) The rinsing solution includes a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.2 M–0.25 M sodium chloride;

[0031] C) The eluent consists of a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.45 M–0.55 M sodium chloride; and,

[0032] D) Isocratic elution was employed;

[0033] Optionally, the equilibration buffer and eluent in the composite anion exchange chromatography have the same composition.

[0034] In some embodiments of this application, hydrophobic chromatography satisfies one or more of the following conditions:

[0035] (I) The equilibrium solution includes a phosphate buffer solution with a pH of 6.9–7.1 and a pH of 17 mM–23 mM and a sodium chloride solution of 0.45 M–0.55 M;

[0036] (II) The eluent consists of a phosphate buffer solution with a pH of 6.9–7.1 and a strength of 17 mM–23 mM;

[0037] (III) The rinsing solution comprises a phosphate buffer solution with a pH of 6.9–7.1, 17 mM–23 mM, and 0.45 M–0.55 M sodium chloride; and,

[0038] (IV) Isocratic elution was employed;

[0039] Optionally, the equilibration buffer and eluent in hydrophobic chromatography have the same composition.

[0040] In some embodiments of this application, weak anion exchange chromatography satisfies one or more of the following conditions:

[0041] Ⅰ) The equilibrium solution includes a phosphate buffer solution with a pH of 7.8–8.1 and a strength of 17 mM–23 mM;

[0042] II) The eluent includes a variety of components; optionally, the eluent includes:

[0043] Eluent 1 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM and a concentration of 0.16 M–0.17 M sodium chloride;

[0044] Eluent 2 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM and a concentration of 0.17 M–0.19 M sodium chloride;

[0045] Eluent 3 comprises a phosphate buffer solution with a pH of 5.8–6.1, 17 mM–23 mM, and 0.2 M–0.23 M sodium chloride; and,

[0046] Eluent 4 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM, and a sodium chloride solution of 0.8 M–1.2 M.

[0047] III) The rinsing solution includes a phosphate buffer solution with a pH of 7.8–8.1 and a strength of 17 mM–23 mM; and,

[0048] IV) Use isocratic elution;

[0049] Optionally, the equilibration buffer and eluent in weak anion exchange chromatography have the same composition.

[0050] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0051] (i) After the composite anion exchange chromatography is completed, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and optionally, a phosphate buffer solution with a pH of 6.8 to 7.2 and a concentration of 17mM to 23mM, including 0.2M to 0.25M sodium chloride, is used for equilibration.

[0052] (ii) After hydrophobic chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and optionally, a phosphate buffer solution with a pH of 6.9 to 7.1 and a pH of 17mM to 23mM containing 0.45M to 0.55M sodium chloride is used for equilibration.

[0053] (iii) After weak anion exchange chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and a phosphate buffer solution with a pH of 7.8 to 8.1 and a pH of 17mM to 23mM is used for equilibration.

[0054] One or more embodiments of this application provide a method for preparing recombinant canine serum albumin, the method comprising the step of purifying recombinant canine serum albumin using the method described above.

[0055] One or more embodiments of this application provide a method for preparing a recombinant canine serum albumin product, the preparation method comprising:

[0056] The method described above was used to purify recombinant canine serum albumin; and,

[0057] Products were prepared using purified recombinant canine serum albumin.

[0058] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0059] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 This is the composite anion exchange chromatography chromatogram from Example 1;

[0061] Figure 2 This is the hydrophobic chromatography chromatogram from Example 1;

[0062] Figure 3 This is the weak anion exchange chromatography chromatogram from Example 1;

[0063] Figure 4This is the composite anion exchange chromatography electrophoresis image from Example 1 (1-Marker, 2-Loading solution, 3-Breakthrough buffer 1, 4-Breakthrough buffer 2, 5-Eluent, 6-Eluent).

[0064] Figure 5 This is the hydrophobic chromatography electrophoresis image from Example 1 (1-Marker, 2-Loading solution, 3-Breakthrough buffer 1 + Breakthrough buffer 2, 4-Eluent 1, 5-Eluent 2, 6-Eluent 3);

[0065] Figure 6 This is the weak anion exchange chromatography electrophoresis image from Example 1 (1-Marker, 2-Loading solution, 3-Eluent 1, 4-Eluent 2, 5-Eluent 3, 6-Eluent 4);

[0066] Figure 7 This is the HPLC spectrum of the product obtained by composite anion exchange chromatography in Example 1;

[0067] Figure 8 This is the HPLC spectrum of the product obtained by hydrophobic chromatography in Example 1;

[0068] Figure 9 This is the HPLC spectrum of the product obtained by weak anion exchange chromatography in Example 1. Detailed Implementation

[0069] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0071] the term

[0072] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0073] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0074] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0075] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0076] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0077] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0078] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0079] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0080] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0081] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0082] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0083] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0084] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0085] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0086] A first aspect of this application provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, the method comprising the following steps:

[0087] The supernatant of Pichia pastoris fermentation broth was subjected to complex anion exchange chromatography to prepare the first chromatographic product;

[0088] The first chromatographic product was subjected to hydrophobic chromatography to prepare a second chromatographic product; and,

[0089] The second chromatographic product was subjected to weak anion exchange chromatography to purify recombinant canine serum albumin.

[0090] The method provided in this application, which sequentially purifies recombinant canine serum albumin through compound anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography, is efficient, simple, and yields recombinant canine serum albumin with the required purity, which has significant practical implications for clinical treatment.

[0091] In this embodiment, composite anion exchange chromatography is used to capture recombinant canine serum albumin in the supernatant of Pichia pastoris fermentation broth. Subsequently, hydrophobic chromatography is used to perform moderate purification of the obtained recombinant canine serum albumin. Finally, weak anion exchange chromatography is used to perform fine purification of the moderately purified recombinant canine serum albumin.

[0092] It should be noted that the embodiments of this application sequentially employ composite anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography to capture and purify recombinant canine serum albumin. The synergistic effect between the three chromatography steps improves the purification effect, ultimately yielding high-purity recombinant canine serum albumin. Changing the order of the three chromatography steps will result in poor purification and failure to obtain a qualified recombinant human serum albumin sample.

