Porous cation exchange polymer microspheres and preparation method and application thereof

Abstract

The present application relates to a cation exchange polymer porous microsphere and its preparation method and application. The cation exchange polymer porous microsphere comprises: a vinyl monomer-polyvinyl crosslinker copolymer mother ball, the vinyl monomer-polyvinyl crosslinker copolymer mother ball is modified with hydroxyl groups; and a cation exchange active group grafted to the vinyl monomer-polyvinyl crosslinker copolymer mother ball via the hydroxyl groups. The above-mentioned cation exchange polymer microsphere is a porous microsphere, on this basis, a vinyl monomer-polyvinyl crosslinker copolymer mother ball modified with hydroxyl groups is combined, and the cation exchange active group is grafted to the hydroxyl group, which can achieve a good separation effect on hemoglobin substances, and can also effectively increase the loading capacity of these substances, thereby realizing the preparation of hemoglobin substances.

Description

Technical Field

[0001] The present application relates to the technical field of polymer microspheres, and in particular to a cation exchange polymer porous microsphere and a preparation method and application thereof. Background Art

[0002] Hemoglobin in adult blood contains hemoglobin A (HbA or HbA0) as the main component, hemoglobin F (HbF) and hemoglobin A2 (HbA2) as minor components, etc. In normal people, the level of HbA2 is between 1.4% and 3.2%. However, in certain diseases, such as β-thalassemia, the level of HbA2 is elevated, usually exceeding 3.5%. Therefore, measuring the elevated HbA2 level is the most practical method for diagnosing β-thalassemia gene carriers. In addition, glycosylated hemoglobin (HbA1c) is also widely used as the gold standard for diagnosing diabetes.

[0003] Methods used by clinical laboratories for quantitative detection of HbA2 include electrophoresis and cation exchange chromatography. High performance liquid chromatography (HPLC) using cation exchange resins is used by laboratories to separate other hemoglobins, but is generally not suitable for quantitative separation of HbA2 and HbA0. This is because the charges of HbA2 and HbA0 are relatively close, and the HbA2 content is low, making it difficult for traditional analytical methods to separate them completely. Therefore, the HbA0 and HbA2 components are usually combined under one peak. At the same time, the HbA0 content is usually much higher than that of HbA2, so it is possible to completely mask any changes in HbA2 levels. There is a method using Bio-Rad DIAMAT TM The 723 fully automatic glycated hemoglobin analyzer system uses cation exchange high performance liquid chromatography to analyze HbA2, using an ID4mm×150mm analytical column. The column is filled with a filler with an average particle size of 6.5 microns (4 to 7 um) and a pore size of Porous, with carboxymethyl functional group cation exchange porous silica microspheres bonded on the surface. However, for protein separation by ion exchange, protein samples are prone to nonspecific adsorption, and it is often necessary to use a strong alkaline solution to regenerate the filler. The filler of the porous silica matrix is ​​not alkali-resistant and is easily decomposed under conditions of pH>10, which is very unfavorable for the regeneration of the filler. The filler has a short life span and high cost for repeated use, which is the fatal disadvantage of the silica matrix filler. There are also methods that use CM-Sepharose FF and Bio-Rex 70 as fillers to separate and purify HbA1c, but the separation performance of the chromatographic columns filled with Bio-Rex 70 and CM-Sepharose FF is not enough, and HbA1c and HbA2 cannot be separated at the same time.

[0004] In addition, the glycosylated hemoglobin analyzer developed by Pumen Technology is a fully automatic glycosylated hemoglobin analyzer system. There is a method that uses this system in combination with a chromatographic column filled with cation exchange chromatography filler prepared by Chinese patent CN113512152A for analysis, which can separate and analyze 13 hemoglobin substances such as HbA1a, HbA1b, LA1c, HbF, HbA1c, P3, P4, HbA0, HbA2, HbE, HbD, HbS, and HbC. However, the cation exchange non-porous microsphere chromatographic filler synthesized by this method has a small particle size, high column pressure, and low loading capacity, making it difficult to achieve large-scale batch chromatography preparation of hemoglobin substances. Summary of the invention

[0005] Based on this, the present application provides a cation exchange polymer porous microsphere and a preparation method and application thereof. The cation exchange polymer porous microsphere can be used as a chromatography filler to effectively separate and batch prepare hemoglobin substances.

[0006] In a first aspect of the present application, a cation exchange polymer porous microsphere is provided, comprising:

[0007] A vinyl monomer-polyvinyl crosslinking agent copolymer mother ball, wherein the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball is modified with a hydroxyl group;

[0008] The cation exchange active groups of the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball are grafted via the hydroxyl group.

