阅读说明：本技术 全血型冻干粉免疫抑制剂质量控制物质及其制备方法和应用 (Quality control substance of whole blood type freeze-dried powder immunosuppressant and preparation method and application thereof ) 是由 邹继华 沈敏 古咏梅 杨晓东 屠敏敏 邹炳德 于 2019-10-30 设计创作，主要内容包括：一种全血型冻干粉免疫抑制剂质量控制物质及其制备方法和应用,该物质主要是以人全血为基质,和免疫抑制剂制成的冻干品。所述的免疫抑制剂包括他克莫司、西罗莫司、依维莫司和环孢素A中的一种或者多种,或者还包括糖皮质激素类免疫抑制剂：氢化可的松、可的松、泼尼松龙、泼尼松、霉酚酸酯经血检测的免疫抑制剂中的一种或者多种。具有均匀性、稳定性良好,不需-70℃低温保存,可用于不同方法学免疫抑制剂试剂盒的校准品,也可作为质控品用于临床监测免疫抑制剂检测系统的优点。(A quality control substance of whole blood type lyophilized powder immunosuppressant is prepared from human whole blood as matrix and lyophilized product of immunosuppressant. The immunosuppressant comprises one or more of tacrolimus, sirolimus, everolimus and cyclosporine A, or further comprises a glucocorticoid immunosuppressant: one or more of hydrocortisone, cortisone, prednisolone, prednisone, mycophenolate mofetil, and immunosuppressant for blood test. Has the advantages of good uniformity and stability, no need of low-temperature storage at-70 ℃, and can be used as a calibrator of immunosuppressant kits of different methodologies and also can be used as a quality control product for a clinical monitoring immunosuppressant detection system.)

1. A quality control substance of a whole blood lyophilized powder immunosuppressant is characterized in that: the substance is a freeze-dried product mainly prepared from human whole blood as a matrix and an immunosuppressant.

2. The whole blood lyophilized powder immunosuppressant quality control substance of claim 1, characterized in that: the immunosuppressant comprises one or more of tacrolimus, sirolimus, everolimus and cyclosporine A, or further comprises a glucocorticoid immunosuppressant: one or more of hydrocortisone, cortisone, prednisolone, prednisone, mycophenolate mofetil, and immunosuppressant for blood test.

3. The whole blood lyophilized powder immunosuppressant quality control substance of claim 2, characterized in that: the quality control substance of the whole blood freeze-dried powder immunosuppressant is prepared into a low-concentration quality control substance or a high-concentration quality control substance, the concentration range of tacrolimus in the low-concentration quality control substance is controlled to be 0-20 ng/mL, the concentration range of sirolimus and everolimus is controlled to be 0-15 ng/mL, and the concentration range of cyclosporine A is controlled to be 0-300 ng/mL; the concentration range of tacrolimus in the high-concentration quality control product is controlled to be 21-50 ng/mL, the concentration range of sirolimus and everolimus is controlled to be 16-50 ng/mL, and the concentration range of cyclosporine A is controlled to be 301-1200 ng/mL.

4. The whole blood lyophilized powder immunosuppressant quality control substance of claim 2, characterized in that: the quality control substance of the whole blood freeze-dried powder immunosuppressant can be prepared into calibrators with different concentration levels and suitable for immunosuppressant detection systems, and the concentration level can be 2-10.

5. A preparation method of a whole blood lyophilized powder immunosuppressant quality control substance is characterized in that: the preparation method comprises the following specific steps:

(1) collect clinical fresh EDTA-K without jaundice, chyle and hemolysis₂Or heparin anticoagulated whole blood, taking a small amount of upper plasma for infectious disease screening, and selecting a whole blood sample with a negative resultCarrying out blood type detection, and mixing whole blood of the same blood type for later use; mixed whole blood of the same blood type is used as a substrate for preparing the quality control substance of the immunosuppressant; if the blood type detection is not carried out on the whole blood sample, adding an anticoagulant into the whole blood after mixing different human parts;

(2) uniformly mixing the whole blood obtained in the step (1), adding a dispersing agent and a solubilizer, and stirring until the whole blood is completely dissolved;

(3) measuring the concentration of the immunosuppressant in the whole blood substrate obtained in the step (2); adding a proper amount of standard stock solution of the immunosuppressant according to the concentration requirement of the quality control substance on the immunosuppressant; before adding the immune inhibitor into whole blood, blow-drying the standard stock solution of the immune inhibitor by nitrogen, and stirring for 1-6 hours to completely dissolve the added immune inhibitor;

(4) Adding a protective agent, an excipient and a preservative into the whole blood in the step (3), and stirring until the whole blood is completely dissolved;

(5) and (4) subpackaging the whole blood in the step (4) according to the specification requirement, and freeze-drying to prepare the lyophilized powder.

6. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 5, characterized in that: the anticoagulant in the step (1) is EDTA-K₂And one or more of heparin, wherein the concentration range is 0-20 mmol/L.

7. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 5, characterized in that: the dispersing agent in the step (2) is one or more of PVP40, PVP360, PEG and Tween 20-80 surfactants, and the concentration range is 0-40%; the solubilizer is one or more of glycol and tert-butyl alcohol polyhydric liquid alcohols, and the concentration range is 0-5%.

8. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 5, characterized in that: the immunosuppressant in the step (3) is cyclosporine A, tacrolimus, sirolimus, everolimus or other types of immunosuppressants.

9. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 8, wherein: adding the cyclosporine A in the step (3) into the whole blood in a mode of dropwise adding a cyclosporine A standard stock solution, and stirring for 1-6 h to completely dissolve the added immunosuppressant.

10. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 5, characterized in that: the protective agent in the step (4) comprises but is not limited to an antioxidant protective agent and a stabilizing agent; the antioxidant protective agent is one or a combination of more of ascorbic acid and salts thereof, sodium sulfite, sodium metabisulfite, cysteine and reductive substances of glutathione, and the concentration range is 0-10%; the stabilizer is mainly protein, sugar, polyol or amino acid substances, is one or a combination of more of bovine serum albumin, human serum albumin, trehalose, glucose and sucrose, and has a concentration range of 0-10%; the excipient is mainly but not limited to one or a combination of a plurality of polyols, saccharides, amino acids and proteins, and the concentration range is 0-10%; the antiseptic is one or more of dehydroacetic acid and its salt, calcium propionate, sodium lactate, benzoic acid and its salt, sodium azide, sorbic acid and its salt.

11. The method for preparing the whole blood lyophilized powder immunosuppressant quality control substance according to claim 5, characterized in that: the quality control substance is prepared into a low-concentration quality control substance and/or a high-concentration quality control substance; the concentration range of tacrolimus, sirolimus and everolimus in the low-concentration quality control product can be controlled to be 0-20 ng/mL, the concentration range of sirolimus and everolimus can be controlled to be 0-15 ng/mL, and the concentration range of cyclosporine A can be controlled to be 0-300 ng/mL; the concentration range of the tacrolimus, the sirolimus and the everolimus in the high-concentration quality control product can be controlled to be 21-50 ng/mL, the concentration range of the sirolimus and the everolimus can be controlled to be 16-50 ng/mL, and the concentration range of the cyclosporine A can be controlled to be 301-1200 ng/mL.

12. A calibrator for calibrating an immunosuppressant detection system adopts a whole blood lyophilized powder immunosuppressant quality control substance.

13. A quality control material for the immunosuppressant in whole blood lyophilized powder is used as a quality control material for clinically monitoring the accuracy and stability of a immunosuppressant detection system.

Technical Field

The invention relates to the technical field of medical inspection, in particular to a quality control substance of a whole blood type freeze-dried powder immunosuppressant, a preparation method and an application thereof.

Background

The immunosuppressant has effects of inhibiting organism immunoreaction, inhibiting proliferation and function of macrophage such as T cell and B cell, reducing antibody immunoreaction, preventing and treating organ transplantation rejection, and treating autoimmune disease and allergic disease. The medicine has strong renal toxicity, narrow treatment window and larger individual difference of pharmacokinetics, and clinically, a patient needs to be monitored (TDM) with a therapeutic medicine to ensure that the use of the immunosuppressant is in a safe and effective treatment range and is not easy to be poisoned.