[0093] The embodiments of this application have the following beneficial effects:

[0094] (1) This application provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The purity of the recombinant canine serum albumin obtained after the first step of using compound anion exchange chromatography can reach 50-60%. The purity of the recombinant canine serum albumin obtained after the second step of using hydrophobic chromatography can reach 90-94%. Finally, the purity of the recombinant canine serum albumin obtained by hydrophobic chromatography can reach 99.5%. The high-purity recombinant canine serum albumin obtained can be used for clinical treatment and has important practical significance.

[0095] (2) The method for purifying recombinant canine serum albumin provided in this application is efficient and simple. The chromatography medium used in the three-step chromatography operation has been commercialized and can be further industrialized, thus being better applied to the medical industry.

[0096] In some embodiments of this application, the supernatant contains a protein stabilizer;

[0097] Optionally, the protein stabilizer includes one or more of caprylic acid and sodium caprylate;

[0098] Optionally, the supernatant is supplemented with 45mM to 55mM (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM) of the protein stabilizer.

[0099] The above pretreatment steps can stabilize the target protein and remove some impurities, facilitating subsequent separation and purification. The stabilizer protects the protein during pretreatment, preventing degradation.

[0100] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0101] (1) The media for composite anion exchange chromatography include NM 90Agarose HAM;

[0102] (2) The media for hydrophobic chromatography include UniHR Butyl-80L; and,

[0103] (3) The media for weak anion exchange chromatography include: UniGel 30DEAE.

[0104] In the embodiments of this application: the NM 90Agarose HAM chromatography medium used is a composite ion exchange chromatography medium with hydrophobic and ionic groups bonded on highly cross-linked agarose microspheres, exhibiting superior selectivity compared to traditional ion exchange chromatography media; the UniHR Butyl-80L chromatography medium has high mechanical strength and high resolution, adsorbs biomolecules under high salt conditions and elutes under low salt conditions, exhibiting excellent adsorption capacity and low nonspecific adsorption, making it suitable for further separation and purification of products after the ion exchange process; the UniGel-30DEAE chromatography medium has excellent mechanical strength, good chemical stability, and uniform particle size. It uses polymethyl methacrylate microspheres as a matrix, and reduces nonspecific adsorption through surface hydrophilic modification, exhibiting chromatographic advantages of high flow rate, high resolution, high loading capacity, and low back pressure.

[0105] In this invention, NM 90Agarose HAM, UniHR Butyl-80L and UniGel-30DEAE chromatography media are used in combination. The three chromatography media are used in a specific order, and the three work together to achieve the effect of elution and purification, and finally obtain recombinant canine serum albumin with high purity and high yield.

[0106] In some embodiments of this application, the second chromatographic product is subjected to ultrafiltration followed by weak anion exchange chromatography;

[0107] Optionally, the ultrafiltration conditions include: using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuging at 5800 rpm to 6200 rpm (e.g., 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200 rpm) for 8 min to 12 min (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 min).

[0108] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0109] 1) The conductivity of the loading solution for composite anion exchange chromatography is 19 mS / cm to 23 mS / cm (e.g., 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23 mS / cm);

[0110] 2) The conductivity of the sample loading solution for hydrophobic chromatography is 45 mS / cm to 49 mS / cm (e.g., 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49 mS / cm); and,

[0111] 3) The conductivity of the loading solution for weak anion exchange chromatography is 2 mS / cm to 4 mS / cm (e.g., 2, 2.5, 3, 3.5, 4 mS / cm).

[0112] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0113] (A) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in the combined anion exchange chromatography process are each independently 6.8 to 7.2 (e.g., 6.8, 6.9, 7, 7.1, 7.2); optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in the combined anion exchange chromatography process are kept consistent.

[0114] (B) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are each independently 6.9–7.1 (e.g., 6.9, 7, 7.1); optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are kept consistent; and,

[0115] (C) In the process of weak anion exchange chromatography, the pH values ​​of the equilibration buffer and the eluent are each independently 7.8 to 8.1 (e.g., 7.8, 7.9, 8, 8.1), and the pH value of the eluent is 5.8 to 6.1 (e.g., 5.8, 5.9, 6.0, 6.1).

[0116] In some embodiments of this application, the composite anion exchange chromatography satisfies one or more of the following conditions:

[0117] A) The equilibrium solution includes a phosphate buffer solution with a pH of 6.8–7.2 (e.g., 6.8, 6.9, 7, 7.1, 7.2) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and a sodium chloride solution of 0.2 M–0.25 M (e.g., 0.2, 0.21, 0.22, 0.23, 0.24, 0.25 M);

[0118] B) The rinsing solution includes a phosphate buffer solution with a pH of 6.8–7.2 (e.g., 6.8, 6.9, 7, 7.1, 7.2) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and a pH of 0.2 M–0.25 M (e.g., 0.2, 0.21, 0.22, 0.23, 0.24, 0.25 M);

[0119] C) The eluent comprises a phosphate buffer solution with a pH of 6.8–7.2 (e.g., 6.8, 6.9, 7, 7.1, 7.2) at 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride at 0.45 M–0.55 M (e.g., 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55 M); and,

[0120] D) Isocratic elution was employed;

[0121] Optionally, the equilibration buffer and eluent in the composite anion exchange chromatography have the same composition.

[0122] In some embodiments of this application, hydrophobic chromatography satisfies one or more of the following conditions:

[0123] (I) The equilibrium solution includes a phosphate buffer solution with a pH of 6.9–7.1 (e.g., 6.9, 7, 7.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride solution with a pH of 0.45 M–0.55 M (e.g., 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55 M);

[0124] (II) The eluent includes a phosphate buffer solution with a pH of 6.9 to 7.1 (e.g., 6.9, 7, 7.1) and a pH of 17 mM to 23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM);

[0125] (III) The rinsing solution comprises a phosphate buffer solution with a pH of 6.9–7.1 (e.g., 6.9, 7, 7.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride at a pH of 0.45 M–0.55 M (e.g., 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55 M); and,

[0126] (IV) Isocratic elution was employed;

[0127] Optionally, the equilibration buffer and eluent in hydrophobic chromatography have the same composition.

