Preparation method of glycosylated brain natriuretic peptide precursor protein, brain natriuretic peptide quality control product, kit and application of brain natriuretic peptide quality control product

Abstract

The invention discloses a preparation method of glycosylated brain natriuretic peptide precursor protein, a brain natriuretic peptide quality control product, a kit and application of the brain natriuretic peptide quality control product. The preparation method comprises the following steps: inserting a target gene of the brain natriuretic peptide precursor protein into a plasmid to construct a recombinant expression vector; transforming the recombinant expression vector into pichia pastoris, performing fermentation culture, and collecting fermentation liquor; and purifying the fermentation liquor to obtain the glycosylated brain natriuretic peptide precursor protein. According to the preparation method, the expressed protein can be subjected to glycosylation modification by using a pichia pastoris expression system, and the expression quantity is high, so that the glycosylation ProBNP with a longer sequence and a longer half-life period is obtained, the problems that in a traditional BNP preparation process, the maturity of the protein expressed by escherichia coli is not high and the protein is different from a natural sample are solved, and the preparation method is suitable for industrial production. And the difficulty in process amplification of mammalian cell expression protein is also avoided.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing a glycosylated brain natriuretic peptide precursor protein, a brain natriuretic peptide quality control product and a kit, and applications thereof. Background Art

[0002] B-type natriuretic peptide, also known as brain natriuretic peptide (BNP), is a biologically active polypeptide synthesized by cardiomyocytes. When the heart dilates due to stress or ventricular wall overload, the gene encoding BNP is activated in cardiomyocytes. This activation results in the intracellular expression of 134 amino acid residues of preproBNP. This molecule removes a 26-amino acid signal peptide at its N-terminus, forming pro-BNP, consisting of 108 amino acid residues. Upon external stimulation of cardiomyocytes, alkaline amino acid proteases / serine proteins cleave pro-BNP, producing the amino-terminal pro-BNP, NT-proBNP, consisting of 76 amino acid residues, and BNP, consisting of 32 amino acid residues. This process is determined by post-translational protein glycosylation. BNP, NT-proBNP, and uncleaved proBNP are all released into the peripheral circulation. Once in the peripheral circulation, BNP is efficiently cleared with a half-life of approximately 20 minutes and poor stability in plasma and serum. Human BNP is a 3.5 kDa peptide with a theoretical isoelectric point of 10.95. NT-proBNP, the N-terminal portion of proBNP, contains seven glycosylation sites and a theoretical isoelectric point of 8.45. Due to glycosylation, its molecular weight is higher than the theoretical molecular weight of 8.5 kDa, and its half-life is 3-6 times longer than that of BNP.

[0003] Human BNP is distributed in tissues such as the brain, lungs, and spinal cord, with the highest concentration in the heart. When the ventricular lining is stimulated by external factors, BNP is rapidly expressed and secreted. For patients suspected of acute heart failure, clinical guidelines recommend the initial use of BNP or NT-proBNP to rule out heart failure. Elevated BNP levels in patients with heart failure indicate worsening disease and increased morbidity and mortality. Furthermore, BNP can inhibit renin-angiotensin-aldosterone production, increase glomerular filtration rate, and reduce cardiac preload and blood pressure. Current methods for preparing BNP typically involve expression in mammalian cells or Escherichia coli systems, followed by affinity chromatography and gel filtration to obtain the BNP protein (amino acid sequence 1-32). This method produces low protein yields, making it difficult to scale up production processes. Furthermore, BNP has a short half-life and poor stability. Summary of the Invention

[0004] Based on this, it is necessary to provide a preparation method of glycosylated brain natriuretic peptide precursor protein, a brain natriuretic peptide quality control product and a kit and their applications.

[0005] The first aspect of the present application provides a method for preparing a glycosylated brain natriuretic peptide precursor protein, comprising the following steps:

[0006] Inserting the target gene of the brain natriuretic peptide precursor protein into a plasmid to construct a recombinant expression vector;

[0007] Transforming the recombinant expression vector into Pichia pastoris and performing fermentation culture, and collecting the fermentation broth; and,

[0008] The fermentation broth is purified to obtain the glycosylated brain natriuretic peptide precursor protein.

[0009] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0010] The plasmid is selected from at least one of pPIC9K and pPICZαA;

[0011] The Pichia pastoris is selected from at least one of Pichia pastoris GS115 and Pichia pastoris KM71;

[0012] The culture medium for the fermentation culture is BMMY culture medium;

[0013] The fermentation culture time is 3 to 5 days; and

[0014] The purification includes ion exchange chromatography and affinity chromatography.