Currently, clinically applied immunosuppressants can be classified into 5 major groups: cytokine inhibitors such as cyclosporin A (CsA), tacrolimus (FK506, etc.); ② DNA synthesis inhibitors such as Mycophenolate Mofetil (MMF), azathioprine (Aza), etc.; ③ adrenocortical hormones, such as prednisone, methylprednisolone, etc.; anti-lymphocyte antibodies, such as anti-lymphocyte globulin (ALG), anti-thymocyte globulin (ATG) and the like; fifthly, other immunosuppressive agents, such as FTY720, cyclophosphamide and the like. Among them, cyclosporin a (csa), tacrolimus (TAC, FK506), Sirolimus (SIR), Everolimus (EVR), etc. are widely used in clinical solid organ transplantation. CsA is lipophilic cyclic polypeptide, TAC is macrocyclic lactam immunosuppressant, and both belong to calmodulin phosphatase inhibitor and act on T lymphocyte; SIR is macrolide isolated from Streptomyces hygroscopicus, EVR is SIR40-O- (2-hydroxyethyl) derivative, both of which bind to mammalian target of rapamycin (mTOR) complex to inhibit the pathway of T cell activation. Due to the narrow therapeutic window and the large intra-individual and inter-individual variation, these immunosuppressive agents are clinically required for Therapeutic Drug Monitoring (TDM), so that more complete therapeutic drug information can be obtained, and the individual drug administration scheme can be conveniently adjusted.

The commonly used analytical methods for monitoring immunosuppressant therapeutic drugs are: spectrophotometry, liquid chromatography tandem mass spectrometry (LC-MS/MS), gas chromatography tandem mass spectrometry (GC-MS/MS), immunization (radioimmunoassay, homogeneous enzyme immunoassay, fluorescence polarization immunoassay, microparticle enzyme immunoassay, chemiluminescence immunoassay), capillary electrophoresis, atomic absorption, etc. The monitoring method commonly used in clinic is a chromatography and an immunoassay, compared with an LC-MS/MS method, the immunoassay has the characteristics of short analysis period, high automation degree, simple operation, suitability for emergency treatment, determination of a large number of samples and the like, but due to the similarity of SIR and EVR structures, mutual interference can be caused when the immunoassay is adopted for detection; it has been reported that the immunoassay for TAC and CsA overestimates by about 30%. The LC-MS/MS method for detecting the immunosuppressant has higher specificity and accuracy, and can accurately guide clinical medication.

CsA, TAC, SIR and EVR are widely present in erythrocytes in human bodies, the distribution ratio of drugs between erythrocytes and blood plasma exceeds 30:1, and EDTA anticoagulated whole blood is often used as a clinical detection sample. The accurate measurement of the immunosuppressant needs a stable quality control substance to calibrate and monitor the state of an instrument and the quality of a reagent, but at present, no patent related report information of a preparation method of the immunosuppressant quality control substance exists in China.

In the 'mass spectrum kit and liquid chromatography tandem mass spectrum detection method for accurately determining the concentrations of four immunosuppressant drugs in human whole blood' patent of the domestic Meikang biological application, quality control products 1 and 2 are included, but the patent does not relate to the information of the preparation method of the quality control products.

In clinical detection of immunosuppressants (CsA, TAC, SIR, EVR, and the like, but not limited to these 4 types, and also glucocorticoid immunosuppressants such as hydrocortisone, cortisone, prednisolone, prednisone, mycophenolate mofetil, and the like), because the detection sample is EDTA anticoagulated whole blood, if the matrix of the quality control substance is the same as or similar to the sample to be detected, the accuracy and stability of the experimental method can be better monitored. Although human whole blood can be used directly as a quality control substance for clinical monitoring of immunosuppressants, since whole blood is a liquid, a cryopreservation device is lacking in clinical laboratories, which makes it unsuitable for long-term storage.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the whole blood lyophilized powder immunosuppressant quality control substance which has good uniformity and stability, does not need low-temperature storage at-70 ℃, can be used as a calibrator of immunosuppressant (CsA, TAC, SIR, EVR and the like) kits of different methodologies, and can also be used as a quality control substance for clinically monitoring the accuracy and stability of a immunosuppressant (CsA, TAC, SIR, EVR and the like) detection system.

In order to solve the technical problems, the invention adopts the technical scheme that: a quality control substance of whole blood lyophilized powder immunosuppressant mainly comprises human whole blood as matrix and lyophilized product of immunosuppressant.

Preferably, the immunosuppressant of the present invention includes one or more of tacrolimus, sirolimus, everolimus and cyclosporine a, but is not limited to these 4 substances, and may also include a mixture of one or more of glucocorticoid immunosuppressants such as hydrocortisone, cortisone, prednisolone, prednisone, mycophenolate mofetil and other blood-detected immunosuppressants; any immunosuppressant that can be detected in whole blood can be prepared by this method and is within the scope of this patent.