[0128] In some embodiments of this application, weak anion exchange chromatography satisfies one or more of the following conditions:

[0129] I) The equilibrium solution includes phosphate buffer solutions with pH values ​​of 7.8–8.1 (e.g., 7.8, 7.9, 8, 8.1) and 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM);

[0130] II) The eluent includes a variety of components; optionally, the eluent includes:

[0131] Eluent 1 comprises a phosphate buffer solution with a pH of 5.8–6.1 (e.g., 5.8, 5.9, 6, 6.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride at a pH of 0.16 M–0.17 M (e.g., 0.16, 0.162, 0.164, 0.166, 0.168, 0.17).

[0132] Eluent 2 comprises a phosphate buffer solution with a pH of 5.8–6.1 (e.g., 5.8, 5.9, 6, 6.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride at a pH of 0.17 M–0.19 M (e.g., 0.17, 0.172, 0.174, 0.176, 0.178, 0.18, 0.182, 0.184, 0.186, 0.188, 0.19 M).

[0133] Eluent 3 comprises a phosphate buffer solution with a pH of 5.8–6.1 (e.g., 5.8, 5.9, 6, 6.1) at 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and sodium chloride at 0.2 M–0.23 M (e.g., 0.2, 0.21, 0.22, 0.23 M); and,

[0134] The eluent 4 comprises a phosphate buffer solution with a pH of 5.8–6.1 (e.g., 5.8, 5.9, 6, 6.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM) and a pH of 0.8 M–1.2 M (e.g., 0.8, 0.9, 1, 1.1, 1.2 M);

[0135] III) The eluent includes a phosphate buffer solution with a pH of 7.8–8.1 (e.g., 7.8, 7.9, 8, 8.1) and a pH of 17 mM–23 mM (e.g., 17, 18, 19, 20, 21, 22, 23 mM); and,

[0136] IV) Use isocratic elution;

[0137] Optionally, the equilibration buffer and eluent in weak anion exchange chromatography have the same composition.

[0138] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0139] (i) After the composite anion exchange chromatography is completed, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M (e.g., 0.4, 0.45, 0.5, 0.55, 0.6M) is used for regeneration, and optionally, a phosphate buffer solution containing 0.2M to 0.25M (e.g., 0.2, 0.21, 0.22, 0.23, 0.24, 0.25M) of sodium chloride with a pH of 6.8 to 7.2 (e.g., 6.8, 6.9, 7, 7.1, 7.2) and 17mM to 23mM (e.g., 17, 18, 19, 20, 21, 22, 23mM) is used for equilibration.

[0140] (ii) After hydrophobic chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a concentration of 0.4M to 0.6M (e.g., 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6M) is used for regeneration. Optionally, a phosphate buffer solution with a pH of 0.45M to 0.55M (e.g., 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55M) and a pH of 6.9 to 7.1 (e.g., 6.9, 7, 7.1) or 17mM to 23mM (e.g., 17, 18, 19, 20, 21, 22, 23mM) is used for equilibration.

[0141] (iii) After weak anion exchange chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a concentration of 0.4M to 0.6M (e.g., 0.4, 0.45, 0.5, 0.55, 0.6M) is used for regeneration, and optional, a phosphate buffer solution with a pH of 7.8 to 8.1 (e.g., 7.8, 7.9, 8, 8.1) or 17mM to 23mM (e.g., 17, 18, 19, 20, 21, 22, 23mM) is used for equilibration.

[0142] Optionally, the collection in hydrophobic chromatography can be flow-through collection. During flow-through collection, the mobile phase remains unchanged, the conditions are singular, the method is simple, and the instrument requirements are low, facilitating the separation and purification process.

[0143] Understandably, each chromatography step typically involves column packing, equilibration, sample loading, elution, elution, regeneration, and equilibration.

[0144] Based on the above operations, the recombinant canine serum albumin obtained in this application has high purity and a wide range of applications, and can be used for clinical treatment and biological research.

[0145] A second aspect of this application provides a method for preparing recombinant canine serum albumin, the method comprising the step of purifying recombinant canine serum albumin using the method described above.

[0146] A third aspect of this application provides a method for preparing a recombinant canine serum albumin product, the method comprising:

[0147] The method described above was used to purify recombinant canine serum albumin; and,

[0148] Products were prepared using purified recombinant canine serum albumin.

[0149] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0150] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0151] Example 1

[0152] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, the specific steps of which are as follows:

[0153] (1) Preprocessing

[0154] Pichia pastoris fermentation broth containing canine serum albumin was mixed with sodium octanoate at a final molar concentration of 55 mM to adjust the pH to 7.0. The broth was then subjected to ultrafiltration using a 10 kDa ultrafiltration tube, followed by filtration through a 0.22 μm filter membrane to obtain the supernatant of the Pichia pastoris fermentation broth.

[0155] (2) Anion exchange chromatography

[0156] Add phosphate buffer solution A (20 mM phosphate buffer solution containing 0.2 M sodium chloride) with a pH of 7.0 to the NM 90Agarose HAM composite anion exchange chromatography column, and use the supernatant of Pichia pastoris fermentation broth that has been purified in step (1) as the loading solution for anion exchange chromatography. The conductivity of the supernatant of Pichia pastoris fermentation broth containing canine serum albumin is in the range of 20-21 mS / cm.

[0157] Subsequently, eluent with pH 7.0 (20 mM phosphate buffer solution containing 0.2 M sodium chloride, consistent with the formulation of phosphate buffer solution A) and elution buffer A (20 mM phosphate buffer solution containing 0.5 M sodium chloride) were added sequentially for isocratic elution, and the eluted liquid was then collected to obtain the first chromatographic product.

[0158] Then, regeneration solution A (0.5M NaOH solution) was added for regeneration, and finally phosphate buffer solution A (20mM phosphate buffer solution containing 0.2M sodium chloride, pH 7.0) was added for equilibration.