[0009] The above-mentioned cation exchange polymer porous microspheres can achieve a good separation effect on hemoglobin-like substances, and can also effectively increase the loading capacity of these substances, thereby realizing the preparation of hemoglobin-like substances.

[0010] The second aspect of the present application provides a method for preparing the cation exchange polymer porous microspheres described in the first aspect, comprising the following steps:

[0011] preparing the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball;

[0012] The cation exchange active groups are grafted onto the vinyl monomer-polyvinyl crosslinker copolymer mother ball through the hydroxyl reaction.

[0013] The third aspect of the present application provides the use of the cation exchange polymer porous microspheres described in the first aspect in chromatographic fillers.

[0014] The fourth aspect of the present application provides the use of the cation exchange polymer porous microspheres described in the first aspect in the preparation of biological components; optionally, the biological component is a hemoglobin biological component; further optionally, the biological component is one or more of HbA2, HbF, HbA1c and HbA0.

[0015] In a fifth aspect of the present application, a method for preparing a hemoglobin biological component is provided, comprising the following steps:

[0016] Obtain the original red blood cell lysate;

[0017] Performing chromatography on the original red blood cell lysate, collecting the eluates of each hemoglobin biological component respectively, and preparing the hemoglobin biological component;

[0018] The filler of the chromatography column used in the chromatography treatment includes the cation exchange polymer porous microspheres described in the first aspect.

[0019] The sixth aspect of the present application provides a quality control material, comprising one or more of the hemoglobin biological components prepared by the preparation method described in the fifth aspect, and a red blood cell stock solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a scanning electron microscope (SEM) image of the PST seed particles prepared in Example 1-1;

[0021] Figure 2 This is a test graph of the particle size distribution of the PST seed particles prepared in Example 1-1;

[0022] Figure 3 is a scanning electron microscope (SEM) image of the porous microspheres P(GMA-DVB) prepared in Example 1-1;

[0023] Figure 4 This is a test graph of the particle size distribution of the porous microspheres P(GMA-DVB) prepared in Example 1-1;

[0024] Figure 5 The test spectrum of the standard protein performance test method for the cation exchange polymer porous microspheres prepared in Examples 1-6;

[0025] Figure 6 The test spectrum of the standard protein performance test method for the cation exchange polymer porous microspheres prepared in Examples 1-7;

[0026] Figure 7 The test spectrum of the standard protein performance test method for the cation exchange polymer porous microspheres prepared in Examples 1-8;

[0027] Figure 8This is a chromatogram of the separation process of preparing hemoglobin biological components in Example 2-1;

[0028] Fig. 9 This is an overlay of the thalassemia sample and the purified sample for preparing the hemoglobin biological component in Example 2-1;

[0029] Fig.10 This is a chromatogram of the separation process of preparing hemoglobin biological components in Example 2-2;

[0030] Fig.11 The chromatogram is a chromatogram of the separation process of preparing hemoglobin biological components in Example 2-3;

[0031] Fig.12 The H100Plus high performance liquid chromatograph thalassemia pattern spectrum of the HbA1c high value quality control material in Example 3-1;

[0032] Fig.13 The H100Plus high performance liquid chromatograph thalassemia mode spectrum of the glycated hemoglobin low value HbA1c quality control material in Example 3-2;

[0033] Fig.14 The H100Plus high performance liquid chromatograph thalassemia pattern spectrum of the HbA2 quality control material for β-thalassemia screening in Example 3-3;

[0034] Fig.15 The thalassemia pattern spectrum of the H100Plus high performance liquid chromatograph of the quality control material HbF for β-thalassemia screening in Example 3-4;

[0035] Fig.16 The thalassemia pattern spectrum of the H100Plus high performance liquid chromatograph of the high level quality control substance IV for β-thalassemia screening in Example 3-5;

[0036] Fig.17 The thalassemia pattern spectrum of the H100Plus high performance liquid chromatograph of the low level quality control substance IV for β-thalassemia screening in Example 3-5;

[0037] Fig.18 The H100Plus high performance liquid chromatograph thalassemia pattern spectrum of the quality control material V of low concentration HbA1c, high concentration HbA2 and HbF in Example 3-6;

[0038] Fig.19 This is the H100Plus high performance liquid chromatograph thalassemia pattern spectrum of the high concentration HbA1c, low concentration HbA2 and HbF quality control materials in Examples 3-6. DETAILED DESCRIPTION

[0039] The following is a further detailed description of the cation exchange polymer porous microspheres of the present application and their preparation methods and applications in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0041] The optional scope of the terms "and / or", "or / and", and "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0042] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0043] In this application, "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0045] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0046] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0047] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0048] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0049] The room temperature in the present application generally refers to 4°C to 30°C, preferably 20±5°C.