[0015] In some embodiments, the ion exchange chromatography and affinity chromatography meet one or more of the following conditions:

[0016] The ion exchange chromatography includes cation exchange chromatography;

[0017] The ion exchange chromatography equilibration buffer comprises 20 mM to 30 mM Tris buffer, and the pH of the equilibration buffer is 6.9 to 7.1;

[0018] The elution buffer of the ion exchange chromatography includes 20mM~30mM Tris buffer and 0.9mol / L~1.1mol / L sodium chloride, and the pH of the elution buffer is 6.9~7.1;

[0019] The affinity chromatography includes Ni column affinity chromatography;

[0020] The equilibration buffer for the affinity chromatography comprises 45 mM to 55 mM Tris buffer and 0.4 mol / L to 0.6 mol / L sodium chloride, and the pH of the equilibration buffer is 7.9 to 8.1;

[0021] The elution buffer of the affinity chromatography includes 45mM~55mM Tris buffer, 0.4 mol / L~0.6 mol / L sodium chloride and 0.4 mol / L~0.6 mol / L imidazole, and the pH of the elution buffer is 7.9~8.1;

[0022] The flow rate of the fermentation broth in the ion exchange chromatography and affinity chromatography is 2.9 mL / min to 3.1 mL / min.

[0023] The second aspect of the present application provides a quality control product of brain natriuretic peptide, which includes a glycosylated brain natriuretic peptide precursor protein prepared by the preparation method described in the first aspect of the present application, Tris-HCl buffer solution, sodium chloride, ProClin300, mannitol, trehalose, bovine serum albumin and PLG-PLL amino acid polymer.

[0024] In some embodiments, the quality control product meets one or more of the following conditions:

[0025] The concentration of the Tris-HCl buffer solution is 10mM~50mM;

[0026] Each 100 mL of the quality control product contains 0.9 g to 1.1 g of sodium chloride;

[0027] Each 100 mL of the quality control product contains 0.05 g to 1 g of ProClin300;

[0028] Each 100 mL of the quality control product includes 5 g to 10 g of mannitol;

[0029] Each 100 mL of the quality control product contains 1 g to 2 g of trehalose;

[0030] Each 100 mL of the quality control product includes 2 g to 4 g of bovine serum albumin;

[0031] Each 100 mL of the quality control product includes 0.1 g to 5 g of the PLG-PLL amino acid polymer;

[0032] The quality control product is a freeze-dried product; and

[0033] The pH of the quality control product is 7.3~7.5.

[0034] In some embodiments, the mass ratio of PLG to PLL in the PLG-PLL amino acid polymer is (3-5):1.

[0035] In some embodiments, the concentration of glycosylated brain natriuretic peptide precursor protein in the quality control product is 80 pg / mL to 500 pg / mL.

[0036] The third aspect of the present application provides a kit for detecting brain natriuretic peptide, comprising the quality control product described in the second aspect of the present application.

[0037] The fourth aspect of the present application provides a method for detecting brain natriuretic peptide in a sample, comprising using the quality control product described in the second aspect of the present application or the kit described in the third aspect of the present application during the detection process.

[0038] The fifth aspect of the present application provides the use of the quality control product described in the second aspect of the present application in the preparation of products for detecting heart diseases.

[0039] The aforementioned preparation method for glycosylated pro-BNP avoids the issues of low protein maturity and differences from native samples in E. coli-expressed proteins, as well as the scale-up challenges associated with mammalian cell-expressed proteins, compared to traditional BNP preparation processes. This method utilizes the Pichia pastoris expression system to glycosylate the expressed protein at high expression levels, resulting in glycosylated proBNP with a longer sequence and a longer half-life. Because BNP is a product of proBNP decomposition, and since BNP undergoes no structural changes before and after decomposition, proBNP can also be recognized by the BNP ligand. Compared to BNP, proBNP contains multiple glycosylation sites and has a longer half-life, making proBNP a superior choice as a raw material for BNP quality control.

[0040] Furthermore, a BNP quality control product was prepared using proBNP as a raw material, using a Tris buffer system, which reduces costs and minimizes batch-to-batch variability. The addition of a poly-L-glutamic acid (PLG)-L-lysine (PLL) amino acid polymer to the quality control product formulation and subsequent freeze-drying makes the product more similar to a natural sample, with a lower freeze-drying loss rate and greater stability than conventional freeze-drying processes, thereby improving the long-term stability and reconstitution stability of the BNP quality control product. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0042] Figure 1Schematic diagram of a process for preparing glycosylated proBNP protein in one embodiment of the present application;

[0043] Figure 2 This is an SDS-PAGE gel image of the glycosylated proBNP protein in one embodiment of the present application;

[0044] Figure 3 This is a general linear regression analysis for verifying the matrix effect in one embodiment of the present application. DETAILED DESCRIPTION

[0045] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0048] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0049] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0050] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0051] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0052] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0053] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0054] The current BNP preparation process results in low BNP antigen yield and purity, making scale-up difficult. Furthermore, current BNP quality control products exhibit poor stability after opening and reconstitution, and performance comparisons between BNP quality control products and clinical samples are difficult to implement without manufacturer-matched comparisons.