[0159] In step (2), the loading solution, breakthrough solution 1 (i.e., the liquid flowing out of the chromatography column during loading), breakthrough solution 2 (i.e., the liquid flowing out of the chromatography column after loading), rinsing solution (i.e., the liquid flowing out of the chromatography column after rinsing), and elution solution 1 (i.e., the elution peak of elution solution A) are collected for gel electrophoresis.

[0160] (3) Hydrophobic Chromatography

[0161] Add phosphate buffer solution B (20 mM phosphate buffer solution containing 0.5 M sodium chloride) with a pH of 7.0 to the UniHR Butyl-80L hydrophobic chromatography column, and use the first chromatography product obtained in step (2) as the loading solution for the hydrophobic chromatography column. The loading solution has a conductivity of 48 mS / cm and a pH of 7.0. Then, add eluent with a pH of 7.0 (20 mM phosphate buffer solution containing 0.5 M sodium chloride, with the same formulation as phosphate buffer solution B) and elution solution B (20 mM phosphate buffer solution) in sequence for isocratic elution. Collect the liquid flowing out during the loading and elution process to obtain the second chromatography product.

[0162] Then, regeneration solution B (0.5M NaOH solution) was added for regeneration, and finally phosphate buffer solution B (20mM phosphate buffer solution containing 0.5M sodium chloride, pH 7.0) was added for equilibration.

[0163] In step (2), the loading solution, breakthrough solution 1 (i.e., the liquid flowing out of the chromatography column during loading) + breakthrough solution 2 (i.e., the liquid flowing out of the chromatography column after loading), elution solution 1 (i.e., the first half of the elution peak of elution solution B), elution solution 2 (i.e., the middle peak of the elution peak of elution solution B), and elution solution 3 (i.e., the second half of the elution peak of elution solution B) are collected for gel electrophoresis.

[0164] (4) Weak anion exchange chromatography

[0165] Add a buffer solution C (20 mM phosphate buffer) with a pH of 8 to the UniGel 30-DEAE weak anion exchange chromatography column, and perform ultrafiltration on the second chromatography product obtained in step (3) using a 10 kDa ultrafiltration centrifuge tube (specifically, by adding an equal amount of the chromatography product from the previous step to the 10 kDa ultrafiltration tube, centrifuging at 6000 rpm for 10 min, followed by adding the equilibration solution for weak anion chromatography, repeating three times. Unless otherwise specified, the same procedure applies to other examples and comparative examples). This solution is used as the loading solution for the weak anion exchange chromatography column, and the conductivity of the loading solution is 3 mS / c. m, then the following solutions were added sequentially: eluent (20 mM phosphate buffer solution, pH 8.0, consistent with the equilibration solution formula), elution buffer C (20 mM phosphate buffer solution containing 0.16 M sodium chloride, pH 6), elution buffer D (20 mM phosphate buffer solution containing 0.18 M sodium chloride, pH 6), elution buffer E (20 mM phosphate buffer solution containing 0.21 M sodium chloride, pH 6), and elution buffer F (20 mM phosphate buffer solution containing 1 M sodium chloride, pH 6) for isocratic elution. The liquid after isocratic elution was collected to obtain purified recombinant human serum albumin.

[0166] Then, regeneration solution C (0.5M NaOH solution) was added for regeneration, and finally buffer solution C (20mM phosphate buffer solution) was added for equilibration.

[0167] In step (3), the loading solution, elution buffer 1 (i.e., elution buffer containing 0.16M sodium chloride in 20mM phosphate buffer solution, pH 6), elution buffer 2 (i.e., elution buffer containing 0.18M sodium chloride in 20mM phosphate buffer solution, pH 6), elution buffer 3 (i.e., elution buffer containing 0.21M sodium chloride in 20mM phosphate buffer solution, pH 6), and elution buffer 4 (i.e., elution buffer containing 1M sodium chloride in 20mM phosphate buffer solution, pH 6) were collected and subjected to gel electrophoresis.

[0168] The chromatograms and electrophoretic results of the products obtained by composite anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography are shown below. Figure 1-6 As shown. Among them, Figure 1 , Figure 2 and Figure 3 The chromatographic results of the product demonstrate the feasibility of the three-step chromatography and its subsequent scale-up. Figure 4 , Figure 5 and Figure 6 The electrophoresis results show that the recombinant canine serum albumin obtained after three-step chromatography has high purity. The HPLC chromatograms of the three-step chromatography are shown below. Figure 7-9As shown, the purities of the three-step chromatography products were 61%, 88%, and 99.5%, respectively. It can be seen that by sequentially separating and purifying the products using composite anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography, high-purity recombinant canine serum albumin can be obtained, which meets industry standards.

[0169] Example 2

[0170] This embodiment provides a method for purifying recombinant canine serum albumin from the supernatant of Pichia pastoris fermentation broth. The specific steps are as follows:

[0171] (1) Preprocessing

[0172] Pichia pastoris fermentation broth containing canine serum albumin was mixed with sodium octanoate at a final molar concentration of 45 mM to adjust the pH to 6.9. The broth was then subjected to ultrafiltration using a 10 kDa ultrafiltration tube, followed by filtration through a 0.22 μm filter membrane to obtain the supernatant of the Pichia pastoris fermentation broth.

[0173] (2) Anion exchange chromatography

[0174] Add pH 6.9 phosphate buffer solution A (20 mM phosphate buffer solution containing 0.24 M sodium chloride) to the NM 90Agarose HAM composite anion exchange chromatography column, and use the supernatant of Pichia pastoris fermentation broth that has been purified in step (1) as the loading solution for anion exchange chromatography. The conductivity range of the supernatant of Pichia pastoris fermentation broth containing canine serum albumin is 20-21 mS / cm.

[0175] Subsequently, eluent with pH 6.9 (20 mM phosphate buffer solution containing 0.24 M sodium chloride, consistent with the formulation of phosphate buffer solution A) and elution buffer A (20 mM phosphate buffer solution containing 0.48 M sodium chloride solution) were added sequentially for isocratic elution, and the eluted liquid was collected to obtain the first chromatographic product.