[0050] The cation exchange chromatography filler provided by the traditional method is difficult to separate hemoglobin substances such as HbA1c, HbA2, and HbA0 at the same time, and it is even more impossible to achieve the preparation of these hemoglobin substances. Although the cation exchange chromatography filler prepared by the Chinese patent CN113512152A can realize the separation and analysis of a variety of hemoglobin substances, it is also difficult to achieve the batch chromatographic preparation of hemoglobin substances. The reason is that: its cation exchange chromatography filler is a non-porous filler with an average particle size D50 of 2μm to 10μm, resulting in high pressure in the chromatographic column, and the pressure requirements for the preparative chromatographic instrument are very high, and the non-porous filler has a small specific surface area, resulting in a low protein loading capacity of the filler, and the sample loading amount is limited when preparing the protein. However, if the non-porous mother core is directly replaced with the porous mother core, the porous microspheres with a small particle size of less than 10um have low pressure resistance compared with the non-porous mother core. Therefore, the current method is difficult to take into account the good separation effect and high loading capacity for hemoglobin substances to achieve the preparation of these hemoglobin substances.

[0051] Based on this, the present invention provides a cation exchange polymer porous microsphere, comprising:

[0052] A vinyl monomer-polyvinyl crosslinking agent copolymer mother ball, wherein the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball is modified with a hydroxyl group;

[0053] The cation exchange active groups of the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball are grafted via the hydroxyl group.

[0054] The above-mentioned cation exchange polymer microspheres are porous microspheres. On this basis, vinyl monomer-polyvinyl crosslinker copolymer mother balls modified with hydroxyl groups are combined, and the cation exchange active groups are grafted to the hydroxyl groups. While achieving a good separation effect on hemoglobin-like substances, it can also effectively increase the loading capacity of these substances, thereby realizing the preparation of hemoglobin-like substances.

[0055] In some examples, the average pore size of the cation exchange polymer porous microspheres is Specifically, the average pore size of the cation exchange polymer porous microspheres includes but is not limited to: Furthermore, the average pore size is

[0056] In some examples, the specific surface area of ​​the cation exchange polymer porous microspheres is 10 m 2 / g~1000m 2 / g. Specifically, the average pore size of the cation exchange polymer porous microspheres includes but is not limited to: 10m 2 / g, 50m 2 / g、100m 2 / g, 150m 2 / g, 200m 2 / g, 250m 2 / g、300m 2 / g, 350m 2 / g, 400m 2 / g, 450m 2 / g、500m 2 / g, 550m 2 / g、600m 2 / g, 650m 2 / g、700m 2 / g, 750m 2 / g、800m 2 / g, 850m 2 / g、900m 2 / g, 950m 2 / g、1000m 2 / g. Furthermore, the specific surface area is 100m 2 / g~800m 2 / g.

[0057] In some examples, the average particle size D50 of the cation exchange polymer porous microspheres is 11 μm to 60 μm. Specifically, the average particle size D50 of the cation exchange polymer porous microspheres includes but is not limited to: 11 μm, 12 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm. Further, the average particle size D50 of the cation exchange polymer porous microspheres is 11 μm to 30 μm.

[0058] Without limitation, the cation exchange active group may be a conventional group having cation exchange activity. In some examples, the cation exchange active group is one or both of a carboxyl functional group and a sulfonic acid functional group.

[0059] In some examples, the vinyl monomer-polyvinyl crosslinker copolymer mother ball includes:

[0060] Vinyl monomer-polyvinyl crosslinker copolymer porous particles, wherein the vinyl monomer-polyvinyl crosslinker copolymer porous particles are modified with epoxy groups; the vinyl monomer-polyvinyl crosslinker copolymer porous particles are formed by crosslinking polystyrene seed microspheres and vinyl monomers with a polyvinyl crosslinker in the presence of a porogen; the vinyl monomer contains an epoxy group and / or the epoxy group is hydrolyzed and then grafted with a compound containing an epoxy group to obtain the epoxy group;

[0061] The hydroxy compound is grafted to the porous particles of the vinyl monomer-multi-vinyl crosslinking agent copolymer via the epoxy group, and the hydroxy compound comprises the hydroxyl group.

