A parathyroid hormone mutant, a liquid quality control containing the parathyroid hormone mutant and application thereof

Abstract

The present application relates to parathyroid hormone, in particular to a kind of parathyroid hormone mutant, liquid quality control containing parathyroid hormone mutant and its application;A kind of parathyroid hormone mutant, it is characterized in that, the parathyroid hormone mutant is based on wild type human parathyroid hormone amino acid sequence modification obtains, specific mutation includes: H9C, M18L, R25A, K26A, K27A, S48C;The amino acid sequence of the parathyroid hormone mutant is as shown in SEQ ID NO.1.Solid-phase polypeptide synthesis process is used, easy to large-scale production, cost controllable.This scheme realizes PTH molecule stabilization by accurate site-directed mutation, obtains the improved molecule with comprehensive performance improvement under the minimum structural disturbance, breaks through the stability bottleneck of traditional quality control product, and provides high-reliable liquid quality control for clinical PTH detection.

Description

Technical Field

[0001] This invention relates to parathyroid hormone, and particularly to a parathyroid hormone mutant, a liquid quality control product containing the parathyroid hormone mutant, and their applications. Background Technology

[0002] Accurate detection of parathyroid hormone (PTH) is crucial for the clinical diagnosis of calcium and phosphorus metabolism disorders and related diseases. To ensure the accuracy, comparability, and long-term traceability of PTH test results across different laboratories and testing platforms, clinical testing relies on calibrators and quality control materials with clearly defined components and stable concentrations. However, developing such highly reliable standard materials faces a fundamental technical bottleneck: the physicochemical instability of the PTH molecule itself. This instability stems from its inherent characteristics as a small polypeptide. PTH molecules contain cleavage sites easily recognized by proteases, making them highly susceptible to peptide chain breakage during production and storage, generating protein fragments with varying immunoreactivity. Furthermore, amino acids such as methionine in the sequence are prone to oxidative modification, and asparagine is susceptible to deamidation; all of these chemical modifications irreversibly alter its immunomodulatory activity. In addition, PTH is prone to intermolecular aggregation and can non-specifically adsorb onto container surfaces, leading to a decrease in the effective concentration of active monomers in the solution. These factors collectively cause calibration drift and poor batch-to-batch consistency in natural PTH quality control materials during storage, severely impacting both internal and interlaboratory quality control effectiveness.

[0003] Currently, the industry primarily employs external additives and optimized preparation processes to enhance PTH stability. For example, protease inhibitors, antioxidants, and high-concentration carrier proteins are added to the quality control matrix, coupled with low-temperature or lyophilization storage. However, these methods have significant drawbacks: the stability of various additives gradually decreases with storage time, and some additives can cause unpredictable signal interference in certain immunoassay systems. More importantly, these methods only provide "external protection" and cannot block endogenous PTH degradation at the molecular level; once the protective system fails or trace amounts of protease contamination are introduced during the raw materials or preparation process, PTH degradation will occur rapidly. Therefore, quality control products prepared using traditional processes have limited shelf life and cannot meet the current clinical testing requirements for highly stable and interoperable quality control products.

[0004] To fundamentally improve peptide stability, the biopharmaceutical field has developed mature protein modification technologies such as polyethylene glycol (PEGylation), fatty acid modification, and Fc fragment fusion. These modification methods can significantly extend the in vivo half-life of pharmaceutical peptides. However, these chemical modification schemes are not suitable for the preparation of raw materials for quality control products: the core requirement for quality control analytes is that their binding ability with antibodies from mainstream commercially available diagnostic reagents and their natural human PTH content must be highly consistent. Large molecular modification groups such as PEG can easily create steric hindrance, obscuring key PTH antigenic epitopes, leading to unpredictable and unstandardized deviations in the reactivity of modified products with antibody reagents from different manufacturers that target different epitopes. Although these macromolecularly modified PTH derivatives exhibit excellent stability, their immunoreactivity characteristics deviate from natural PTH, failing to meet the requirements for reference materials and thus unsuitable for calibration and quality control in existing detection systems.