[0176] Then, regeneration solution A (0.5M NaOH solution) was added for regeneration, and finally buffer solution A (20mM phosphate buffer solution containing 0.24M sodium chloride) was added for equilibration.

[0177] (3) Hydrophobic Chromatography

[0178] Add phosphate buffer solution B (20 mM phosphate buffer solution containing 0.48 M sodium chloride) with a pH of 7.1 to the UniHR Butyl-80L hydrophobic chromatography column, and use the first chromatography product obtained in step (2) as the loading solution for the hydrophobic chromatography column. The loading solution has a conductivity of 46 mS / cm and a pH of 7.1. Then, add eluent with a pH of 7.1 (20 mM phosphate buffer solution containing 0.48 M sodium chloride, with the same formulation as phosphate buffer solution B) and elution solution B (20 mM phosphate buffer solution) in sequence for isocratic elution. Collect the liquid flowing out during the loading and elution process to obtain the second chromatography product.

[0179] Then, regeneration solution B (0.5M NaOH solution) was added for regeneration, and finally buffer solution B (20mM phosphate buffer solution containing 0.48M sodium chloride, pH 7.1) was added for equilibration.

[0180] (4) Weak anion exchange chromatography

[0181] Add buffer solution C (20 mM phosphate buffer) with a pH of 8.1 to the UniGel 30DEAE weak anion exchange chromatography column. The second chromatographic product obtained in step (3) is then ultrafiltered using a 10 kDa ultrafiltration centrifuge tube, and this solution is used as the loading solution for the weak anion exchange chromatography column. The conductivity of the loading solution is 2.8 mS / cm. Subsequently, eluent (20 mM phosphate buffer, pH 8.1, consistent with the formulation of buffer solution C) and elution buffer C (20 mM phosphate buffer) are added sequentially. Eluent M (containing 0.16M sodium chloride, pH 6.1) and eluents D (containing 0.18M sodium chloride, pH 6.1) and E (containing 0.21M sodium chloride, pH 6.1) and F (containing 1M sodium chloride, pH 6.1) were used for isocratic elution. The liquids after isocratic elution were collected to obtain purified recombinant human serum albumin.

[0182] Then, regeneration solution C (0.5M NaOH solution) was added for regeneration, and finally buffer solution C (20mM phosphate buffer solution) was added for equilibration.

[0183] Example 3

[0184] This embodiment provides a method for purifying recombinant human serum albumin from pig blood, and the specific steps are as follows:

[0185] (1) Preprocessing

[0186] Pichia pastoris fermentation broth containing canine serum albumin was mixed with sodium octanoate at a final molar concentration of 50 mM to adjust the pH to 7.1. The broth was then subjected to ultrafiltration using a 10 kDa ultrafiltration tube, followed by filtration through a 0.22 μm filter membrane to obtain the supernatant of the Pichia pastoris fermentation broth.

[0187] (2) Anion exchange chromatography

[0188] Add phosphate buffer solution A (20 mM phosphate buffer solution containing 0.25 M sodium chloride) with a pH of 7.1 to the NM 90AgaroseHAM composite anion exchange chromatography column, and use the supernatant of Pichia pastoris fermentation broth removed in step (1) as the loading solution for anion exchange chromatography. The conductivity range of the supernatant of Pichia pastoris fermentation broth containing canine serum albumin is 20-21 mS / cm.

[0189] Subsequently, eluent with pH 7.1 (20 mM phosphate buffer solution containing 0.25 M sodium chloride, consistent with the formulation of phosphate buffer solution A) and elution buffer A (20 mM phosphate buffer solution containing 0.52 M sodium chloride) were added sequentially for isocratic elution, and the eluted liquid was collected to obtain the first chromatographic product.

[0190] Then, regeneration solution A (0.5M NaOH solution) was added for regeneration, and finally buffer solution A (20mM phosphate buffer solution containing 0.25M sodium chloride) was added for equilibration.

[0191] (3) Hydrophobic Chromatography

[0192] Add phosphate buffer solution B (20 mM phosphate buffer solution containing 0.52 M sodium chloride) with a pH of 6.9 to the UniHR Butyl-80L hydrophobic chromatography column, and use the first chromatography product obtained in step (2) as the loading solution for the hydrophobic chromatography column. The loading solution has a conductivity of 50 mS / cm and a pH of 6.9. Then, add eluent with a pH of 6.9 (20 mM phosphate buffer solution containing 0.52 M sodium chloride, with the same formulation as phosphate buffer solution B) and elution solution B (20 mM phosphate buffer solution) in sequence for isocratic elution. Collect the liquid flowing out during the loading and elution process to obtain the second chromatography product.

[0193] Then, regeneration solution B (0.5M NaOH solution) was added for regeneration, and finally buffer solution B (20mM phosphate buffer solution containing 0.52M sodium chloride) was added for equilibration.

[0194] (4) Weak anion exchange chromatography

[0195] Add buffer solution C (20 mM phosphate buffer) with a pH of 7.8 to the UniGel 30DEAE weak anion exchange chromatography column. The second chromatographic product obtained in step (3) is then subjected to ultrafiltration using a 10 kDa ultrafiltration centrifuge tube, and this solution is used as the loading solution for the weak anion exchange chromatography column. The conductivity of the loading solution is 2.8 mS / cm. Subsequently, eluent (20 mM phosphate buffer, with the same formulation as buffer solution C) and elution buffer C (20 mM phosphate buffer) are added sequentially. The following solutions were used for isocratic elution: a salt buffer solution containing 0.17 M sodium chloride (pH 5.8), eluent D (20 mM phosphate buffer solution containing 0.18 M sodium chloride (pH 5.8), eluent E (20 mM phosphate buffer solution containing 0.21 M sodium chloride (pH 5.8)), and eluent F (20 mM phosphate buffer solution containing 1 M sodium chloride (pH 5.8)). The liquids after isocratic elution were collected to obtain purified recombinant human serum albumin.

[0196] Then, regeneration solution C (0.5M NaOH solution) was added for regeneration, and finally buffer solution C (20mM phosphate buffer solution) was added for equilibration.

