Preparation method of recombinant canine albumin and production method of recombinant canine albumin preparation

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of recombinant canine albumin and a production method of a recombinant canine albumin preparation. The preparation method comprises the following steps: purifying fermentation liquid supernatant of genetically engineered bacteria for expressing recombinant canine albumin through cation exchange chromatography, ultrafiltration and hydrophobic chromatography in sequence; wherein the conditions of the cation exchange chromatography are as follows: a chromatographic column filler comprises UniGel 65SP HC; the hydrophobic chromatography conditions are as follows: a chromatographic column filler comprises 80L of UniHR Butyl. The purity of the preparation method reaches 99%, the steps are simple, efficient and rapid, and industrialization is facilitated. And the high-purity recombinant canine albumin can be used for clinical treatment, so that the high-purity recombinant canine albumin can be better applied to the pet medical industry and has important practical significance.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and particularly relates to a method for preparing recombinant canine albumin and a method for producing a recombinant canine albumin preparation. Background Art

[0002] With the economic development, people's living standards are getting richer and richer, and more and more people keep pets to accompany themselves and their families. Dogs are the most numerous among pet species. With the increasing number of dogs, the occurrence of dog diseases is inevitable. In the treatment of dog diseases, the use of canine blood albumin is common and effective. At present, canine blood albumin is mainly prepared by fermentation of engineering bacteria. Therefore, it is crucial to extract high-purity recombinant canine blood albumin from the fermentation products.

[0003] Currently, reports on recombinant albumin mainly focus on recombinant human albumin. For example:

[0004] CN100463921C describes a method for purifying recombinant human albumin, which includes subjecting the supernatant of the fermentation broth containing recombinant human albumin to the following chromatography steps: (a) cation exchange chromatography based on dual functions and tolerant to high salt; (b) hydrophobic chromatography; and (c) anion exchange chromatography. The conductivity of the supernatant of the fermentation broth applied to step (a) is higher than 10 mS / cm, further higher than 20 mS / cm, and more specifically 25 - 50 mS / cm.

[0005] CN101768601B describes a method for producing recombinant human serum albumin-interferon α2b. By constructing a Pichia pastoris engineering strain of the human serum albumin-interferon α2b fusion protein gene, fermenting and expressing it, and then purifying it. It is characterized in that the construction of the engineering strain is to fuse the C-terminus of the human serum albumin cDNA with the N-terminus of the IFN cDNA, connect it with the yeast expression vector pPICZα, and introduce it into Pichia pastoris X33 for expression to become the engineering strain of recombinant human serum albumin-interferon α2b; the purification after fermentation and expression is to collect the fermentation supernatant by centrifugation, and then obtain recombinant human serum albumin-interferon α2b through cation exchange chromatography, hydrophobic chromatography and molecular sieve chromatography in sequence; for the cation exchange chromatography, it needs to be adjusted with glacial acetic acid to make the pH 4.6, and then dilute the fermentation broth with distilled water to make the conductivity ≤20 mS / cm, and directly load the fermentation supernatant onto the capto MMC column; in the hydrophobic chromatography step, add Na2SO4 to the cation target protein collection solution to make the final concentration of Na2SO4 reach 0.7 M, directly load it onto the Phenyl Sepharose High Performance column, and collect the target protein peak; in the molecular sieve chromatography step, pass the target protein peak collected by hydrophobic chromatography through a Superdex 200 prep grand molecular sieve chromatography column, elute it with 20 mM phosphate buffer solution, and collect the target protein peak to obtain recombinant human serum albumin-interferon α2b.