[0055] Based on this, the embodiments of the present application at least provide a method for preparing a glycosylated brain natriuretic peptide precursor protein, a brain natriuretic peptide quality control product and a kit and their applications.

[0056] In some embodiments, because BNP is a decomposition product of proBNP, and BNP undergoes no structural changes before and after decomposition, proBNP can also be recognized by BNP ligands. Compared to BNP, proBNP contains multiple glycosylation sites and has a longer half-life, making proBNP a preferred raw material for BNP quality control. Pichia pastoris is an ideal host for proBNP production due to its low cost, ease of operation, and ease of separation and purification of fermentation products. Its ability to undergo relatively mild post-translational protein modifications makes it an ideal host for proBNP production. The pAOX1-αMF-ProBNP plasmid was constructed, and a bacterial culture containing glycosylated proBNP protein was obtained using the Pichia pastoris expression system. Glycosylated proBNP was then purified by cation exchange column and Ni column affinity chromatography.

[0057] In some embodiments, poly-L-glutamic acid (PLG)-poly-L-lysine (PLL) amino acid polymer is added to the quality control product formula and freeze-dried, so that the quality control product is closer to the natural sample, the freeze-drying loss rate is smaller, and the stability is higher than the conventional freeze-drying process, thereby improving the long-term stability and re-dissolution stability of the BNP quality control product.

[0058] In some embodiments, a PLG-PLL amino acid polymer is obtained through rapid amino acid cyclic anhydride (NCA) polymerization initiated by lithium bis(trimethylsilyl)amide (LiHMDS). This polymer exhibits moisture insensitivity and a fast reaction rate, significantly improving protein stability. The average molecular weight of the PLG-PLL amino acid polymer is 280-300. Without limitation, the average molecular weight of the PLG-PLL amino acid polymer can be, but is not limited to, 280, 285, 290, 295, 300, or any number or range between any two of these values. Furthermore, the average molecular weight of the PLG-PLL amino acid polymer is 293.3.

[0059] In the first aspect of the present application, a method for preparing glycosylated brain natriuretic peptide precursor protein is provided, and the flow chart thereof is as follows: Figure 1 As shown, the following steps are included:

[0060] S100: Insert the target gene of brain natriuretic peptide precursor protein into the plasmid to construct a recombinant expression vector;

[0061] S200: transforming the recombinant expression vector into Pichia pastoris and performing fermentation culture, and collecting the fermentation broth; and,

[0062] S300: Purify the fermentation broth to obtain glycosylated brain natriuretic peptide precursor protein.

[0063] In some embodiments, in step S100 , the plasmid is selected from at least one of pPIC9K and pPICZαA.

[0064] In some embodiments, in step S200, the Pichia pastoris is selected from at least one of Pichia pastoris GS115 and Pichia pastoris KM71.

[0065] In some embodiments, in step 200, the fermentation culture medium may be BMMY medium, and the fermentation culture time may be 3 days, 4 days, or 5 days.

[0066] In some embodiments, in step 300, purification can be a conventional purification method in the art, including sequential purification using a cation exchange medium and Ni column affinity chromatography.

[0067] The steps for purifying the target protein using cation exchange media include:

[0068] S1: Equilibrating the medium with an equilibration buffer, wherein the equilibration buffer is 20 mM to 30 mM Tris buffer with a pH of 6.9 to 7.1; further, the equilibration buffer is 25 mM Tris buffer with a pH of 7.0.

[0069] S2: loading the fermentation broth, wherein the flow rate of loading the fermentation broth is 2.9 mL / min~3.1 mL / min; further, the flow rate of loading the fermentation broth is 3 mL / min.

[0070] S3: Gradient elution of the target protein is performed using an elution buffer, wherein the elution buffer comprises 20 mM to 30 mM Tris buffer and 0.9 mol / L to 1.1 mol / L sodium chloride, with a pH of 6.9 to 7.1; further, the elution buffer comprises 25 mM Tris buffer and 1 mol / L sodium chloride, with a pH of 6.9 to 7.1.