Preferably, the whole blood lyophilized powder immunosuppressant quality control substance is used for monitoring the accuracy and stability of a detection system, and generally comprises a low-concentration quality control substance or a high-concentration quality control substance, wherein the concentration range of tacrolimus in the low-concentration quality control substance is controlled to be 0-20 ng/mL, the concentration range of sirolimus and everolimus is controlled to be 0-15 ng/mL, and the concentration range of cyclosporine A is controlled to be 0-300 ng/mL; the concentration range of tacrolimus in the high-concentration quality control product is controlled to be 21-50 ng/mL, the concentration range of sirolimus and everolimus is controlled to be 16-50 ng/mL, and the concentration range of cyclosporine A is controlled to be 301-1200 ng/mL; the quality control substance preparation concentration of the immunosuppressive agent with low concentration and high concentration can be adjusted according to the actual clinical needs.

Further preferably, when the whole blood lyophilized powder immunosuppressant quality control substance is used for a calibrator of an immunosuppressant detection system, different concentration levels, such as 2-10, can be prepared according to different methodological requirements, the number is not limited in the range, and the specific range is prepared according to different methodological requirements.

Further, the invention also provides a preparation method of the whole blood lyophilized powder immunosuppressant quality control substance, which comprises the following specific preparation steps:

(1) collect clinical fresh EDTA-K without jaundice, chyle and hemolysis₂Or heparin anticoagulated whole blood, taking a small amount of upper plasma for infectious disease screening (HIV, hepatitis B, syphilis and hepatitis C), selecting a whole blood sample with a negative result for blood type detection, and mixing whole blood with the same blood type for later use; mixed whole blood of the same blood type is used as a substrate for preparing the quality control substance of the immunosuppressant; if the blood type detection is not carried out on the whole blood sample, adding an anticoagulant into the whole blood after mixing different human parts;

(2) uniformly mixing the whole blood obtained in the step (1), adding a dispersing agent and a solubilizer, and stirring until the whole blood is completely dissolved;

(3) Measuring the concentration of the immunosuppressant in the whole blood substrate obtained in the step (2); adding a proper amount of standard stock solution of the immunosuppressant according to the concentration requirement of the quality control substance on the immunosuppressant; before adding whole blood, blow-drying the standard stock solution of the immunosuppressant by nitrogen, and stirring for 1-6 h to completely dissolve the added immunosuppressant;

(4) adding a protective agent, an excipient and a preservative into the whole blood in the step (3), and stirring until the whole blood is completely dissolved;

(5) and (4) subpackaging the whole blood in the step (4) according to the specification requirement, and freeze-drying to prepare the lyophilized powder.

When in use, the freeze-dried powder is re-dissolved.

Preferably, the anticoagulant in step (1) of the present invention may be EDTA-K₂Heparin, etc., in a concentration range of 0 to 20mmol/L (final concentration in whole blood).

Preferably, the dispersant in step (2) of the present invention may be a surfactant, such as PVP40, PVP360, PEG, tween 20-80, etc., to ensure that the formed components in the whole blood are uniformly dispersed, and the concentration range is 0-40% (mass percentage content); the solubilizer can be polyhydric liquid alcohols, such as ethylene glycol, tertiary butanol and the like, can be used for promoting the dissolution of the added immunosuppressant in the whole blood matrix, and has a concentration range of 0-5% (mass percentage content).

Preferably, the immunosuppressant in the step (3) of the present invention may be one or more of cyclosporin a, tacrolimus, sirolimus and everolimus, but is not limited to these 4 substances, and may also include one or more of glucocorticoid immunosuppressants such as blood-test immunosuppressants such as hydrocortisone, cortisone, prednisolone, prednisone and mycophenolate mofetil; any immunosuppressant that can be detected in whole blood can be prepared by this method and is within the scope of this patent.

Further preferably, the solubility of the cyclosporine A in the step (3) is low, the cyclosporine A is added into the whole blood in a mode of dropwise adding a cyclosporine A standard stock solution, and the mixture is stirred for 1-6 hours to completely dissolve the added immunosuppressant.

Preferably, the protective agent in step (4) of the present invention includes, but is not limited to, antioxidant protective agent, stabilizer, etc.; the antioxidant protective agent can be a substance with reducibility, such as ascorbic acid and salts thereof, sodium sulfite, sodium metabisulfite, cysteine, glutathione and the like, with the concentration range of 0-10% (mass percentage concentration); the stabilizer is mainly protein, saccharide, polyalcohol or amino acids, and can be one or more of Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), trehalose, glucose, sucrose, etc., with concentration of 0-10%.