[0006] CN110092827A describes a purification method for obtaining high-purity recombinant human serum albumin, including the following steps: (1) centrifugally treating the fermentation broth containing recombinant human serum albumin, and then heating it with a heat stabilizer to obtain a recombinant human serum albumin supernatant; (2) sequentially performing primary ultrafiltration and secondary ultrafiltration on the recombinant human serum albumin supernatant to obtain a recombinant human serum albumin ultrafiltrate; (3) adding the recombinant human serum albumin ultrafiltrate to a pre-equilibrated Ni-NTA chromatography column, sequentially rinsing the Ni-NTA chromatography column with a protein impurity removal solution, a impurity removal solution, and a equilibration solution, then eluting it with a protein elution solution, and dialyzing the eluted protein with a dialysis solution to obtain a recombinant human serum albumin dialysis solution; (4) sequentially performing cation exchange chromatography, anion exchange chromatography, and hydrophobic chromatography on the recombinant human serum albumin dialysis solution to obtain the high-purity recombinant human serum albumin.

[0007] CN112210002B describes a method for purifying recombinant human serum albumin, which includes: sequentially subjecting the fermentation broth containing recombinant human serum albumin to impurity removal, first concentration and buffer exchange, first cation exchange chromatography, hydrophobic chromatography, second concentration and buffer exchange, anion exchange chromatography, affinity chromatography, third concentration and buffer exchange, and second cation exchange chromatography; wherein, the packing material used for the first cation exchange chromatography is Cellufine MAX s-h packing material; the packing material used for the hydrophobic chromatography is Cellufine MAX Phenyl packing material; the packing material used for the anion exchange chromatography is Cellufine MAX DEAE packing material; the packing material used for the affinity chromatography is Cellufine PB packing material; and the packing material used for the second cation exchange chromatography is Cellufine MAX GS packing material.

[0008] However, these current purification methods have cumbersome steps and cannot achieve the efficient preparation of recombinant canine albumin, which is not conducive to marketization. Therefore, there is an urgent need for a process method for purifying albumin from canine serum quickly, simply, and with high purity. Summary of the Invention

[0009] Based on this, one or more embodiments of the present application provide a method for preparing recombinant canine albumin.

[0010] One or more embodiments of the present application provide a method for preparing recombinant canine albumin, which includes the steps of sequentially purifying the supernatant of the fermentation broth of the genetically engineered bacteria expressing recombinant canine albumin through cation exchange chromatography, ultrafiltration, and hydrophobic chromatography; wherein,

[0011] The conditions for the cation exchange chromatography include: the packing material of the chromatography column includes UniGel 65SP HC;

[0012] The conditions for the hydrophobic chromatography include: the packing material of the chromatography column includes UniHR Butyl 80L.

[0013] In some embodiments of the present application, the conditions for the cation exchange chromatography further include one or more of the conditions shown in (1) to (3):

[0014] (1) The equilibration buffer includes 20 - 25 mM acetate buffer with a pH value of 4 - 5;

[0015] (2) The elution buffer includes 25 - 35 mM sodium chloride and 18 - 22 mM acetic acid - sodium acetate buffer with a pH value of 4 - 5; and,

[0016] (3) The elution buffer includes 0.4 - 0.6 mol sodium chloride and 18 - 22 mM phosphate buffer with a pH value of 4 - 5.

[0017] In some embodiments of the present application, the conditions for hydrophobic chromatography further include one or more of the conditions shown in (A) to (C):

[0018] (A)The equilibration buffer comprises 0.18 - 0.22 mol sodium chloride and 18 - 22 mM phosphate buffer, with a pH value of 6.5 - 7.5;

[0019] (B)The elution buffer comprises 0.18 - 0.22 mol sodium chloride and 18 - 22 mM phosphate buffer, with a pH value of 6.5 - 7.5;

[0020] (C)The elution buffer comprises 18 - 22 mM phosphate buffer, with a pH value of 6.5 - 7.5; and,

[0021] (D)The regeneration buffer comprises 0.45 - 0.55 M NaOH and water.

[0022] In some embodiments of the present application, the conductivity of the supernatant of the fermentation broth is 5 - 7 mS / cm.

[0023] In some embodiments of the present application, the conductivity of the sample loading solution for hydrophobic chromatography is 20 - 25 mS / cm.