[0071] The steps of Ni column affinity chromatography purification include:

[0072] S10: Equilibrating the medium with an equilibration buffer, wherein the equilibration buffer comprises 45 mM to 55 mM Tris buffer and 0.4 mol / L to 0.6 mol / L sodium chloride, with a pH of 7.9 to 8.1; further, wherein the equilibration buffer comprises 50 mM Tris buffer and 0.5 mol / L sodium chloride, with a pH of 8.0.

[0073] S20: loading the fermentation broth, wherein the flow rate of loading the fermentation broth is 2.9 mL / min to 3.1 mL / min; further, the flow rate of loading the fermentation broth is 3 mL / min.

[0074] S30: Gradient elution of the target protein is performed using an elution buffer, wherein the elution buffer includes 45 mM to 55 mM Tris buffer, 0.4 mol / L to 0.6 mol / L sodium chloride, and 0.4 mol / L to 0.6 mol / L imidazole, with a pH of 7.9 to 8.1; further, the elution buffer includes 50 mM Tris buffer, 0.5 mol / L sodium chloride, and 0.5 mol / L imidazole, with a pH of 8.0.

[0075] In the second aspect of the present application, a quality control product of brain natriuretic peptide is provided, comprising a glycosylated brain natriuretic peptide precursor protein prepared by the preparation method of the first aspect of the present application, Tris-HCl buffer solution, sodium chloride, ProClin300, mannitol, trehalose, bovine serum albumin and PLG-PLL amino acid polymer.

[0076] In this application, unless otherwise specified, mannitol and trehalose are used as excipients to give the quality control product a certain form.

[0077] In this application, unless otherwise specified, sodium chloride and bovine serum albumin are used as stabilizers to maintain the stability of quality control products.

[0078] In this application, unless otherwise stated, ProClin300 is used as a preservative to extend the shelf life of quality control products.

[0079] In this application, unless otherwise specified, PLG and PLL amino acid polymers are composed of two single chiral L-amino acids. L-amino acids are not only cheaper than racemic amino acids but also easily degrade into essential amino acids. Furthermore, due to the water displacement mechanism, PLG can form hydrogen bonds with proteins and displace water molecules during the freeze-drying process to prevent protein aggregation. The glass transition mechanism also indicates that the amorphous structure of PLL can lower the glass transition temperature of proteins.

[0080] In some embodiments, the concentration of the Tris-HCl buffer solution can be 10 mM to 50 mM. In a non-limiting manner, the concentration of the Tris-HCl buffer solution can be, but is not limited to, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or a value or range between any two of the above values.

[0081] In some embodiments, each 100 mL of the quality control product includes 0.9 g to 1.1 g of sodium chloride; without limitation, each 100 mL of the quality control product includes but is not limited to 0.9 g, 1.0 g, 1.1 g or a value or range between any two of the above values ​​of sodium chloride.

[0082] In some embodiments, each 100 mL of the quality control product includes 0.05 g to 1 g of ProClin300; without limitation, each 100 mL of the quality control product includes but is not limited to 0.05 g, 0.08 g, 1 g, or a value or range between any two of the above values ​​of ProClin300.

[0083] In some embodiments, each 100 mL of the quality control product includes 5 g to 10 g of mannitol; without limitation, each 100 mL of the quality control product includes but is not limited to 5 g, 6 g, 7 g, 8 g, 9 g, 10 g of mannitol or a value or range between any two of the above values.

[0084] In some embodiments, each 100 mL of the quality control product includes 1 g to 2 g of trehalose; without limitation, each 100 mL of the quality control product includes but is not limited to 1 g, 1.5 g, 2 g, or a value or range of trehalose between any two of the above values.

[0085] In some embodiments, each 100 mL of the quality control product includes 2 g to 4 g of bovine serum albumin; without limitation, each 100 mL of the quality control product includes but is not limited to 2 g, 3 g, 4 g or a value or range between any two of the above values ​​of bovine serum albumin.

[0086] In some embodiments, each 100 mL of the quality control product includes, but is not limited to, 0.1 g to 5 g of the PLG-PLL amino acid polymer; without limitation, each 100 mL of the quality control product includes 0.1 g, 1 g, 2 g, 3 g, 4 g, 5 g or a value or range between any two of the above values ​​of PLG-PLL amino acid polymer.

[0087] In some embodiments, the mass ratio of PLG to PLL in the PLG-PLL amino acid polymer can be (3-5):1; without limitation, the mass ratio of PLG to PLL in the PLG-PLL amino acid polymer can be, but is not limited to, 3:1, 4:1, 5:1, or a ratio or range between any two of the above ratios.

[0088] In some embodiments, the pH of the quality control product is 7.3-7.5. Further, the pH of the quality control product is 7.4.

[0089] In some embodiments, the quality control product is a freeze-dried product. Setting the quality control product as a freeze-dried product can further increase the stability of the quality control product.