[0005] In summary, there are pressing technical challenges in this field: the insufficient structural stability of natural PTH hinders the development of high-performance quality control products; existing chemical modification methods that can significantly improve stability, however, can disrupt the essential innate immune response characteristics of quality control references. The industry urgently needs a novel PTH molecular modification scheme to enhance its resistance to enzymatic and chemical degradation at the molecular structural level, while fully preserving the spatial conformation and all antigenic epitopes of natural PTH. Summary of the Invention

[0006] Purpose of the invention

[0007] This invention aims to solve the following problems and provides a solution, as detailed below:

[0008] (1) Solve the technical problem that natural PTH molecules are easily enzymatically decomposed, oxidized, and aggregated, leading to the instability of quality control products;

[0009] (2) Solve the technical problems of short shelf life, concentration drift and decreased immunoreactivity of natural PTH in liquid matrix;

[0010] (3) Provide a liquid quality control product containing stabilized PTH that can be stored stably for a long time and has the same immunoreactivity as natural PTH;

[0011] (4) Provide a method for preparing the above-mentioned quality control products so that they can be industrialized and applied.

[0012] Technical solution

[0013] This invention obtains a modified PTH mutant with high stability and high immunocompatibility through site-directed mutagenesis, and uses it to prepare liquid quality control products.

[0014] A parathyroid hormone mutant, characterized in that the parathyroid hormone mutant is obtained by modifying the amino acid sequence of wild-type human parathyroid hormone, and the specific mutations include: H9C, M18L, R25A, K26A, K27A, S48C; the amino acid sequence of the parathyroid hormone mutant is shown in SEQ ID NO.1.

[0015] The parathyroid hormone mutant is characterized in that the parathyroid hormone mutant is a purified protein prepared by artificial synthesis, recombinant expression, or chemical modification.

[0016] A liquid quality control product containing a parathyroid hormone mutant, characterized in that it comprises the parathyroid hormone mutant and a matrix solution.

[0017] The liquid quality control containing the parathyroid hormone mutant is characterized in that, based on mass-volume concentration, the concentration of the parathyroid hormone mutant added to the liquid quality control is 15 pg / mL to 20 ng / mL.

[0018] The liquid quality control product containing the parathyroid hormone mutant is characterized in that the matrix solution is a biological buffer matrix selected from any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer, and the pH value of the matrix solution is 6.0 to 8.0.

[0019] The liquid quality control product containing the parathyroid hormone mutant is characterized in that it further comprises at least one of a stabilizer, a preservative, and an osmotic pressure regulator; the stabilizer is selected from one or more of sucrose, trehalose, and bovine serum albumin; and the preservative is selected from one or two of sodium azide and ProClin300.

[0020] The application of the parathyroid hormone mutant in the preparation of parathyroid hormone quality control products.

[0021] The application is characterized in that the quality control product is a liquid quality control product for in vitro diagnostics, suitable for quality control of parathyroid hormone detection on chemiluminescence immunoassay, enzyme-linked immunosorbent assay, colloidal gold assay, and electrochemical immunoassay platforms.

[0022] PTH mutant design concept:

[0023] (1) Anti-enzymatic mutation: For the enzyme cleavage sensitive sites at positions 25-26-27, R25 is replaced with K or A, and K26 / K27 is replaced with A, thereby reducing the efficiency of protease recognition and cleavage.

[0024] (2) Antioxidant mutation: Replace the easily oxidized methionine (M) at position 18 with leucine (L) or oroleucine (Nle) to eliminate the oxidation site.

[0025] (3) Conformation stabilization mutation: Introduce cysteine ​​at appropriate sites to form intramolecular disulfide bonds to lock the conformation, reducing unfolding and aggregation.

[0026] (4) Construction of combinatorial mutants:

[0027] - M1: R25K + M18L (basic stable type)

[0028] - M2: R25A + K26A + M18L (Strongly resistant to enzymatic hydrolysis)

[0029] - M3: H9C / S48C + M18L (Conformation-locked type)

[0030] - M4: Multiple combination mutation (comprehensive enhancement type), which is a combination of the above three M1-M3 mutations.

[0031] Preparation of liquid quality control products

[0032] (1) Quality control matrix: buffer system containing preservatives and osmotic pressure regulators.

[0033] (2) Preparation: Add the preferred PTH mutant to the matrix at the clinical test concentration, mix well and make up to volume.

[0034] (3) Filtration and sterilization and dispensing: 0.22 μm filter membrane filtration and dispensing into inert plastic bottles.

[0035] (4) Sealed storage: Store in a cool, dark place or freeze.

[0036] Key indicators of quality control products

[0037] 1. Appearance: Clear liquid, without sediment or turbidity;

[0038] 2. Stability: 7 days at 2-8 degrees Celsius after opening, 3 years at -20 degrees Celsius without opening;

[0039] 3. Immunoreactivity: The curves are parallel to those of mainstream PTH detection kits, with a slope ratio of 0.9–1.1.