[0024] In some embodiments of the present application, the elution method used in cation exchange chromatography includes linear elution; the program of linear elution includes: within 14 - 16 column volumes, the volume ratio of the elution buffer rises from 0% to 100%.

[0025] In some embodiments of the present application, ultrafiltration uses a membrane package with a molecular weight cut-off above 30 KD.

[0026] In some embodiments of the present application, the concentration of the recombinant canine albumin in the supernatant of the fermentation broth is 2 - 5 g / L.

[0027] In some embodiments of the present application, the supernatant of the fermentation broth of the genetically engineered bacteria expressing recombinant canine albumin is from Pichia pastoris.

[0028] One or more embodiments of the present application also provide a production method of a recombinant canine albumin preparation, which includes the following steps:

[0029] Prepare recombinant canine albumin using the described preparation method;

[0030] Prepare a recombinant canine albumin preparation using the canine recombinant albumin.

[0031] Compared with the traditional technology, the present application has the following beneficial effects:

[0032] The preparation method of recombinant canine albumin provided by this application purifies the supernatant of the fermentation broth of the genetically engineered bacteria expressing recombinant canine albumin through cation exchange chromatography on a chromatography column with appropriate packing materials, ultrafiltration, and hydrophobic chromatography on a chromatography column with appropriate packing materials in sequence, and can achieve a purity of 99%. The steps are simple, efficient, and fast, which is conducive to industrialization. And this highly pure recombinant canine albumin can be used for clinical treatment, and thus can be better applied to the pet medical industry, which has important practical significance. Description of the Drawings

[0033] 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 required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0034] Figure 1 It is the cation exchange chromatography diagram in Example 1;

[0035] Figure 2 It is the cation exchange chromatography electrophoresis diagram in Example 1 (M - Marker, 1 - loading solution, 2 - breakthrough solution 1, 3 - breakthrough solution 2, 4 - breakthrough solution 3, 5 - elution solution 1, 6 - elution solution 2, 7 - elution solution 3, 8 - elution solution 4, 9 - elution solution 5);

[0036] Figure 3 It is the hydrophobic chromatography diagram in Example 1;

[0037] Figure 4 It is the hydrophobic electrophoresis diagram in Example 1 (M - Marker, 1 - loading solution, 2 - breakthrough solution);

[0038] Figure 5 It is the HPLC spectrum diagram of the product obtained by anion exchange chromatography in Example 1;

[0039] Figure 6 It is the HPLC spectrum diagram of the product obtained by hydrophobic chromatography in Example 1. Detailed Embodiments

[0040] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. 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 changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing the embodiments and examples and are not intended to limit this application.

[0042] Term

[0043] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0044] The term "and / or", "or / and", "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, D, that is, includes the combination of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (that is, the technical solution connected by "logical and").

[0045] In this application, the terms "a plurality of", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, mean greater than 2 or equal to 2 in quantity. For example, "one or more types" means one type or greater than or equal to two types.

[0046] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0047] In this application, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] In this application, "preferred", "better", "more preferable", "as appropriate" are only used to describe the implementation modes or embodiments with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0049] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of this application.

[0050] In this application, "optionally", "optional", "optional" mean that it can be either present or absent, that is, it refers to any one of the two alternative options of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual constraints, each "optional" is independent.

[0051] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing description, and it should be understood that they do not constitute a closed limitation on quantity.

[0052] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open-ended technical solution containing the listed features.

[0053] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0054] For the temperature parameters in this application, unless otherwise specified, both constant temperature treatment and variation within a certain temperature range are allowed. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0055] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0056] All the documents mentioned in this application are cited as references in this application, just as if each document was cited separately as a reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents related to this application are cited in their entirety and for all purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0057] The canine albumin production process provided by the present invention is simple, low-cost, has a high protein yield, and a purity of over 99%, and is suitable for large-scale industrial production. The recombinant canine albumin obtained by the present invention has a relatively high purity and can be used for clinical treatment and biological research.