[0090] In some embodiments, the concentration of glycosylated brain natriuretic peptide precursor protein in the quality control sample is 80 pg / mL to 500 pg / mL. Without limitation, the concentration of glycosylated brain natriuretic peptide precursor protein in the quality control sample can be, but is not limited to, 80 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, or a value or range between any two of the above values.

[0091] In a third aspect of the present application, a kit for detecting brain natriuretic peptide is provided, comprising the quality control product of the second aspect of the present application.

[0092] In a fourth aspect of the present application, a method for detecting brain natriuretic peptide in a sample is provided, comprising using the quality control product of the second aspect of the present application or the kit of the third aspect of the present application during the detection process.

[0093] In the fifth aspect of the present application, a performance testing method for a quality control product is provided, namely a method for comparing the consistency of quality control and clinical samples, including a double verification method, which is matrix effect verification and reagent acceleration verification.

[0094] In some embodiments, matrix effect validation and reagent accelerated validation are combined, and if the results of the two are consistent, the consistency requirements of the quality control product and clinical samples are met.

[0095] It should be noted that the above-mentioned quality control and clinical sample consistency comparison method is verified through a double verification scheme. In the absence of a reagent comparison manufacturer, it can effectively verify the consistency of quality control and clinical samples, and the two perform consistently.

[0096] In the sixth aspect of the present application, there is provided an application of the quality control product of the second aspect of the present application in the preparation of a product for detecting heart diseases.

[0097] Some examples are provided below.

[0098] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0099] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0100] Example 1

[0101] 1. Preparation of glycosylated proBNP protein:

[0102] The proBNP target gene was inserted into the expression vector pPIC9K to obtain the pAOX1-αMF-ProBNP plasmid, which was linearized by restriction endonuclease Sal I. The linearized plasmid p AOX1-αMF-ProBNP was electroporated into Pichia pastoris GS115 and screened using the culture medium YND (YNB 6.7 g / L, glucose 10 g / L) to obtain the Pichia pastoris strain GS-p AOX1-αMF-ProBNP containing the target gene. The desired yeast strain was activated in YPD medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) for 2 days and then inoculated into fresh YPD liquid medium and cultured overnight to logarithmic phase (OD600 nm = 4–8). The culture was then centrifuged at 5000 g at 4°C for 5 min, washed twice with sterile water, and inoculated into BMMY fermentation medium (20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB, 100 mmol / L pH 6.0 potassium phosphate buffer, 5 mL / L methanol) at an OD600 nm of 1. Methanol was added every 24 h during fermentation for 4 days.

[0103] After shake flask fermentation, the strain was centrifuged at 10,000 g and 4°C for 10 minutes, and the supernatant was collected. After filtration through a 0.45 μm filter, the target protein was purified using cation exchange media (SP). The media was first equilibrated with equilibration buffer 1 (25 mM Tris buffer, pH 7.0) at a flow rate of 4 mL / min. The fermentation supernatant was then loaded at a flow rate of 3 mL / min. The target protein was then gradient eluted using elution buffer 1 (25 mM Tris buffer + 1 mol / L sodium chloride, pH 7.0) at a flow rate of 3 mL / min. The elution peak was collected and the purification was analyzed by SDS-PAGE. The purified protein sample was collected and further purified using Ni-TED media. First, the medium was equilibrated with equilibration buffer 2 (50 mmol / L Tris + 500 mmol / L sodium chloride, pH 8.0) at a flow rate of 4 mL / min, and then the fermentation supernatant was loaded at a flow rate of 3 mL / min. The target protein was then gradient eluted with elution buffer 2 (50 mmol / L Tris + 500 mmol / L sodium chloride + 500 mmol / L imidazole, pH 8.0) at a flow rate of 3 mL / min. Finally, the elution peak was collected and the protein purification effect was detected by SDS-PAGE. The results are shown in Figure 2. Figure 2 shown.

[0104] The amino acid sequence of the prepared glycosylated proBNP protein is shown in SEQ ID NO: 1:

[0105] SHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH.

[0106] Comparative Example 1

[0107] The proBNP target gene was inserted into the expression vector pET-28a to construct the pET-28a-DsbA-proBNP expression plasmid. The plasmid was transformed into the competent BL21 (DE3) Escherichia coli strain and cultured overnight in LB medium containing 50 μg / ml kanamycin sulfate. Positive clones were screened and further fermented to obtain recombinant proBNP protein. The target protein was purified and isolated using the purification method of Example 1 to obtain a purified product.