[0040] Beneficial effects

[0041] (1) Significantly improved stability: It blocks the three major degradation pathways of enzymatic hydrolysis, oxidation and aggregation at the molecular level, and the liquid state is stable for a long time. It does not require freeze drying and is convenient to use.

[0042] (2) High fidelity of immune reactivity: The mutation site avoids the core antigen epitope and is consistent with the immune recognition characteristics of natural PTH, making it compatible with mainstream detection platforms.

[0043] (3) Wide range of applications: It can be used in fully automated chemiluminescence, electrochemiluminescence and other immunoassay systems to meet clinical quality control needs.

[0044] (4) Mature preparation process: It adopts recombinant expression and conventional formulation process, which is easy to scale up production and the cost is controllable.

[0045] This approach achieves PTH molecule stabilization through precise site-directed mutagenesis, obtaining improved molecules with comprehensively enhanced performance under minimal structural perturbation. It breaks through the stability bottleneck of traditional quality control products and provides highly reliable liquid quality control products for clinical PTH testing. Attached Figure Description

[0046] Figure 1 HPLC results for the PTH mutant;

[0047] Figure 2 To obtain the stability test results of M4 for this invention;

[0048] Figure 3 The results of the stability study of commercially available PTH are for Comparative Example 1.

[0049] Figure 4 The results of the long-term stability study of M4 were obtained for this invention. Detailed Implementation

[0050] Example 1: Preparation of PTH mutant

[0051] 1. Molecular design of modified PTH mutants

[0052] PTH mutant design concept:

[0053] (1) Anti-enzymatic mutation: For the enzyme cleavage sensitive sites at positions 25-27, replace R25 with K or A, and replace K26 / K27 with A to reduce the efficiency of protease recognition and cleavage.

[0054] (2) Antioxidant mutation: Replace the easily oxidized methionine (M) at position 18 with leucine (L) or oroleucine (Nle) to eliminate the oxidation site.

[0055] (3) Conformation stabilization mutation: Introduce cysteine ​​at appropriate sites to form intramolecular disulfide bonds to lock the conformation, reducing unfolding and aggregation.

[0056] Therefore, the following strategy is formed:

[0057] M1: R25K + M18L (basic stable type)

[0058] M2: R25A + K26A + M18L (Strongly resistant to enzymatic hydrolysis)

[0059] M3: H9C / S48C + M18L (Conformation-locked type)

[0060] M4: Multiple combination mutation (comprehensive enhancement type), which is a combination of the above three M1-M3 mutations.

[0061] Specifically as follows:

[0062] To address the stability bottleneck in PTH-containing liquid quality control products, this invention employs a minimal mutation strategy: replacing the methionine at position 18 with leucine (M18L) to eliminate the major oxidation-sensitive site in the middle; mutating the arginine / lysine tandem sequence at positions 25-27 to alanine (R25A / K26A / K27A) to disrupt the specific recognition and cleavage of trypsin-like enzymes; and simultaneously introducing a pair of cysteine ​​residues (H9C / S48C) at positions 9 and 48 to form intramolecular disulfide bonds, locking the native conformation and inhibiting unfolding. This design, through precise modification of only a few sites, significantly improves the molecule's resistance to degradation and conformational stability while preserving the immune recognition properties of wild-type PTH to the greatest extent possible, making it suitable for the development of liquid quality control products for long-term storage. The design strategy follows three points: (1) Mutate the arginine / lysine (R25, K26, K27) at positions 25-27 to alanine (A) to eliminate the cleavage site of trypsin-like enzymes; (2) Mutate the methionine (M18) at position 18 to leucine (Nle) or leucine (L) to eliminate the oxidative degradation pathway; (3) Introduce a pair of cysteine ​​(H9C / S48C) at positions 9 and 48 to construct intramolecular disulfide bonds to stabilize the spatial conformation.

[0063] Finally, the M4 strategy was used to obtain the modified PTH mutant (named PTH-Mut), whose complete amino acid sequence is as follows (single-letter code):

[0064] M4: PTH-Mut full sequence (84 aa), see SEQ ID NO.1:

[0065] SVSEIQLM C NLGKHLNS L ERVEWL AAA LQDVHNFVALGAPLAPRDAG C QRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

[0066] Note: Compared to wild type (WT), specific mutations include: H9C (introduction of cysteine ​​at position 9), M18L (antioxidant mutation at position 18), R25A, K26A, K27A (anti-enzymatic mutation at positions 25-27), and H9C / S48C (introduction of cysteine ​​at position 9 or 48).