[0058] The UniGel 65SP HC chromatography medium used in the present invention uses a polymethacrylate-based sphere, which has excellent mechanical strength, good chemical stability, and uniform particle size. It has the chromatography advantages of high flow rate, high resolution, and low backpressure. The manufacturer has designed its salt tolerance, and it can still maintain a loading capacity of 120 mg / ml at a conductivity of 10 ms / cm, and it is widely used in the albumin project; the UniHR Butyl 80L chromatography medium is also a polymer-based filler with excellent pressure resistance, and it is more suitable for large-scale albumin production. It adsorbs biomolecules under high-salt conditions and elutes under low-salt conditions, with excellent adsorption capacity and low non-specific adsorption, and is suitable for the further separation and purification of products after the ion exchange process.

[0059] In the present invention, UniGel 65SP HC and UniHR Butyl 80L are used in combination. After the protein is captured by UniGel 65SP HC, a recombinant canine albumin with higher purity and higher yield can be obtained only through one-step hydrophobic chromatography.

[0060] The first aspect of the present application

[0061] The present application provides a method for preparing recombinant canine albumin, which includes the steps of sequentially purifying the supernatant of the fermentation broth of the genetically engineered bacteria expressing recombinant canine albumin through cation exchange chromatography, ultrafiltration, and hydrophobic chromatography; wherein,

[0062] The conditions for cation exchange chromatography include: the filler of the chromatography column includes UniGel 65SP HC;

[0063] The conditions for hydrophobic chromatography include: the filler of the chromatography column includes UniHR Butyl 80L.

[0064] In some embodiments of the present application, the conditions for cation exchange chromatography further include one or more of the conditions shown in (1) to (3):

[0065] (1) The equilibration buffer includes 20-25 mM (such as 20, 21, 22, 23, 24, 25 mM) acetic acid buffer, and the pH value is 4-5 (such as 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5);

[0066] (2) The eluent includes 25 - 35 mM (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 mM) sodium chloride and 18 - 22 mM (such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 mM) acetic acid - sodium acetate buffer solution, with a pH value of 4 - 5 (such as 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5); and,

[0067] (3) The eluate includes 0.4 - 0.6 mol (such as 0.4, 0.42, 4.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6 mol) sodium chloride and 18 - 22 mM (such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 mM) phosphate buffer solution, with a pH value of 4 - 5 (such as 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5).

[0068] In some embodiments of the present application, the conditions for hydrophobic chromatography further include one or more of the conditions shown in (A) to (C):

[0069] (A) The equilibration buffer includes 0.18 - 0.22 mol (such as 0.18, 0.19, 0.20, 0.21, 0.22 mol) sodium chloride and 18 - 22 mM (such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 mM) phosphate buffer solution, with a pH value of 6.5 - 7.5 (such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5);

[0070] (B) The eluent includes 0.18 - 0.22 mol (such as 0.18, 0.19, 0.20, 0.21, 0.22 mol) sodium chloride and 18 - 22 mM (such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 mM) phosphate buffer solution, with a pH value of 6.5 - 7.5 (such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5);

[0071] (C) The eluent comprises a phosphate buffer solution of 18 - 22 mM (such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 mM), with a pH value of 6.5 - 7.5 (such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5); and,

[0072] (D) The regeneration solution comprises 0.45 - 0.55 M (such as 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 M) NaOH and water.

[0073] In some embodiments of the present application, the conductivity of the supernatant of the fermentation broth is 5 - 7 mS / cm (such as 5, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7 mS / cm).

[0074] In some embodiments of the present application, the conductivity of the sample loading solution for hydrophobic chromatography is 20 - 25 mS / cm (such as 20, 21, 22, 23, 24, 25 mS / cm).

[0075] In some embodiments of the present application, the elution method used in cation exchange chromatography includes linear elution; the program of linear elution includes: within 14 - 16 (such as 14, 15, 16) column volumes, the volume ratio of the eluent rises from 0% to 100%.