[0197] Example 4

[0198] This embodiment provides a method for purifying recombinant human serum albumin from pig blood, and the specific steps are as follows:

[0199] (1) Preprocessing

[0200] The supernatant of Pichia pastoris fermentation broth containing canine serum albumin was obtained by adding sodium caprylate to a final molar concentration of 55 mM, adjusting the pH to 7.0, ultrafiltration using a 10 kDa ultrafiltration tube, and then filtering through a 0.22 μm filter membrane.

[0201] (2) Anion exchange chromatography

[0202] Add phosphate buffer solution A (20 mM phosphate buffer solution containing 0.24 M sodium chloride) with a pH of 7.0 to the NM90AgaroseHAM composite anion exchange chromatography column, and use the supernatant of Pichia pastoris fermentation broth removed in step (1) as the loading solution for anion exchange chromatography. The conductivity range of the supernatant of Pichia pastoris fermentation broth containing canine serum albumin is 20-21 mS / cm.

[0203] Subsequently, eluent with pH 6.8 (20 mM phosphate buffer solution containing 0.25 M sodium chloride) and elution buffer A with pH 6.9 (20 mM phosphate buffer solution containing 0.52 M sodium chloride) were added sequentially for isocratic elution. The eluted liquid was then collected to obtain the first chromatographic product.

[0204] Then, regeneration solution A (0.5M NaOH solution) was added for regeneration, and finally buffer solution A (20mM phosphate buffer solution containing 0.24M sodium chloride) was added for equilibration.

[0205] (3) Hydrophobic Chromatography

[0206] Add phosphate buffer solution B (20 mM phosphate buffer solution containing 0.52 M sodium chloride) with a pH of 7.0 to the UniHR Butyl-80L hydrophobic chromatography column, and use the first chromatography product obtained in step (2) as the loading solution for the hydrophobic chromatography column. The loading solution has a conductivity of 50 mS / cm and a pH of 6.9. Then, add eluent with a pH of 6.9 (20 mM phosphate buffer solution containing 0.52 M sodium chloride) and elution solution B (20 mM phosphate buffer solution) in sequence for isocratic elution. Collect the liquid flowing out during the loading and elution process to obtain the second chromatography product.

[0207] Then, regeneration solution B (0.5M NaOH solution) was added for regeneration, and finally buffer solution B (20mM phosphate buffer solution containing 0.52M sodium chloride) was added for equilibration.

[0208] (4) Weak anion exchange chromatography

[0209] Add buffer solution C (20 mM phosphate buffer) with a pH of 8.0 to the UniGel 30DEAE weak anion exchange chromatography column. The second chromatographic product obtained in step (3) is then subjected to ultrafiltration using a 10 kDa ultrafiltration centrifuge tube, and this solution is used as the loading solution for the weak anion exchange chromatography column. The conductivity of the loading solution is 2.8 mS / cm. Subsequently, eluent (20 mM phosphate buffer) and elution buffer C (20 mM phosphate buffer) are added sequentially. The solution was eluted isocratically with eluent D (20 mM phosphate buffer containing 0.17 M sodium chloride, pH 5.8), eluent E (20 mM phosphate buffer containing 0.21 M sodium chloride, pH 5.8), and eluent F (20 mM phosphate buffer containing 1 M sodium chloride, pH 5.8). The liquid after isocratization was collected to obtain purified recombinant human serum albumin.

[0210] Then, regeneration solution C (0.5M NaOH solution) was added for regeneration, and finally buffer solution C (20mM phosphate buffer solution) was added for equilibration.

[0211] Example 5

[0212] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The only difference from Example 1 is that sodium caprylate is not added in the pretreatment step. The specific steps are as follows:

[0213] (1) Preprocessing

[0214] The supernatant of Pichia pastoris fermentation broth containing canine serum albumin was obtained by adjusting the pH to 7.0, ultrafiltration using a 10 kDa ultrafiltration tube, and then filtering through a 0.22 μm filter membrane.

[0215] The other steps are the same as in Example 1.

[0216] Example 6

[0217] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The only difference from Example 1 is that the composite ion exchange chromatography medium is NW Rose HAM, the hydrophobic chromatography medium is UniHRPhenyl-80L, and the anion exchange chromatography medium is UniGel 80Q. All other process parameters and steps are the same as in Example 1.

[0218] Example 7

[0219] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The only difference from Example 1 is that ultrafiltration is not performed during the weak anion exchange chromatography in step (4). All other process parameters and steps are consistent with Example 1.

[0220] Example 8

[0221] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The only difference from Example 1 is that the conductivity of the loading solution is 3 mS / cm during the composite anion exchange chromatography, 100 mS / cm during the hydrophobic chromatography, and 10 mS / cm during the weak anion exchange chromatography; all other process parameters and steps are consistent with Example 1.

[0222] Example 9

[0223] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The only difference from Example 1 is that the conductivity of the loading solution is 10 mS / cm during the composite anion exchange chromatography, 70 mS / cm during the hydrophobic chromatography, and 15 mS / cm during the weak anion exchange chromatography; all other process parameters and steps are the same as in Example 1.

[0224] Example 10

[0225] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, with the only difference from Example 1 being:

[0226] The pH of buffer solution A, eluent, and elution solution A during the complex anion exchange chromatography process is 8;

[0227] During hydrophobic chromatography, the pH of buffer solution B and elution buffer B is 6.8;

[0228] During weak anion exchange chromatography, the pH of buffer solution C, eluent C, eluent D, eluent E, and eluent F is 7.

[0229] All other process parameters and steps are consistent with those in Example 1.

[0230] Example 11

[0231] This embodiment provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, with the only difference from Example 1 being:

[0232] The pH of buffer solution A, eluent, and elution solution A during the complex anion exchange chromatography process is 6;

[0233] During hydrophobic chromatography, the pH of buffer solution B and eluent B is 8;

[0234] During weak anion exchange chromatography, the pH of buffer solution C, eluent, elution solution C, elution solution D, elution solution E, and elution solution F is 5.0.

[0235] All other process parameters and steps are consistent with those in Example 1.