[0062] In some examples, the crosslinking degree of the vinyl monomer-polyvinyl crosslinker copolymer mother ball is 20% to 80%. Specifically, the crosslinking degree of the vinyl monomer-polyvinyl crosslinker copolymer mother ball includes but is not limited to: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%. Further, the crosslinking degree of the vinyl monomer-polyvinyl crosslinker copolymer mother ball is 40% to 70%.

[0063] In some examples, the vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether, 4-vinylbenzyl glycidyl ether and methacrylic acid glycidyl ether.

[0064] In some examples, the epoxy group-containing compound is selected from at least one of epichlorohydrin, glycidyl ether and ethylene glycol diglycidyl ether.

[0065] In some examples, the average particle size D50 of the polystyrene seed microspheres is 1 μm to 10 μm. Specifically, the average particle size D50 of the polystyrene seed microspheres includes but is not limited to: 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm. Further, the average particle size D50 of the polystyrene seed microspheres is 2 μm to 5 μm.

[0066] In some examples, the hydroxy compound is selected from at least one of ethylene glycol, 1,3-propylene glycol, and 1,4-butanediol.

[0067] In some examples, the polyvinyl crosslinking agent is selected from at least one of divinylbenzene, divinyltoluene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trivinylbenzene, divinylnaphthalene and trivinylnaphthalene. Further, the polyvinyl crosslinking agent is divinylbenzene.

[0068] In some examples, the amount of the vinyl monomer is 10% to 80% of the total weight of the vinyl monomer and the polyvinyl crosslinker. Specifically, the amount of the vinyl monomer includes but is not limited to: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% of the total weight of the vinyl monomer and the polyvinyl crosslinker. Further, the amount of the vinyl monomer is 20% to 60% of the total weight of the vinyl monomer and the polyvinyl crosslinker. Furthermore, the amount of the vinyl monomer is 30% to 50% of the total weight of the vinyl monomer and the polyvinyl crosslinker.

[0069] In some of the examples, the porogen is one or more of toluene, xylene, diethylbenzene, dodecylbenzene, hexane, heptane, isopentanol, cyclohexanol, n-hexanol, octanol, diethyl phthalate and n-butyl phthalate. Further, the porogen is one or more of toluene, xylene and n-hexanol.

[0070] In some examples, the amount of the porogen is 50% to 300% of the total weight of the vinyl monomer and the polyvinyl crosslinker. Specifically, the amount of the porogen includes but is not limited to: 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 180%, 200%, 250%, 300% of the total weight of the vinyl monomer and the polyvinyl crosslinker. Further, the amount of the porogen is 80% to 150% of the total weight of the vinyl monomer and the polyvinyl crosslinker.

[0071] Some examples of the present application also provide a method for preparing the cation exchange polymer porous microspheres as described above, comprising the following steps:

[0072] preparing the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball;

[0073] The cation exchange active groups are grafted onto the vinyl monomer-polyvinyl crosslinker copolymer mother ball through the hydroxyl reaction.

[0074] In some examples, the method for preparing the vinyl monomer-polyvinyl crosslinker copolymer mother ball comprises the following steps:

[0075] Preparation of porous particles of vinyl monomer-polyvinyl crosslinker copolymer, wherein the porous particles of the vinyl monomer-polyvinyl crosslinker copolymer are modified with epoxy groups, and the preparation steps include: using polystyrene seed microspheres as seeds, emulsifying together with vinyl monomer and polyvinyl crosslinker in the presence of porogen, stabilizer and emulsifier, and then polymerizing in a medium by seed swelling method; the vinyl monomer contains epoxy groups and / or the epoxy groups are hydrolyzed and then grafted with epoxy-containing compounds to obtain the epoxy groups;

[0076] The hydroxyl compound is grafted onto the porous particles of the vinyl monomer-multi-vinyl crosslinking agent copolymer through the epoxy reaction, and the hydroxyl compound comprises the hydroxyl group.

[0077] In one example, the initiator used in the polymerization is an oily initiator. Further, one of an oil-soluble peroxide and an oil-soluble azo compound. Specifically, the oil-soluble peroxide may be exemplified by dibenzoyl peroxide, dioctanoyl peroxide, lauroyl peroxide, etc.; the oil-soluble azo compound may be exemplified by 2,2'-azobisisobutyronitrile, 2,2'-azobisisocapronitrile, etc.

[0078] In one example, the amount of the initiator used is 0.1% to 10% by weight of the vinyl monomer. Further, the amount of the initiator used is 0.5% to 5% by weight of the vinyl monomer.