[0076] In some embodiments of the present application, the ultrafiltration uses a membrane package with a cut-off molecular weight above 30 KD.

[0077] In some embodiments of the present application, the concentration of the recombinant canine albumin in the supernatant of the fermentation broth is 2 - 5 g / L (such as 2, 2.5, 3, 3.5, 4, 4.5, 5 g / L).

[0078] In some embodiments of the present application, the supernatant of the fermentation broth of the genetically engineered bacterium expressing recombinant canine albumin is from Pichia pastoris.

[0079] In the second aspect of the present application, there is provided a production method of a recombinant canine albumin preparation, which comprises the following steps:

[0080] Prepare recombinant canine albumin by using the described preparation method;

[0081] Prepare a recombinant canine albumin preparation with the canine recombinant albumin.

[0082] The implementation scheme of the present application will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application is preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0083] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special indication, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0084] Example 1

[0085] This example provides a method for purifying recombinant canine albumin expressed by yeast, and the specific steps are as follows:

[0086] (1) Add 20 mM acetate buffer solution with a pH value of 4.5 to the UniGel 65SP HC cation exchange chromatography column, and use the supernatant of the fermentation broth with a concentration of 3 g / L as the loading solution for cation exchange chromatography, and the conductivity of the sample is 6 mS / cm;

[0087] Subsequently, add the eluent with a pH value of 4.5 (20 mM HAC NaAC + NaCl, 30 mM sodium chloride solution) and the elution solution (20 mM PB, 0.5 mol sodium chloride solution, pH value of 4.5) in sequence. Among them, the elution is linear elution, and the proportion of the elution solution rises from 0% to 100% within 15 column volumes;

[0088] In step (1), collect the loading solution, breakthrough liquid 1 (i.e., the liquid flowing out of 1 / 3 of the chromatography column during loading), breakthrough liquid 2 (i.e., the liquid flowing out of 2 / 3 of the chromatography column during loading), breakthrough liquid 3 (i.e., the liquid flowing out of the chromatography column after the first elution), eluate 1 (i.e., the first peak of the elution peak), eluate 2 (i.e., the second peak of the elution peak), eluate 3 (the first half peak of the third elution peak), eluate 4 (i.e., the second half peak of the third elution peak), and eluate 5 (i.e., the trailing part after the third elution peak) for gel electrophoresis.

[0089] (2) Add a phosphate buffer solution with a pH of 7.0 (20 mM PB, 0.2 mol sodium chloride solution) to the UniHR Butyl80L hydrophobic chromatography column. For the first chromatographic product (a mixture of eluate 2 and eluate 3) obtained in step (1), use a 20 mM phosphate buffer solution for ultrafiltration and buffer exchange with a 30 KD membrane package as the sample solution for the hydrophobic chromatography column. The conductivity of the ultrafiltered solution is 23 mS / cm. Subsequently, add a washing solution with a pH of 7.0 (20 mM PB, 0.2 mol sodium chloride solution), an eluent with a pH of 7.0 (20 mM PB), and a regeneration solution (i.e., the sodium hydroxide elution peak, 0.5 M sodium hydroxide solution) for gel electrophoresis.

[0090] In step (2), collect the sample solution and the breakthrough liquid 1 (i.e., the liquid flowing out of the chromatography column during sample loading) respectively for gel electrophoresis.

[0091] The chromatograms and electrophoresis results of the products obtained by cation exchange chromatography and hydrophobic chromatography are shown respectively as Figures 1-4 follows. Among them, Figure 1 and Figure 3 are the chromatographic results of the products, indicating the operability and subsequent scalability of the two-step chromatography; Figure 2 , Figure 4 are the electrophoresis results of the products. It can be seen that the purity of the recombinant canine albumin obtained through the two-step chromatography is relatively high. The HPLC chromatograms of the two-step chromatography are shown as Figure 5 , Figure 6 follows. The purities of the products obtained by the two-step chromatography are 94% and 99% respectively, and the total recovery rate of the two-step chromatography is 85%. It can be seen that through cation exchange chromatography and hydrophobic chromatography separation and purification in sequence, high-purity recombinant canine albumin can be obtained, which can meet the industry use standards.