[0236] Comparative Example 1

[0237] This comparative example provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. Except for omitting hydrophobic chromatography, the other process parameters and steps are consistent with those in Example 1.

[0238] Comparative Example 2

[0239] This comparative example provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. Except for omitting the complex anion exchange chromatography, the other process parameters and steps are consistent with those in Example 1.

[0240] Comparative Example 3

[0241] This comparative example provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The chromatography sequence in this method differs from that in Example 1. Specifically, this comparative example sequentially performs complex anion exchange chromatography, weak anion exchange chromatography, and hydrophobic chromatography, while the process parameters in each chromatography step remain consistent with those in Example 1.

[0242] Comparative Example 4

[0243] This comparative example provides a method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth. The chromatography sequence in this method differs from that in Example 1. Specifically, this comparative example uses weak anion exchange chromatography, hydrophobic chromatography, and complex ion exchange chromatography in sequence, while the remaining process parameters and steps are consistent with those in Example 1.

[0244] The yield, purity, and appearance of the recombinant canine serum albumin obtained in Examples 1-11 and Comparative Examples 1-4 are shown in Table 1. The appearance of the recombinant canine serum albumin was compared using standard colorimetric solutions. This application prepared standard colorimetric solutions according to the "Solution Color Inspection Method" in Appendix 2005 of the Chinese Pharmacopoeia, with 50 vials per set (including 8 blank ampoules), comprising the following five hues: YG-1-10 representing yellow-green standard colorimetric solutions 1-10; Y-1-10 representing yellow standard colorimetric solutions 1-10; OY-1-10 representing orange-yellow standard colorimetric solutions 1-10; OR-1-10 representing orange-red standard colorimetric solutions 1-10; and BR-1-10 representing reddish-brown standard colorimetric solutions 1-10. Yield is the same as the production rate. Yield and purity were determined using HLPC according to the methods in the pharmacopoeia, and purity and yield were calculated based on peak area.

[0245] Table 1

[0246] serial number yield purity Appearance Example 1 51.3％ 99.5％ Pale yellow solution (color number less than Y3) Example 2 50.1％ 99.3％ Pale yellow solution (color number less than Y3) Example 3 50.4％ 99.1％ Pale yellow solution (color number less than Y3) Example 4 50.2％ 97.2％ Pale yellow solution (color number less than Y3) Example 5 50.2％ 94.7％ Pale yellow solution (color number less than Y3) Example 6 60.9％ 86.1％ Pale yellow solution (color number less than Y3) Example 7 49.1％ 97.8％ Pale yellow solution (color number less than Y3) Example 8 65.7％ 91.1％ Pale yellow solution (color number less than Y3) Example 9 70.5％ 89.7％ Pale yellow solution (color number less than Y3) Example 10 51.1％ 88.9％ Pale yellow solution (color number less than Y3) Example 11 52.5％ 91.3％ Pale yellow solution (color number less than Y3) Comparative Example 1 21.1％ 54.1％ Pale yellow solution (color number less than Y3) Comparative Example 2 17.4％ 68.2％ Pale yellow solution (color number less than Y3) Comparative Example 3 30.8％ 62.6％ Pale yellow solution (color number less than Y3) Comparative Example 4 31.7％ 51.3％ Pale yellow solution (color number less than Y3)

[0247] As can be seen from the table above, compared with Comparative Examples 1 and 2, the purity of recombinant canine serum albumin obtained by Comparative Examples 1 and 2 is lower. This indicates that the separation by anion-complex anion exchange chromatography, hydrophobic chromatography and weak anion exchange chromatography are complementary. Omitting any one of the chromatography processes will affect the efficiency of the entire separation and purification process.

[0248] Compared with Comparative Examples 3-4, the recombinant canine serum albumin obtained by sequentially separating and purifying using composite anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography in Example 1 has higher purity, indicating that the three chromatography processes work synergistically and the order of the three chromatography processes cannot be arbitrarily changed.

[0249] Compared with Example 4, the recombinant canine serum albumin obtained in Example 1 has higher purity, indicating that when the pH values ​​of the equilibration buffer and the elution buffer are the same during the chromatography process, the chromatography process can achieve better separation results.

[0250] Compared with Example 5, the purity of the recombinant canine serum albumin obtained in Example 5 was significantly lower, indicating that the pretreatment operation before the chromatography process plays an important role in the separation and purification process, which can enhance the stability of the target protein and facilitate the subsequent chromatography process to obtain high-purity recombinant canine serum albumin.

[0251] Compared with Example 6, Example 1 showed higher yield and purity of recombinant canine serum albumin, indicating that the three chromatography media NM 90Agarose HAM, UniHR Butyl-80L and UniGel-30DEAE are the optimal combination for separating and purifying pig blood to obtain recombinant canine serum albumin.

[0252] Compared with Example 7, the purity of the recombinant canine serum albumin obtained in Example 7 was lower, indicating that the ultrafiltration buffer exchange operation in the weak anion exchange chromatography process is indispensable. Ultrafiltration buffer exchange can improve the selectivity of weak anion exchange chromatography, remove some impurities, and improve protein purity.

[0253] Compared with Examples 8-9, the recombinant canine serum albumin obtained in Example 1 has higher purity, indicating that the conductivity of the loading solution needs to be within a certain range during the chromatography process to ensure the orderly progress of the chromatography process and achieve better separation and purification effects.

[0254] Compared with Examples 10-11, the recombinant canine serum albumin obtained in Example 1 has higher purity, indicating that the pH of the buffer solution, elution solution and elution solution needs to be maintained within a certain range during the chromatography process to ensure the stability of the protein, thereby ensuring the efficiency of the separation and purification process and obtaining high-purity recombinant canine serum albumin.

[0255] In summary, by sequentially performing composite anion exchange chromatography, hydrophobic chromatography, and weak anion exchange chromatography, and through the synergistic effect of the sample loading solution within a specific conductivity range and the pH of the buffer solution, eluent, and elution solution within a specific pH range during the chromatography process, the recombinant canine serum albumin obtained after separation and purification has high purity, which is of great significance for clinical drug use and biochemical research.