[0079] In one example, the stabilizer is a water-soluble polymer. Further, the stabilizer is at least one of polyvinyl alcohol, polyvinyl pyrrolidone and modified cellulose.

[0080] In one example, the amount of the stabilizer is 0.5% to 15% by weight of the vinyl monomer. Further, the amount of the stabilizer is 1% to 8% by weight of the vinyl monomer.

[0081] In one example, the emulsifier refers to a suitable surfactant, such as at least one of a cationic surfactant, a nonionic surfactant, an amphoteric surfactant, and an anionic surfactant.

[0082] Specifically, the cationic surfactant is selected from at least one of quaternary ammonium salts, alkyl pyridinium salts and amine salts; the nonionic surfactant is selected from at least one of alkyl ether polymers and alkylphenol polyalkoxy compounds (such as alkylphenol polyethoxylates); the amphoteric surfactant is selected from at least one of amino acid type amphoteric surfactants (such as dodecylaminopropionic acid) and betaine type amphoteric surfactants (such as sulfobetaine); the anionic surfactant is selected from at least one of carboxyl salt surfactants, sulfonate surfactants, sulfate surfactants and phosphate surfactants.

[0083] In one example, the amount of the emulsifier used is 0.05% to 10% by weight of the vinyl monomer. Further, the amount of the emulsifier used is 0.1% to 5% by weight of the vinyl monomer.

[0084] Without limitation, the emulsification method may be one or more of a high-speed shearing emulsifier, an ultrasonic emulsifier, and a high-pressure average emulsifier.

[0085] Some examples of the present application also provide the use of the cation exchange polymer porous microspheres as described above in chromatography packing materials.

[0086] Some examples of the present application also provide the use of the cation exchange polymer porous microspheres as described above in the preparation of biological components. Further, the biological component is a hemoglobin biological component. Further, the biological component is one or more of HbA2, HbF, HbA1c and HbA0.

[0087] By using the cation exchange polymer porous microspheres as chromatographic fillers as described above, it is possible to purify and prepare hemoglobin-like biological components, especially one or more of HbA2, HbF, HbA1c and HbA0, and use them as quality control materials for diabetes diagnosis, β-thalassemia screening, etc., filling the gap in related quality control materials in China.

[0088] Some examples of the present application also provide a method for preparing a hemoglobin-based biological component, comprising the following steps:

[0089] Obtaining a first red blood cell lysate;

[0090] Performing chromatography on the red blood cell lysate, collecting the eluates of each hemoglobin biological component respectively, and preparing the hemoglobin biological component;

[0091] The filler of the chromatography column used in the chromatography treatment includes the cation exchange polymer porous microspheres as described above.

[0092] The above preparation method can achieve the purification and preparation of hemoglobin biological components, especially one or more of HbA2, HbF, HbA1c and HbA0.

[0093] It is understandable that the first red blood cell lysing solution should be a freshly prepared red blood cell lysing solution. Specifically, the first red blood cell lysing solution can be a fresh human whole blood red blood cell lysing solution, which can be obtained by lysing a fresh whole blood sample with physiological saline according to a traditional method.

[0094] In one example, the hemoglobin biological component is one or more of HbA2, HbF, HbA1c and HbA0. It is understandable that, according to actual needs, one of HbA2, HbF, HbA1c and HbA0 can be separated and prepared by the above preparation method, or two or more of HbA2, HbF, HbA1c and HbA0 can be separated and prepared simultaneously.

[0095] Further, the purity of each of the hemoglobin-like biological components prepared is ≥70%. Further, the purity of each of the hemoglobin-like biological components prepared is ≥80%. Further, the purity of each of the hemoglobin-like biological components prepared is ≥90%. Specifically, the purity of each of the hemoglobin-like biological components prepared includes but is not limited to: 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. It can be understood that the purity of each hemoglobin-like biological component can be detected and analyzed by HPLC chromatograph (such as Agilent 1260II).

[0096] In one example, the mobile phase used in the chromatography treatment includes mobile phase A and mobile phase B; the mobile phase A includes 1mM to 20mM phosphate buffer with a pH of 5.3 to 6; the mobile phase B includes 30mM to 100mM phosphate buffer with a pH of 6 to 7.2.

[0097] Furthermore, the mobile phase A comprises 5 mM to 15 mM phosphate buffer with a pH of 5.6 to 5.8.

[0098] Furthermore, the mobile phase B comprises 70 mM to 90 mM phosphate buffer with a pH of 6.5 to 6.7.

[0099] In one example, the chromatography process uses mobile phase A and mobile phase B sequentially for isocratic elution.