[0092] Example 2

[0093] This example is a variant of Example 1. The differences compared with Example 1 include:

[0094] In step (1), the conditions for cation exchange chromatography include:

[0095] The equilibration solution includes 25 mM acetate buffer with a pH of 5;

[0096] The washing solution includes 35 mM sodium chloride and 18 mM acetate-acetate buffer with a pH of 5;

[0097] The eluent includes 0.6 mol sodium chloride and 20 mM phosphate buffer with a pH of 5;

[0098] The elution methods used in cation exchange chromatography include linear elution; the program of linear elution includes: within 14 column volumes, the volume ratio of the eluent rises from 0% to 100%;

[0099] The conductivity of the supernatant of the fermentation broth is 7 mS / cm.

[0100] In step (2), the conditions for hydrophobic chromatography include:

[0101] The equilibration buffer includes 0.22 mol of sodium chloride and 18 mM of phosphate buffer, with a pH value of 7.5;

[0102] The washing buffer includes 0.18 mol of sodium chloride and 22 mM of phosphate buffer, with a pH value of 6.5;

[0103] The eluent includes 22 mM of phosphate buffer, with a pH value of 7.5; and,

[0104] The conductivity of the sample loading solution for hydrophobic chromatography is 25 mS / cm;

[0105] Ultrafiltration and buffer exchange with a 35KD membrane package using a phosphate buffer solution is used as the sample loading solution for the hydrophobic chromatography column.

[0106] After analysis by high performance liquid chromatography and electrophoresis, the purity of the pure canine albumin product is 98.5%. The total recovery rate of the two-step chromatography is 83%.

[0107] Example 3

[0108] This example is a variant of Example 1. The differences compared to Example 1 include: in step (1), the conductivity of the sample loading solution is 7.5 mS / cm. After analysis by high performance liquid chromatography and electrophoresis, the purity of the pure canine albumin product is 95%, and the recovery rate is 80%.

[0109] Example 4

[0110] This example is a variant of Example 1. The differences compared to Example 1 include: in step (2), the conductivity of the sample loading solution is 30 mS / cm. After analysis by high performance liquid chromatography and electrophoresis, the purity of the pure canine albumin product is 99%, and the recovery rate is 82%.

[0111] Example 5

[0112] This example is a variant of Example 1. The differences compared to Example 1 include: in step (1), the pH value of the equilibration buffer is 5.5. After analysis by high performance liquid chromatography and electrophoresis, the purity of the pure canine albumin product is 99%, and the recovery rate is 75%.

[0113] Example 6

[0114] This example is a variant of Example 1. The differences from Example 1 include: in step (1), the pH value of the eluent is 5.5. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 99%, and the recovery rate is 80%.

[0115] Example 7

[0116] This example is a variant of Example 1. The differences from Example 1 include: in step (1), the pH value of the elution liquid is 5.5. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 96%, and the recovery rate is 88%.

[0117] Example 8

[0118] This example is a variant of Example 1. The differences from Example 1 include: in step (2), the pH value of the equilibration liquid is 8. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 98%, and the recovery rate is 93%.

[0119] Example 9

[0120] This example is a variant of Example 1. The differences from Example 1 include: in step (2), the pH value of the eluent is 6. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 87%, and the recovery rate is 89%.

[0121] Example 10

[0122] This example is a variant of Example 1. The differences from Example 1 include: in step (2), the pH value of the elution liquid is 7. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 85%, and the recovery rate is 92%.

[0123] Comparative Example 1

[0124] This comparative example is a comparative example of Example 1. The differences from Example 1 include: using NanoGel50SP instead of UniGel 65SP HC cation exchange chromatography column. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 90%, and the recovery rate is 82%.