[0256] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples 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.

[0257] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth, characterized in that, The method includes the following steps: The supernatant of Pichia pastoris fermentation broth was subjected to complex anion exchange chromatography to prepare the first chromatographic product; The first chromatographic product was subjected to hydrophobic chromatography to prepare a second chromatographic product; and, The second chromatographic product was subjected to weak anion exchange chromatography to purify recombinant canine serum albumin.

2. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to claim 1, characterized in that, The supernatant contains a protein stabilizer; Optionally, the protein stabilizer includes one or more of caprylic acid and sodium caprylate; Optionally, the supernatant is supplemented with 45 mM to 55 mM of the protein stabilizer.

3. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to claim 1, characterized in that, The method satisfies one or more of the following conditions: (1) The media for composite anion exchange chromatography include NM 90Agarose HAM; (2) The media used for hydrophobic chromatography include UniHR Butyl-80L; as well as, (3) The media for weak anion exchange chromatography include: UniGel 30DEAE.

4. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to claim 1, characterized in that, The second chromatographic product was subjected to ultrafiltration followed by weak anion exchange chromatography. Optionally, the ultrafiltration treatment conditions include: using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuging at 5800 rpm to 6200 rpm for 8 min to 12 min.

5. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to claim 1, characterized in that, The method satisfies one or more of the following conditions: 1) The conductivity of the loading solution for composite anion exchange chromatography is 19 mS / cm to 23 mS / cm; 2) The conductivity of the sample loading solution for hydrophobic chromatography is 45 mS / cm to 49 mS / cm; and, 3) The conductivity of the loading solution for weak anion exchange chromatography is 2 mS / cm to 4 mS / cm.

6. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to claim 1, characterized in that, The method satisfies one or more of the following conditions: (A) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in the composite anion exchange chromatography process are each independently 6.8 to 7.2; optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in the composite anion exchange chromatography process are kept consistent. (B) The pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are each independently 6.9–7.1; optionally, the pH values ​​of the equilibration buffer, eluent, and elution buffer used in hydrophobic chromatography are kept consistent; and, (C) In the process of weak anion exchange chromatography, the pH values ​​of the equilibration buffer and the eluent are each independently set to 7.8–8.1, and the pH value of the elution buffer is 5.8–6.

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7. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to any one of claims 1 to 6, characterized in that, Composite anion exchange chromatography satisfies one or more of the following conditions: A) The equilibrium solution consists of a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.2 M–0.25 M sodium chloride; B) The rinsing solution includes a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.2 M–0.25 M sodium chloride; C) The eluent consists of a phosphate buffer solution with a pH of 6.8–7.2, 17 mM–23 mM, and 0.45 M–0.55 M sodium chloride; as well as, D) Isocratic elution was employed; Optionally, the equilibration buffer and eluent in the composite anion exchange chromatography have the same composition.

8. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to any one of claims 1 to 6, characterized in that, Hydrophobic chromatography satisfies one or more of the following conditions: (I) The equilibrium solution includes a phosphate buffer solution with a pH of 6.9–7.1 and a pH of 17 mM–23 mM and a sodium chloride solution of 0.45 M–0.55 M; (II) The eluent consists of a phosphate buffer solution with a pH of 6.9–7.1 and a strength of 17 mM–23 mM; (III) The rinsing solution comprises a phosphate buffer solution with a pH of 6.9–7.1, 17 mM–23 mM, and 0.45 M–0.55 M sodium chloride; and, (IV) Isocratic elution was employed; Optionally, the equilibration buffer and eluent in hydrophobic chromatography have the same composition.

9. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to any one of claims 1 to 6, characterized in that, Weak anion exchange chromatography satisfies one or more of the following conditions: Ⅰ) The equilibrium solution includes a phosphate buffer solution with a pH of 7.8–8.1 and a strength of 17 mM–23 mM; II) The eluent includes a variety of components; optionally, the eluent includes: Eluent 1 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM and a concentration of 0.16 M–0.17 M sodium chloride; Eluent 2 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM and a concentration of 0.17 M–0.19 M sodium chloride; Eluent 3 comprises a phosphate buffer solution with a pH of 5.8–6.1, 17 mM–23 mM, and 0.2 M–0.23 M sodium chloride; and, Eluent 4 comprises a phosphate buffer solution with a pH of 5.8–6.1 and a concentration of 17 mM–23 mM, and a sodium chloride solution of 0.8 M–1.2 M. III) The rinsing solution includes a phosphate buffer solution with a pH of 7.8–8.1 and a strength of 17 mM–23 mM; and, IV) Use isocratic elution; Optionally, the equilibration buffer and eluent in weak anion exchange chromatography have the same composition.

10. The method for purifying recombinant canine serum albumin from Pichia pastoris fermentation broth according to any one of claims 1 to 6, characterized in that, The method satisfies one or more of the following conditions: (i) After the composite anion exchange chromatography is completed, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and optionally, a phosphate buffer solution with a pH of 6.8 to 7.2 and a concentration of 17mM to 23mM, including 0.2M to 0.25M sodium chloride, is used for equilibration. (ii) After hydrophobic chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and optionally, a phosphate buffer solution with a pH of 6.9 to 7.1 and a pH of 17mM to 23mM containing 0.45M to 0.55M sodium chloride is used for equilibration. (iii) After weak anion exchange chromatography, the chromatography column is regenerated and equilibrated. Optionally, a sodium hydroxide solution with a sodium hydroxide concentration of 0.4M to 0.6M is used for regeneration, and a phosphate buffer solution with a pH of 7.8 to 8.1 and a pH of 17mM to 23mM is used for equilibration.

11. A method for preparing recombinant canine serum albumin, characterized in that, The preparation method includes the step of purifying recombinant canine serum albumin using the method described in any one of claims 1 to 10.

12. A method for preparing a recombinant canine serum albumin product, characterized in that, The preparation method includes: Purify recombinant canine serum albumin using the method described in any one of claims 1 to 10; and, Products were prepared using purified recombinant canine serum albumin.

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