Preferably, the preservative in step (4) of the present invention is one or more of dehydroacetic acid and salts thereof, calcium propionate, sodium lactate, benzoic acid and salts thereof, sodium azide, sorbic acid and salts thereof, and the like.

Preferably, the excipient in step (4) of the present invention includes, but is not limited to, one or more combinations of polyols (such as mannitol, inositol, etc.), sugars (such as glucose, lactose, etc.), amino acids (such as cysteine), proteins (BSA, HSA, etc.), etc., at a concentration ranging from 0 to 10%. Excipients may also sometimes act as stabilizers.

Preferably, the quality control substance comprises a low-concentration quality control substance and/or a high-concentration quality control substance; the concentration range of tacrolimus in the low-concentration quality control product can be controlled to be 0-20 ng/mL, the concentration ranges of sirolimus and everolimus can be controlled to be 0-15 ng/mL, and the concentration range of cyclosporine A can be controlled to be 0-300 ng/mL; the concentration range of the tacrolimus in the high-concentration quality control product can be controlled to be 21-50 ng/mL, the concentration range of the sirolimus and the everolimus can be controlled to be 16-50 ng/mL, and the concentration range of the cyclosporine A can be controlled to be 301-1200 ng/mL.

The quality control products of the low-concentration and high-concentration immunosuppressants are not distinguished by clear concentration limits, and the preparation concentration can be adjusted according to the actual clinical needs.

Preferably, the quality control materials of the present invention also include immunosuppressant calibrators at various concentration levels, and the concentration levels and specific ranges can be prepared according to the requirements of various methodologies.

The concentrations of the various substances mentioned above in the present invention are the concentrations thereof in the substances obtained after addition to the respective substances. The concentration ranges of various substances in the invention are understood as mass percentage contents without unit limitation and special description.

The invention can obtain the quality control substance of the whole blood lyophilized powder immunosuppressant, can be suitable for the calibrator of immunosuppressant kits of different methodologies, and can also be used as the quality control substance for monitoring the accuracy and stability of immunoassay reagent detection systems of different methodologies in clinic.

The freeze drying conditions in step (5) of the present invention are: pre-freezing for 2-8 hours at-45 to-55 ℃, slowly heating to-40 ℃, stabilizing for 1-3 hours, and then cooling to-55 ℃ and stabilizing for 2-4 hours to completely freeze; and in the freeze drying stage, the temperature is gradually increased at the temperature increasing rate of 1-3 ℃, the temperature is gradually increased until a certain temperature is balanced, the temperature is stabilized for 0.5-3 hours, the temperature is gradually increased until the temperature reaches-35 ℃ and then is kept for 12-25 hours, the temperature increasing rate in the analysis drying stage is 2-5 ℃ per hour, the highest temperature cannot exceed 15 ℃, and the temperature is stabilized for 4-15 hours after being increased to 15 ℃.

The invention has the advantages and beneficial effects that:

1. the invention takes whole blood as a substrate for the first time, and combines with the immunosuppressant to prepare the whole blood lyophilized powder immunosuppressant quality control substance in the form of lyophilized powder; the quality control substance has good uniformity and stability, does not need to be stored at the low temperature of-70 ℃, can be used as a calibrator of an immunosuppressant (CsA, TAC, SIR, EVR and the like) kit, and can also be used as a quality control substance of a whole blood lyophilized powder immunosuppressant quality control substance for clinically monitoring the accuracy and stability of a immunosuppressant (CsA, TAC, SIR, EVR and the like) detection system.

2. The invention provides a preparation method of a complete whole blood freeze-dried powder immunosuppressive agent quality control substance, which comprises the following steps: in the clinical detection of immunosuppressants (CsA, TAC, SIR, EVR and the like), because a detection sample is EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood, if a matrix of a quality control substance is the same as or similar to that of a sample to be detected, the accuracy and the stability of an experimental method can be better monitored, although the whole blood of a user can be directly used as the quality control substance for clinical monitoring of the immunosuppressants, the whole blood is liquid, and a clinical laboratory lacks an ultralow temperature storage device, so that the immunosuppressants are not suitable for long-term storage; therefore, in order to overcome the defects, the whole blood is used as a substrate for the first time and is combined with the immunosuppressant to prepare a product with the property of freeze-dried powder, and the defects that the quality control substance of fresh whole blood and/or frozen whole blood immunosuppressant is unstable and is not easy to store for a long time are overcome; the quality control substance of the immunosuppressive agent prepared by the invention is biased within +/-15% compared with a target value after 7 days of accelerated test at 37 ℃, and the quality control substance is stable in quality and can be stored for a long time (the quality control substance can be stored for 1 year at 2-8 ℃).