[0067] 2. Solid-phase synthesis of polypeptides

[0068] 2.1 Reagents and Instruments

[0069] Main reagents: Fmoc protected amino acids (Novabiochem), Rink Amide MBHA resin, HATU, HBTU, DIEA, TFA, acetonitrile (HPLC grade), reduced glutathione (GSH), oxidized glutathione (GSSG).

[0070] Main instruments: fully automated peptide synthesizer (CSBio CS336X), preparative high performance liquid chromatograph (Waters AutoPurification System), analytical HPLC (Agilent 1260), electrospray ionization mass spectrometer (ESI-MS, Thermo Scientific).

[0071] 2.2 Solid-phase peptide synthesis (SPPS)

[0072] The standard Fmoc / tBu strategy was used for synthesis.

[0073] Resin pretreatment and deprotection: Weigh Fmoc-Rink Amide MBHA resin (degree of substitution 0.3 mmol / g) and place it in the reactor. Swell with dichloromethane (DCM) for 30 min. Remove the Fmoc protecting group using 20% ​​piperidine / DMF solution, deprotecting for 15 min each time, repeating twice, followed by DMF washing.

[0074] Amino acid coupling: The amino acids were added sequentially from the C-terminus to the N-terminus according to the PTH-Mut sequence. Key steps: At positions 48 and 9 (cysteine ​​sites), Fmoc-Cys(Trt)-OH (triphenylmethyl protecting the thiol group) was used. The amino acids (4 eq), HATU (3.9 eq), and DIEA (8 eq) were activated in DMF for 2 min and then added to the reactor. The reaction was carried out with shaking at room temperature for 60 min.

[0075] Capping: After each coupling step, capping reaction was performed for 5 min using acetic anhydride / DIEA / DMF (5:2:93, v / v / v) to reduce the impurities of the missing peptide.

[0076] Washing cycle: After each reaction, the resin is thoroughly washed with DMF, methanol, and DCM in sequence.

[0077] 2.3 Cleavage and Crude Peptide Acquisition

[0078] After synthesis, the resin was transferred to a lysis flask, and pre-cooled cleavage buffer (TFA:TIS:H2O:phenol = 94:2.5:2.5:1, v / v) was added. The mixture was magnetically stirred at room temperature for 2 h. The filtrate was collected by filtration, precipitated with ice-cold anhydrous diethyl ether, centrifuged (4000 rpm, 10 min, 4 ℃), and the precipitate was washed three times with diethyl ether and dried under vacuum to obtain the crude peptide.

[0079] 2.4 Intramolecular disulfide bond formation (oxidative folding)

[0080] The crude peptide was dissolved in 0.1 M Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride, and the peptide concentration was adjusted to 0.5 mg / mL. GSH and GSSG were added, and the final concentration ratio was controlled at peptide:GSH:GSSG = 1:10:1. After purging with nitrogen, the mixture was sealed and stirred at room temperature in the dark for 24 h. The reaction solution was monitored by HPLC, and the reaction was terminated when the main peak (oxidized form) no longer increased.

[0081] 2.5 Purification and Structure Identification

[0082] Preparation and purification: A C18 reversed-phase preparative column (Waters XBridge Prep C18, 5 µm, 19 × 150 mm) was used. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution. Gradient elution (5% → 40% B, 40 min) was performed at a flow rate of 10 mL / min. The target peak fraction was collected (see...). Figure 1 (As shown), after being combined, the product was freeze-dried to obtain a white powdery pure product.

[0083] Example 2: Detection of Actual PTH Concentration

[0084] Prepare a 40 mL quality control diluent. Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and add it to the buffer solution, stirring to dissolve. Add 0.04 mL of ProClin 300 to the solution and mix gently. Make up to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, and then add the synthesized PTH according to Table 1. Aliquot and store. Finally, detect the actual concentration using a Cobase 411 chemiluminescence immunoassay analyzer and a New Industries MAGLUMI X8 chemiluminescence immunoassay analyzer, respectively.

[0085] Table 1. Three levels of sample concentration (theoretical concentration) for the modified PTH mutant.

[0086]

[0087] Table 2. Detection results of actual concentration of the modified PTH mutant

[0088]

[0089] Comparative Example 1: Actual Concentration Detection of Commercially Available PTH

[0090] Prepare a 40 mL quality control diluent. Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and add it to the buffer solution, stirring to dissolve. Add 0.04 mL of ProClin 300 to the solution and mix gently. Make up to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, and then add commercially available PTH according to Table 1. Aliquot and store. Finally, detect the actual concentration using a Cobase 411 chemiluminescence immunoassay analyzer and a New Industries MAGLUMI X8 chemiluminescence immunoassay analyzer, respectively.