[0108] Comparative Example 2

[0109] The BNP target gene was inserted into the expression vector pET-28a to construct the pET-28a-DsbA-proBNP expression plasmid, which was transformed into the competent BL21 (DE3) Escherichia coli strain and cultured overnight in LB medium containing 50 μg / ml kanamycin sulfate. Positive clones were screened and further fermented to obtain BNP protein. The target protein was purified and separated using the purification method of Example 1 to obtain a purified product.

[0110] The amino acid sequence of the BNP protein is shown in SEQ ID NO: 2:

[0111] SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH.

[0112] Comparative Example 3

[0113] BNP antigen was prepared using the method of Example 1, except that the BNP target gene was inserted into the expression vector pPIC9K to obtain the pAOX1-αMF-ProBNP plasmid. The amino acid sequence of the BNP antigen is shown in SEQ ID NO: 2.

[0114] Antigen concentration test

[0115] The proBNP antigen prepared in Example 1 and the proBNP antigens or BNP antigens prepared in Comparative Examples 1 to 3 were tested for concentration, and the results are shown in Table 1 below. The results show that the glycosylated proBNP antigen prepared by the preparation method of Example 1 of the present application has a higher concentration and higher purity than the BNP antigens or proBNP antigens prepared in the comparative examples.

[0116] Table 1 Verification results of proBNP and BNP antigen concentrations in different processes

[0117]

[0118] Example 2

[0119] 1. Formula of BNP quality control product:

[0120] Solution preparation: Freeze-dried solution is prepared in a 100,000-level production workshop. The applicable proportions of excipients added during the general preparation process are as follows (the percentages of excipients added are all weight-to-volume ratios):

[0121] Tris-HCl buffer solution: 10mM~50mM;

[0122] Sodium chloride (W / V): 0.9%~1.1%;

[0123] ProClin300 (W / V): 0.05%~1%;

[0124] Mannitol (W / V): 5%~10%;

[0125] Anhydrous trehalose (W / V): 1%~2%;

[0126] Bovine serum albumin (BSA): 2%~4%;

[0127] PLG-PLL amino acid polymer (customized product, PLG-PLL mass fraction ratio: 4:1): 0.1%~5%.

[0128] 2. Experimental group: During the specific preparation, the proportions of each excipient were as follows: Tris-HCl buffer solution: 50 mM; sodium chloride (W / V): 0.9%; PC300 (W / V): 0.05%; mannitol (W / V): 8%; anhydrous trehalose (W / V): 2%; bovine serum albumin (BSA): 4%; PLG-PLL amino acid polymer: 1%;

[0129] Calculate the amount according to the content of each component, first add Tris-HCl buffer solution and sodium chloride to 800 mL of pure water, and after the materials are dissolved, add PC300, mannitol, and anhydrous trehalose in sequence. After the materials are dissolved, measure the solution pH, which should be within the range of 7.4±0.5. Then add bovine serum albumin and PLG-PLL amino acid polymer, and make up to 1 L with pure water. After stirring and mixing, measure the solution pH, and the final pH should be within the range of 7.4±0.1.

[0130] 3. Control group: When preparing the control group, the proportions of the excipients are as follows: Tris-HCl buffer solution: 50 mM; sodium chloride (W / V): 0.9%; PC300 (W / V): 0.05%; mannitol (W / V): 8%; anhydrous trehalose (W / V): 2%; bovine serum albumin (BSA): 4%;

[0131] Calculate the amount according to the content of each component, first add Tris-HCl buffer solution and sodium chloride to 800 mL of pure water, and after the materials are dissolved, add PC300, mannitol, and anhydrous trehalose in sequence. After the materials are dissolved, measure the pH of the solution. The pH should be within the range of 7.4±0.5. Then add bovine serum albumin and make up to 1 L with pure water. After stirring and mixing, measure the pH of the solution. The final pH should be within the range of 7.4±0.1.

[0132] 4. Confirm the raw material concentration: Pre-dilute the raw material in two 1.5 mL centrifuge tubes. Add 990 μL of Tris-HCl buffer and 10 μL of BNP or proBNP antigen to tube 1. Add 990 μL of Tris-HCl buffer and 10 μL of the solution from tube 1 to tube 2. Mix thoroughly, and then use tube 2 as the base solution (pre-dilution factor 10,000) for serial dilutions. In five other 1.5 mL centrifuge tubes, add 500 μL of Tris-HCl buffer to each of these tubes. Then, pipette 500 μL, 250 μL, 125 μL, 62.5 μL, and 31.25 μL of the base solution from tube 2, respectively. Analyze these five samples on an APHILANTHROPY iFlash series instrument (e.g., iFlash 3000-A) using the APHILANTHROPY B-type natriuretic peptide assay kit (chemiluminescence).