3. Compared with the prior art, the prior art does not relate to a specific preparation method, and the quality control materials I and II mentioned in the prior art are mainly characterized by comprising quality control material objects; the patent focuses on how to obtain a preparation method of an immunosuppressant quality control substance, and the two methods are different in focus; the concentration ranges of the quality control substance I and the quality control substance II in the kit in the prior art are set in consideration of the reference interval of the clinical immunosuppressant, and only two concentration levels are set; in this patent, "quality control substance of immunosuppressant" if used as a quality control substance, the concentration range can be set according to the clinical reference interval, because the reference interval is fixed, the two set ranges of the quality control substance overlap, because the clinical reference interval and abnormal value, generally prepare two levels, but this patent is not limited to two concentration levels, the concentration level can be multiple; when the quality control substance is used as a calibrator to calibrate a detection system, the quality control substance can be prepared into a plurality of concentration levels, 2-10 according to needs (the concentration levels are slightly different according to the requirements of different methodology kits).

Clinical reference interval:

tacrolimus: the whole blood grain concentration is 5-20 ng/mL, and 20ng/mL has poisoning risk

Sirolimus: the whole blood grain concentration is 5-15 ng/mL, and the concentration is more than 15ng/mL, so that the whole blood grain has poisoning risk

Everolimus: the whole blood grain concentration is 5-15 ng/mL, and the concentration is more than 15ng/mL, so that the whole blood grain has poisoning risk

Cyclosporin A: the whole blood has a trough concentration of 100-300 ng/mL, and a poisoning risk when the whole blood is more than 300 ng/mL; the peak concentration of whole blood (blood taken 2 hours after administration) is 500-1200 ng/mL

In clinic, the immunosuppressant can generate a treatment effect only when reaching a certain concentration, and a sample with a concentration lower than the valley concentration exists in clinic, so the concentration range of the immunosuppressant tacrolimus in the quality control substance is set to be 0-20 ng/mL, the concentration ranges of the sirolimus and the everolimus are set to be 0-15 ng/mL, and the concentration of the cyclosporine A can be set to be 0-300 ng/mL; the concentration ranges of the tacrolimus, the sirolimus and the everolimus in the high-concentration quality control product are controlled to be 20-50 ng/mL, and the concentration range of the cyclosporine A is controlled to be 300-1200 ng/mL.

In addition, the prior art differs from the present invention in the scope of application: the immunosuppressant quality control substance in the application can be suitable for a mass spectrometry kit, and can also be suitable for other methodology kits, not only the mass spectrometry kit; whereas the prior art contains only the quality control substances I and II in the mass spectrometric kit. In addition, the present invention includes a range, a variety of immunosuppressants different from the prior art: in the present application, 4 clinically commonly used immunosuppressive agent quality control substances are taken as examples, but the present invention is not limited to the 4 substances, and all immunosuppressive agents capable of being detected in whole blood can be prepared by the method, and the method also falls into the scope of the present invention, and the scope is wider. The prior art only aims at the 4 kinds of immunosuppressants, and does not include other kinds of immunosuppressants.

Detailed Description

The invention is further illustrated by the following specific examples, but is not limited to the following examples:

the freeze drying condition control of the invention comprises the following steps: the conditions of freeze drying are that the mixture is pre-frozen for 2 to 8 hours at the temperature of minus 45 ℃ to minus 55 ℃, the mixture is slowly heated to minus 40 ℃ and then stabilized for 1 to 3 hours, and then the mixture is cooled to minus 55 ℃ and stabilized for 2 to 4 hours to be completely frozen; and in the freeze drying stage, the temperature is gradually increased at the temperature increasing rate of 1-3 ℃, the temperature is gradually increased until a certain temperature is balanced, the temperature is stabilized for 0.5-3 hours, the temperature is gradually increased until the temperature reaches-35 ℃ and then is kept for 12-25 hours, the temperature increasing rate in the analysis drying stage is 2-5 ℃ per hour, the highest temperature cannot exceed 15 ℃, and the temperature is stabilized for 4-15 hours after being increased to 15 ℃.