[0125] Comparative Example 2

[0126] This comparative example is a comparative example of Example 1. The differences from Example 1 include: using UniHRPheny80L instead of UniHR Butyl80L hydrophobic chromatography column. After high-performance liquid chromatography and electrophoresis analysis, the purity of the pure canine albumin is 95%, and the recovery rate is 68%.

[0127] In summary, through cation exchange chromatography and hydrophobic chromatography in sequence, and by the synergistic compounding effect of the loading solution within a specific conductivity range during the chromatography process and the pH values of the buffer solution, eluent, and elution solution within specific ranges, the recombinant canine albumin obtained by separation and purification has a high purity, which is of great significance for clinical medication and biochemical research.

[0128] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity in description, 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, it should be considered to be within the scope described in this specification.

[0129] The above-described embodiments only represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for preparing recombinant canine albumin, which comprises the steps of successively purifying the supernatant of the fermentation broth of a genetically engineered bacterium expressing recombinant canine albumin by cation exchange chromatography, ultrafiltration and hydrophobic chromatography; wherein, The conditions for cation exchange chromatography include: the packing material of the chromatography column includes UniGel 65SP HC; The conditions for hydrophobic chromatography include: the packing material of the chromatography column includes UniHR Butyl 80L.

2. The preparation method of the recombinant canine albumin according to claim 1, wherein, The conditions for cation exchange chromatography further include one or more of the conditions shown in (1) to (3): (1) The equilibration buffer includes 20 - 25 mM acetate buffer with a pH value of 4 - 5; (2) The elution buffer includes 25 - 35 mM sodium chloride and 18 - 22 mM acetate - sodium acetate buffer with a pH value of 4 - 5; and, (3) The elution solution includes 0.4 - 0.6 mol sodium chloride and 18 - 22 mM phosphate buffer with a pH value of 4 - 5.

3. The preparation method of the recombinant canine albumin according to claim 1, wherein, The conditions for hydrophobic chromatography further include one or more of the conditions shown in (A) to (C): (A) The equilibration buffer includes 0.18 - 0.22 mol sodium chloride and 18 - 22 mM phosphate buffer with a pH value of 6.5 - 7.5; (B) The elution buffer includes 0.18 - 0.22 mol sodium chloride and 18 - 22 mM phosphate buffer with a pH value of 6.5 - 7.5; (C) The elution solution includes 18 - 22 mM phosphate buffer with a pH value of 6.5 - 7.5; and, (D) The regeneration solution includes 0.45 - 0.55 M NaOH and water.

4. The preparation method of the recombinant canine albumin according to claim 1, wherein, The conductivity of the supernatant of the fermentation broth is 5 - 7 mS / cm.

5. The preparation method of the recombinant canine albumin according to claim 1, wherein, The conductivity of the sample loading solution for hydrophobic chromatography is 20 - 25 mS / cm.

6. The preparation method of the recombinant canine albumin according to any one of claims 1 to 5, wherein, The elution method used in cation exchange chromatography includes linear elution; the program of linear elution includes: within 14 - 16 column volumes, the volume ratio of the elution solution rises from 0% to 100%.

7. The method for preparing recombinant canine albumin according to any one of claims 1 to 5, wherein, Ultrafiltration uses a membrane package with a molecular weight cut-off of more than 30 KD.

8. The preparation method of the recombinant canine albumin according to any one of claims 1 to 5, wherein, The concentration of the recombinant canine albumin in the supernatant of the fermentation broth is 2 - 5 g / L.

9. The preparation method of the recombinant canine albumin according to any one of claims 1 to 5, wherein, The supernatant of the fermentation broth of the genetically engineered bacterium expressing recombinant canine albumin is from Pichia pastoris.

10. A method for producing a recombinant canine albumin preparation, which comprises the following steps: Preparing recombinant canine albumin by using the preparation method according to any one of claims 1 to 9; Preparing a recombinant canine albumin preparation with the canine recombinant albumin.

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