[0100] Further, the mobile phase A is used for isocratic elution until the eluate no longer contains one or more of HbA1a, HbA1b, HbF, LA1c and HbA1c. It can be understood that the elution volume of the mobile phase A and the corresponding length of time for being pumped through the column can be adjusted accordingly, and whether the corresponding components are contained can be detected and analyzed by an HPLC chromatograph (such as Agilent 1260II).

[0101] Further, the mobile phase B is used for isocratic elution until the eluate no longer contains one or more of hemoglobin P3, hemoglobin P4, HbA0 and HbA2. It can be understood that the elution volume of the mobile phase B and the corresponding length of time for being pumped through the column can be adjusted accordingly, and whether the corresponding components are contained can be detected and analyzed by an HPLC chromatograph (such as Agilent 1260II).

[0102] It is understood that the chromatography column can be adjusted according to the amount of pre-preparation, and a slender column for HPLC equipment can be selected. In one example, the inner diameter of the chromatography column used in the chromatography treatment is 10mm to 100mm and the length is 50mm to 1000mm. Further, the length of the chromatography column is 100mm to 500mm. Accordingly, the pressure drop on the chromatography column will vary according to the length, inner diameter, pore size and particle size of the chromatographic filler, and is generally in the range of about 0.1MPa to about 5MPa.

[0103] In one embodiment, the chromatography is performed at room temperature.

[0104] In one example, the flow rate of the mobile phase used in the chromatography treatment is 1 mL / min to 30 mL / min. Further, the flow rate is 2 mL / min to 5 mL / min.

[0105] In one example, the chromatography treatment is performed using a high performance liquid preparative chromatography system. Without limitation, a HPLC medium and high pressure preparative liquid chromatography system with a proportional valve capable of generating an automatic linear / isocratic gradient can be used.

[0106] Thalassemia is a hemoglobin abnormality caused by a hereditary disorder of peptide chain synthesis. According to WHO estimates, about 4.5% (about 340 million) of the world's population carries genes that cause hemoglobin diseases, and at least 300,000 children with severe thalassemia are born each year. So far, hemoglobin diseases have become the top three most common birth defects in the world. The most serious types of thalassemia genes include Hb Bart's fetal edema syndrome, severe β-thalassemia, and hemoglobin HbH disease. Thalassemia screening has now become a routine test item before marriage and pregnancy.

[0107] Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin or the body cannot effectively use the insulin it produces. Hyperglycemia, or elevated blood sugar, is a common consequence of uncontrolled diabetes and can cause serious damage to many systems in the body, especially the nerves and blood vessels, over time. Currently, glycated hemoglobin (HbA1c) is widely used as the gold standard for diagnosing diabetes.

[0108] Therefore, the separation, identification and quantitative analysis of hemoglobin play an important role in the screening and diagnosis of diseases such as thalassemia and diabetes, especially the quantitative analysis of HbA1c, HbF and HbA2. However, at present, the relevant quality control technology has been monopolized by a few brands, including Bio-rad in the United States, Sebia in France and Helena in the United States. In particular, there is very little research and development and production of hemoglobin HbF and HbA2 quality control materials used for β-thalassemia screening.

[0109] Based on this, some examples of the present application also provide a quality control material, including one or more of the hemoglobin biological components prepared by the preparation method described above, and a second red blood cell lysate. Based on the above method, high-purity HbF, HbA1c, HbA2 and HbA0 are purified and prepared, and then HbA1c quality control materials for diabetes diagnosis gold standard and hemoglobin HbF / HbA2 quality control materials for β-thalassemia screening can be prepared, and the quality control materials can be stored for a long time, filling the gap of such quality control materials, and can be widely used in diabetes diagnosis and β-thalassemia screening.

[0110] It is understandable that the second red blood cell lysing solution should be a freshly prepared red blood cell lysing solution. Specifically, the second red blood cell lysing solution can be one or both of a fresh red blood cell lysing solution derived from normal human whole blood and a fresh red blood cell lysing solution derived from newborn whole blood, and can be obtained by lysing a fresh whole blood sample with physiological saline according to a conventional method.

[0111] In one example, the quality control material also includes a lyoprotectant and a preservative.

[0112] Furthermore, the mass percentage of the lyophilization protectant in the quality control material is 5% to 15%. Specifically, the mass percentage of the lyophilization protectant in the quality control material includes but is not limited to: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or a range between any two of the foregoing.

[0113] Furthermore, the lyoprotectant is one or more of trehalose, glucose, sucrose, lactose and mannitol.