[0133] The added BNP antigen or proBNP antigen specifically includes: ① the proBNP antigen of Comparative Example 1, ② the BNP antigen of Comparative Example 2, ③ the BNP antigen of Comparative Example 3, and ④ the glycosylated proBNP protein of Example 1 of the present application.

[0134] 5. Preparation of quality control products: Based on the concentration confirmation results, the antigens of the three comparative examples and Example 1 of the present application were respectively added to the experimental group formula, and the glycosylated proBNP of the present application was added to the control group formula. The low value was added at a ratio of 1 / 115145, and the high value was added at a ratio of 1 / 18550. The target concentrations of the quality control products were set within the range of 80 pg / mL (±15%) and 500 pg / mL (±15%), thereby obtaining 5 sets of BNP quality control products.

[0135] 6. Lyophilization and Lyophilization Loss Measurement of Quality Controls: All five sets of quality control solutions prepared above were dispensed into 7 mL low-borosilicate brown glass vials in 2 mL aliquots and placed in a freeze dryer for lyophilization. After lyophilization, the vials were capped under vacuum and the samples collected. The quality control solutions were reconstituted with 2 mL of water and compared to the prepared values. The lyophilization loss rate, defined as the ratio of the reconstituted concentration to the prepared value, was calculated. The results are shown in Table 2 below. The results demonstrated that the glycosylated proBNP prepared in this application had a lower lyophilization loss rate than the control solutions prepared with the experimental formulations.

[0136] Table 2 Comparison of freeze-drying loss rate

[0137]

[0138] 7. Stability Verification: To verify the performance of BNP quality control products in extreme environments such as high temperatures, this application conducted accelerated stability testing on these products at 37°C. Products heat-treated at 37°C for 10 days and stored at 2-8°C served as controls. Furthermore, to verify the stability of reconstituted BNP quality control products, the present invention stored the reconstituted products at 20-25°C, 2-8°C, and -20°C for 8 days, 35 days, and 100 days, respectively. Products stored as lyophilized powder at 2-8°C served as controls. Calibration tests were performed using a B-type natriuretic peptide assay kit (chemiluminescence). The test results are shown in Tables 3 and 4 below. The results showed that the relative deviations of the BNP quality control concentrations under different conditions were within ±10% compared to the control group, indicating good stability of the BNP quality control products.

[0139] Table 3 Thermal stability verification results

[0140]

[0141] Table 4 Reconstitution stability verification results

[0142]

[0143]

[0144] The above results indicate that the glycosylated proBNP prepared in Example 1 of the present application and the quality control product prepared from the experimental group formula have a lower freeze-drying loss rate, accelerated stability can reach 10 days, and long-term stability estimated by thermal deduction can be stored for approximately 18 months. In terms of reconstitution stability, after reconstitution, the product can be stored at 20-25°C for up to 8 days, at 2-4°C for up to 35 days, and at ≤-20°C for up to 100 days. These performance indicators are superior to those of the quality control products prepared from the raw materials of other comparative examples.

[0145] Example 3

[0146] Quality control and clinical sample consistency comparison method

[0147] 1. Matrix effect verification: The test substance (glycosylated proBNP antigen prepared in Example 1) that covers the measurement range was added to the quality control matrix without BNP (i.e., the solution formula of the experimental group in Example 2) and clinical physical examination blood samples in parallel, with the concentration distributed as evenly as possible. 20 processed samples were prepared for each. Calibration tests were performed using the B-type natriuretic peptide assay kit (chemiluminescence method). 20 quality control matrix solutions and clinical serum samples with different concentrations of the test substance were tested simultaneously. The concentration values ​​of the clinical serum samples were used as the X-axis and the concentration values ​​of the quality control matrix solution were used as the Y-axis. A scatter plot was drawn and ordinary linear regression analysis was performed. The results are shown in Tables 5 and 6 below. Figure 3 The correlation coefficient (r) is ≥ 0.975, and the slope is 0.9 ≤ ≤ 1.1, indicating that the quality control product has good consistency with the clinical sample and can simulate the clinical sample to test the precision and accuracy of the instrument and reagent.

[0148] Table 5 Matrix effect verification results

[0149]

[0150] 2. Reagent acceleration verification: Take one box of reagent and place it at 37°C for thermal acceleration for 7 days, and store one box of reagent at 4°C. The accelerated reagent and the unaccelerated reagent placed at 4°C are loaded on the same iFlash series instrument of Yahuilong (such as iFlash 3000-A) at the same time. They are calibrated with their respective calibrators. The BNP quality control product (glycosylated proBNP prepared by the present application in Example 2 and the quality control product prepared by the experimental group formula) and the BNP clinical sample are tested. The test results of the accelerated reagent and the unaccelerated reagent are compared. The results are shown in Table 6 below. The test values ​​of the BNP quality control product and the BNP clinical sample on the accelerated reagent are consistent with the test values ​​of the unaccelerated reagent. Both are negative deviations and are within 15%, indicating that the performance of the BNP quality control product and the BNP clinical sample in terms of raw materials combined with the reagent components is consistent. Comprehensive matrix effect verification results, if both verification results meet the requirements, then the product requirements are met.