[0091] Table 3. Actual concentration test results of commercially available PTH

[0092]

[0093] As shown in Tables 2 and 3, compared with commercially available PTH, the modified PTH mutant prepared in this invention has a higher degree of agreement with the theoretical reference value when used as a quality control product, and the product uniformity is better.

[0094] Example 3: Stability assessment of the modified PTH mutant synthesized in this invention

[0095] The stability of the three levels of liquid immunoassay control products containing PTH (synthesized in this invention) as described in Table 1 was tested:

[0096] Seven bottles of quality control samples were opened, reconstituted, and stored at 2-8°C in the dark for 6 days. One bottle was randomly selected on days 1, 2, 3, 4, 5, and 6 for testing. Each bottle was tested three times on a suitable immunoassay analyzer, and the mean was calculated.

[0097] Comparative Example 2: Market-Sold Stability Study

[0098] Stability tests were conducted on the above three levels of liquid immunoassay control products containing PTH (commercially available).

[0099] Seven bottles of quality control samples were opened, reconstituted, and stored at 2-8°C in the dark for 6 days. One bottle was randomly selected on days 1, 2, 3, 4, 5, and 6 for testing. Each bottle was tested three times on a suitable immunoassay analyzer, and the mean was calculated.

[0100] Result: As Figure 2 and Figure 3 As shown,

[0101] When the PTH synthesized in this invention was added to the quality control sample, after remelting and storage at 2-8°C for 7 days, its detection values ​​remained within acceptable range. Compared to commercially available PTH, the quality control sample of this invention did not show significant drift or trend changes. In contrast, the commercially available PTH in Comparative Example 2 showed shifts at all three levels, especially a large shift at level 3.

[0102] Example 4: Long-term stability study of the modified PTH mutant synthesized in this invention

[0103] The stability of the three levels of liquid immunoassay control products containing PTH (synthesized in this invention) as described in Table 1 was tested:

[0104] Two vials of PTH-containing liquid quality control samples at three different concentrations were collected at months 0, 6, 12, 18, 24, 30, 36, and 38. Each vial was tested three times on a suitable immunoassay analyzer, and the mean was calculated.

[0105] The long-term stability test results of liquid quality control products containing PTH are as follows: Figure 4 As shown, the horizontal axis represents the time span of 0-38 months, and the vertical axis represents the concentration of PTH at different levels; this indicates that the prepared liquid quality control product containing PTH can be stably stored for 38 months under low temperature storage conditions (-80~-20℃), and its shelf life covers the shelf life of the quality control product.

Claims

1. A parathyroid hormone mutant, characterized in that, The parathyroid hormone mutant was obtained by modifying the amino acid sequence of wild-type human parathyroid hormone. The specific mutations include: H9C, M18L, R25A, K26A, K27A, and S48C; a pair of cysteine ​​residues were introduced at positions 9 and 48 to form an intramolecular disulfide bond; the amino acid sequence of the parathyroid hormone mutant is shown in SEQ ID NO.

1.

2. The parathyroid hormone mutant according to claim 1, characterized in that, The parathyroid hormone mutant is a purified protein prepared by artificial synthesis, recombinant expression, or chemical modification.

3. A liquid quality control product containing a parathyroid hormone mutant, characterized in that, It includes the parathyroid hormone mutant as described in claim 1 or 2 and the matrix fluid.

4. The liquid quality control product containing a parathyroid hormone mutant according to claim 3, characterized in that, The concentration of the parathyroid hormone mutant in the liquid quality control product is 15 pg / mL to 20 ng / mL, based on mass-volume concentration.

5. The liquid quality control product containing a parathyroid hormone mutant according to claim 3, characterized in that, The matrix solution is a biological buffer matrix, selected from any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer, and the pH value of the matrix solution is 6.0 to 8.

0.

6. The liquid quality control product containing a parathyroid hormone mutant according to claim 3, characterized in that, The liquid quality control product also contains at least one of the following additives: stabilizer, preservative, and osmotic pressure regulator; the stabilizer is selected from one or more of sucrose, trehalose, and bovine serum albumin; the preservative is selected from one or two of sodium azide and ProClin300.

7. The use of the parathyroid hormone mutant according to claim 1 in the preparation of parathyroid hormone quality control products.

8. The application according to claim 7, characterized in that, The quality control material is a liquid quality control material for in vitro diagnostics, suitable for quality control of parathyroid hormone detection on chemiluminescence immunoassay, enzyme-linked immunosorbent assay, colloidal gold assay, and electrochemical immunoassay platforms.

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