[0114] Furthermore, the mass percentage of the preservative in the quality control material is 0.1% to 0.5%. Specifically, the mass percentage of the preservative in the quality control material includes but is not limited to: 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or a range between any two of the foregoing.

[0115] Furthermore, the preservative is one or more of sodium azide, gentamicin, sodium benzoate, potassium sorbate and benzoic acid.

[0116] In one example, the control material is a control material for screening β-thalassemia, and the control material includes control material I and / or control material II; the control material I includes HbA2 prepared by the preparation method described above, and red blood cell lysate derived from normal whole blood; the control material II includes HbF prepared by the preparation method described above or red blood cell lysate derived from newborn whole blood, and red blood cell lysate derived from normal whole blood.

[0117] Furthermore, the mass percentage of HbA2 in the high-level quality control material I is 4% to 10%; the mass percentage of HbA2 in the low-level quality control material I is 2% to 3.5%. It can be understood that "high level" refers to a high concentration level, and "low level" refers to a low concentration level, and the same reason will not be repeated below.

[0118] Furthermore, the mass percentage of HbF in the high-level quality control substance II is 4% to 16%; the mass percentage of HbF in the low-level quality control substance II is 1% to 3%.

[0119] In one example, the quality control material is quality control material III for diagnosing diabetes; the quality control material III includes HbA1c prepared by the preparation method described above, and red blood cell lysate derived from normal whole blood.

[0120] Furthermore, the mass percentage of HbA1c in the high-level quality control substance III is 8.0% to 11.5%; and / or, the mass percentage of HbA1c in the low-level quality control substance III is 4% to 6.5%.

[0121] In one example, the quality control material is quality control material IV for β-thalassemia screening, and the quality control material IV includes HbA2 and HbF prepared by the preparation method described above, and red blood cell lysate derived from normal whole blood.

[0122] Furthermore, in the high-level quality control substance IV, the mass percentage of HbA2 is 4% to 10%, and the mass percentage of HbF is 4% to 16%.

[0123] Furthermore, in the low-level quality control substance IV, the mass percentage of HbA2 is 2% to 3.5%, and the mass percentage of HbF is 1% to 3%.

[0124] In one example, the quality control material is a dual quality control material V for diabetes diagnosis and β-thalassemia screening, and the quality control material V includes HbA1c, HbA2, HbF prepared by the preparation method described above, and red blood cell lysate derived from normal whole blood.

[0125] Furthermore, in the quality control material V of low-concentration HbA1c, high-concentration HbA2 and HbF, the mass percentage of HbA1c is 4% to 6.5%, the mass percentage of HbA2 is 4% to 10%, and the mass percentage of HbF is 4% to 16%.

[0126] Furthermore, in the quality control material V having high concentration of HbA1c, low concentration of HbA2 and HbF, the mass percentage of HbA1c is 8.5% to 11.5%, the mass percentage of HbA2 is 2% to 3.5%, and the mass percentage of HbF is 1% to 3%.

[0127] In addition, without limitation, the above-mentioned quality control material is in the form of freeze-dried powder.

[0128] The following are specific examples.

[0129] The tests performed below do not include components other than inevitable impurities unless otherwise specified. The chemicals and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods without specific conditions in the examples are carried out according to conventional conditions, such as those described in the literature, books, or methods recommended by the manufacturer.

[0130] Unless otherwise specified, in the following examples, the samples to be tested for the detection of glycosylated hemoglobin are all fresh blood samples taken from Peking University Shenzhen Hospital; the samples to be tested for the detection of thalassemia are all fresh blood samples taken from Guangxi Baise People's Hospital. The diluent is deionized water.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0132] Unless otherwise specified, the meanings of the English abbreviations in the following content are as shown in Table 1:

[0133] Table 1

[0134]

[0135] The characterization methods involved in the embodiments are described as follows:

[0136] (1) Characterization of microsphere surface morphology: Scanning electron microscopy (SEM) was used to characterize the surface morphology of microspheres; (2) Determination of microsphere particle size: A commercially available laser particle size analyzer was used to determine the particle size of microspheres;

[0137] (3) Determination of microsphere epoxy value:

[0138] The epoxy value of microspheres was determined by perchloric acid titration method. The specific operation is as follows: 0.05g of microspheres were immersed in 10mL of chloroform and ultrasonically dispersed in an ultrasonic cleaner in a conical flask for 30min. 10mL of tetraethylammonium bromide solution and 2 drops of crystal violet indicator were added to the conical flask. The solution in the conical flask was titrated with V mL of calibrated perchloric acid standard solution (0.1M) until the color of the solution changed from blue to bright green and remained unchanged for 60min. At the same time, a blank control experiment was performed, consuming V0mL of perchloric acid standard solution. The calculation formula of the epoxy value (E) of the microspheres is as follows: E = (V-V0) * 0.1 / 10.