Example 1

Collecting whole blood without jaundice, chyle and hemolysis, selecting a whole blood sample with an infectious disease screening result of negative (HIV, hepatitis B, syphilis and hepatitis C) for blood type detection, selecting 100mL of A-type blood, and uniformly mixing for quality control product preparation; adding 5g of PVP40, and stirring until the PVP is completely dissolved; 0.5g of t-butanol was added thereto, and the mixture was stirred until it was completely dissolved.

Through detection, the whole blood does not contain cyclosporin A, tacrolimus, sirolimus and everolimus, the prepared quality control product contains cyclosporin A35 ng/mL, tacrolimus, sirolimus and everolimus 6ng/mL, and a proper amount of standard stock solution needs to be added.

Calculating the volume of the standard storage solution to be added according to the concentration of the standard storage solution of tacrolimus, sirolimus and everolimus, the volume of the whole blood and the concentration requirement of a quality control product, transferring the standard storage solution into a clean beaker by using a pipette, drying the clean beaker by using nitrogen, adding the treated whole blood, transferring a proper amount of the cyclosporine A standard storage solution, dropwise adding the cyclosporine A standard storage solution into the whole blood while stirring; then sequentially adding 2g of glutathione, 4g of bovine serum albumin and 3g of trehalose, stirring for 4h until the glutathione, the bovine serum albumin and the trehalose are completely dissolved, subpackaging according to the specification of 0.5 mL/bottle, pre-freezing for 4h at the temperature of minus 45 ℃, raising the temperature to minus 40 ℃ at the temperature rise rate of 5 ℃/h, stabilizing for 2 h, and then cooling to minus 50 ℃ for freezing for 2 h; in the freeze drying stage, the temperature is raised to-35 ℃ at the temperature rise rate of 2 ℃/h, and the temperature is stabilized for 18 hours; and then heating to 10 ℃ at the heating rate of 3 ℃/h for resolution drying, stabilizing for 8 hours after heating to 10 ℃, finishing freeze-drying, and taking out through a vacuum gland to obtain freeze-dried powder.

Example 2

Collecting whole blood without jaundice, chyle and hemolysis, selecting whole blood sample with negative infectious disease screening result (HIV, hepatitis B, syphilis and hepatitis C), mixing, adding 20mg EDTA-K₂Stirring until the mixture is completely dissolved, adding 10g of PVP40, and stirring until the mixture is completely dissolved; 2.5g of t-butanol was added thereto, and the mixture was stirred until it was completely dissolved.

Through detection, the whole blood does not contain cyclosporin A, tacrolimus, sirolimus and everolimus, the prepared quality control product contains cyclosporin A50 ng/mL, tacrolimus, sirolimus and everolimus 10ng/mL, and a proper amount of standard stock solution needs to be added.

Calculating the volume of the standard storage solution to be added according to the concentration of the standard storage solution of tacrolimus, sirolimus and everolimus, the volume of the whole blood and the concentration requirement of a quality control product, transferring the standard storage solution into a clean beaker by using a pipette, drying the clean beaker by using nitrogen, adding the treated whole blood, transferring a proper amount of the cyclosporine A standard storage solution, dropwise adding the cyclosporine A standard storage solution into the whole blood while stirring; then sequentially adding 5.0g of sodium metabisulfite, 2.5g of human serum albumin and 5.0g of mannitol, stirring for 6h until the sodium metabisulfite, sub-packaging according to the specification of 1.0 mL/bottle, pre-freezing at-55 ℃ for 3 h, heating to-40 ℃ at the heating rate of 4 ℃/h, stabilizing for 1 h, and then cooling to-55 ℃ for freezing for 4 h; in the freeze drying stage, the temperature is raised to-35 ℃ at the temperature rise rate of 4 ℃/h, and the temperature is stabilized for 20 hours; and then heating to 10 ℃ at the heating rate of 3 ℃/h for resolution drying, stabilizing for 12 hours after heating to 15 ℃, finishing freeze-drying, and taking out through a vacuum gland to obtain freeze-dried powder.

Example 3

Collecting whole blood without jaundice, chyle and hemolysis, selecting a whole blood sample with negative infectious disease screening result (HIV, hepatitis B, syphilis and hepatitis C), mixing, adding 113mg heparin, stirring to completely dissolve, adding 10g PVP40, stirring to completely dissolve; 2.5g of t-butanol was added thereto, and the mixture was stirred until it was completely dissolved.