[0151] Table 6 Reagent accelerated validation results

[0152]

[0153] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person of ordinary skill in the art may make a number of variations and improvements without departing from the spirit of the present invention, and these variations and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings shall be used to interpret the scope of the claims.

Claims

1. A method for preparing a glycosylated brain natriuretic peptide precursor protein, characterized in that: The following steps are involved: Inserting the target gene of the brain natriuretic peptide precursor protein into a plasmid to construct a recombinant expression vector; Transforming the recombinant expression vector into Pichia pastoris and performing fermentation culture, and collecting the fermentation broth; and, The fermentation broth is purified to obtain the glycosylated brain natriuretic peptide precursor protein.

2. The preparation method according to claim 1, wherein One or more of the following conditions are met: The plasmid is selected from at least one of pPIC9K and pPICZαA; The Pichia pastoris is selected from at least one of Pichia pastoris GS115 and Pichia pastoris KM71; The fermentation culture medium is BMMY medium; The fermentation culture time is 3 to 5 days; and The purification includes ion exchange chromatography and affinity chromatography.

3. The preparation method according to claim 2, wherein The ion exchange chromatography and affinity chromatography meet one or more of the following conditions: The ion exchange chromatography includes cation exchange chromatography; The ion exchange chromatography equilibration buffer comprises 20 mM to 30 mM Tris buffer, and the pH of the equilibration buffer is 6.9 to 7.1; The elution buffer of the ion exchange chromatography includes 20mM~30mM Tris buffer and 0.9mol / L~1.1mol / L sodium chloride, and the pH of the elution buffer is 6.9~7.1; The affinity chromatography includes Ni column affinity chromatography; The equilibration buffer for the affinity chromatography comprises 45 mM to 55 mM Tris buffer and 0.4 mol / L to 0.6 mol / L sodium chloride, and the pH of the equilibration buffer is 7.9 to 8.1; The elution buffer of the affinity chromatography includes 45mM~55mM Tris buffer, 0.4 mol / L~0.6 mol / L sodium chloride and 0.4 mol / L~0.6 mol / L imidazole, and the pH of the elution buffer is 7.9~8.1; The flow rate of the fermentation broth in the ion exchange chromatography and affinity chromatography is 2.9 mL / min to 3.1 mL / min.

4. A quality control product for brain natriuretic peptide, characterized in that: The quality control product includes glycosylated brain natriuretic peptide precursor protein prepared by the preparation method according to any one of claims 1 to 3, Tris-HCl buffer solution, sodium chloride, ProClin300, mannitol, trehalose, bovine serum albumin and PLG-PLL amino acid polymer.

5. The quality control product according to claim 4, characterized in that: The quality control product meets one or more of the following conditions: The concentration of the Tris-HCl buffer solution is 10mM~50mM; Each 100 mL of the quality control product contains 0.9 g to 1.1 g of sodium chloride; Each 100 mL of the quality control product contains 0.05 g to 1 g of ProClin300; Each 100 mL of the quality control product includes 5 g to 10 g of mannitol; Each 100 mL of the quality control product contains 1 g to 2 g of trehalose; Each 100 mL of the quality control product includes 2 g to 4 g of bovine serum albumin; Each 100 mL of the quality control product includes 0.1 g to 5 g of the PLG-PLL amino acid polymer; The quality control product is a freeze-dried product; and The pH of the quality control product is 7.3~7.

5.

6. The quality control product according to claim 5, characterized in that: The mass ratio of PLG to PLL in the PLG-PLL amino acid polymer is (3-5):

1.

7. The quality control product according to claim 6, characterized in that: The concentration of glycosylated brain natriuretic peptide precursor protein in the quality control product is 80 pg / mL~500 pg / mL.

8. A kit for detecting brain natriuretic peptide, characterized in that: Including the quality control product as described in any one of claims 4 to 7.

9. A method for detecting brain natriuretic peptide in a sample, characterized in that: The method comprises using the quality control product according to any one of claims 4 to 7 or the kit according to claim 8 during the detection process.

10. Use of the quality control product according to any one of claims 4 to 7 in the preparation of a product for detecting heart diseases.

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