Through detection, the whole blood does not contain cyclosporin A, tacrolimus, sirolimus and everolimus, the prepared quality control product contains cyclosporin A100 ng/mL, tacrolimus, sirolimus and everolimus 20ng/mL, and a proper amount of standard stock solution needs to be added.

Calculating the volume of the standard storage solution to be added according to the concentration of the standard storage solution of tacrolimus, sirolimus and everolimus, the volume of the whole blood and the concentration requirement of a quality control product, transferring the standard storage solution into a clean beaker by using a pipette, drying the clean beaker by using nitrogen, adding the treated whole blood, transferring a proper amount of the cyclosporine A standard storage solution, dropwise adding the cyclosporine A standard storage solution into the whole blood while stirring; then adding 3.5g of cysteine, 5.0g of bovine serum albumin and 5.0g of glucose in sequence, stirring for 6h until the cysteine, the bovine serum albumin and the glucose are completely dissolved, subpackaging according to the specification of 5.0 mL/bottle, pre-freezing at-50 ℃ for 5 h, heating to-40 ℃ at the heating rate of 5 ℃/h, stabilizing for 3 h, and then cooling to-50 ℃ for freezing for 3 h; in the freeze drying stage, the temperature is raised to-35 ℃ at the temperature rise rate of 2 ℃/h, and the temperature is stabilized for 25 hours; and then heating to 10 ℃ at the heating rate of 4 ℃/h for desorption drying, stabilizing for 15 hours after heating to 15 ℃, finishing freeze-drying, and taking out through a vacuum gland to obtain freeze-dried powder.

Quality control Material uniformity examination of immunosuppressants (Cyclosporin A, Tacrolimus, sirolimus, Everolimus)

Taking lyophilized powder of immunosuppressant (cyclosporin A, tacrolimus, sirolimus, everolimus) quality control substances, randomly extracting 10 bottles, accurately adding 1.0mL of purified water, detecting by liquid chromatography-tandem mass spectrometry, and calculating the concentrations, mean values, SD and the like of cyclosporin A, tacrolimus, sirolimus and everolimus, wherein the results are shown in Table 1.

TABLE 1 immunosuppressant Freeze-drying quality control substance homogeneity investigation (Unit: ng/mL)

The above quality control substances were mixed in 3 bottles at will, continuously measured 10 times, and the mean values and SD of the measurement results of cyclosporin a, tacrolimus, sirolimus, and everolimus were calculated, and the results are shown in table 2.

TABLE 2 measurement results (unit: ng/mL) of immunosuppressant quality control substance after 3 bottles are mixed well

Calculate% CV for repeatability between bottles according to the following equation:

when S is₁<S₂Let CV be_{Bottle room}＝0

In the formula:is mean difference

S is the standard deviation

n is the number of measurements

X_iThe ith measurement for a given parameter

Through investigation, the quality control substances of the immunosuppressant prepared by the method, namely cyclosporine A, tacrolimus, sirolimus and everolimus, are subjected to the inter-bottle S treatment _{Bottle room}1.552, 0.209, 0.236, 0.253, respectively, corresponding to repetitive CV between bottles_{Bottle room}4.8%, 4.0%, 4.1%, 4.4%, less than 10% respectively.

Quality control substance for immunosuppressant (cyclosporin A, tacrolimus, sirolimus, everolimus) accelerates stability test

Taking 6 bottles of freeze-dried powder, putting the 6 bottles of freeze-dried powder in a water bath at 37 ℃, taking 2 bottles of freeze-dried powder after 3 days, 5 days and 7 days respectively, redissolving, detecting cyclosporine A, tacrolimus, sirolimus and everolimus for 2 times in each bottle, comparing the result with the average values in the table 1, and detailing the result in the table 3.

TABLE 3 immunosuppressive quality control substance 37 ℃ accelerated stability test results (in ng/mL)

As can be seen from the data in Table 3, the mass control substance of the immunosuppressive agent prepared by the invention is biased within +/-15% compared with the target value after 7 days of accelerated examination at 37 ℃, which indicates that the mass control substance of the immunosuppressive agent is stable in quality and can be stored for a long time (the mass control substance can be stored for 1 year at 2-8 ℃).

The starting materials used in the examples are, unless otherwise indicated, commercially available products.

The foregoing is only a preferred embodiment of the invention. It should be noted that, under the core technology of the present invention, improved optimization can be made, and these improved optimization shall also belong to the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.