[0139] (4) Microsphere ion exchange capacity (IEC) test:

[0140] Take about 5g of dry filler, weigh it and place it in a 250ml beaker, add about 150mL of 0.2M hydrochloric acid solution, stir and soak overnight. Filter the suspension with filter paper, wash once with 10mL of 0.2M hydrochloric acid solution, then wash with purified water until the pH is neutral, and drain. Wash it three times with about 10mL of ethanol and drain it, pour it into a watch glass, and dry it in a vacuum oven at 80℃ for 2 hours. Weigh mg of the dried filler (accurate to 0.0001) in a 100mL conical flask, accurately measure 80mL (V NaOH )0.05M(C NaOH ) Add standard sodium hydroxide solution to the conical flask, shake evenly, place in a 30℃ water bath for one hour, and drain. Measure about 25mL of 0.5M sodium chloride solution and add it to the funnel to wash the filler three times, and drain. Collect the filtrates from the above two steps, mix them evenly, and accurately measure three portions of 25mL of filtrate and place them in a 150mL conical flask, add 3 drops of phenolphthalein indicator, and use 0.05M (C HCl ) standard hydrochloric acid solution until the solution changes from purple to colorless and does not change color within 0.5 min, which means the end point is reached. Record the volume of 0.05 M standard hydrochloric acid consumed (V HCl ), and calculate the ion exchange capacity (IEC).

[0141] Ion exchange capacity (IEC) = (C NaOH *V NaOH -8*C HCl *V HCl ) / m(mmol / g)

[0142] C NaOH ——0.05M standard sodium hydroxide solution concentration, mol / L; V NaOH ——0.05M standard sodium hydroxide solution volume, 80mL; C HCl ——0.05M standard hydrochloric acid solution concentration, mol / L; V HCl——The volume of 0.05M standard hydrochloric acid solution consumed, mL; m——The mass of the filler after precise weighing, g.

[0143] (5) Standard protein performance test method:

[0144] The cation exchange filler was dispersed evenly with ultrapure water and then loaded into a 4.6*35mm (column volume 0.581mL) chromatographic column using a high-pressure pneumatic pump. The separation performance of standard protein ribonucleic acid A, cytochrome C and lysozyme was tested by high performance liquid chromatography (Agilent 1260 Infinity II HPLC). At a flow rate of 1.0mL / min and an injection volume of 10μL, at room temperature, buffer A: 40mmol, pH5.8 PB (Phosphate Buffer) and buffer B: A phase (i.e., buffer A) + 1M NaCl (pH5.8) were used as eluents for gradient elution. The elution gradient was 0min: 100% (v / v) A, 25min: 30% (v / v) A and 70% (v / v) B, and a UV detector (wavelength 214nm) was used for detection. "0min: 100% (v / v) A, 25min: 30% (v / v) A and 70% (v / v) B" means that from 0min to 25min, the ratio of buffer A and buffer B in the eluent changes from 100% (v / v) A and 0% (v / v) B to 30% (v / v) A and 70% (v / v) B.

[0145] The chromatographic conditions were as follows:

[0146] Test column type: cation exchange filler, column tube: 4.6x 35mm;

[0147] Mobile phase A was 40 mM PB, pH 5.8; mobile phase B was phase A + 1 M NaCl, pH 5.8;

[0148] Flow rate: 1 mL / min, elution gradient: 0-25 min using phase B from 0 (v / v) to 70% (v / v); detector wavelength: 214 nm, column temperature: 25°C; injection volume: 10 μL; test sample: a mixture of ribonuclease A (0.5 mg / mL), cytochrome C (0.5 mg / mL), and lysozyme (0.5 mg / mL);

[0149] The detection equipment was Agilent 1260 Infinity II high performance liquid chromatograph.

[0150] (6) Test method for standard protein dynamic loading:

[0151] The test was conducted using a general liquid chromatography instrument, with a detection wavelength of 214 nm and a flow rate of 1.5 mL / min. The prepared cation exchange filler was filled into a 4.6*35 mm (column volume 0.581 mL) chromatographic column, and the chromatographic column was balanced with a 40 mM PB (pH = 5.8) eluent. The mobile phase in the system was replaced with a 2.5 mg / mL lysozyme solution until the pipeline in front of the chromatographic column was filled with the sample solution. A penetration curve was made with a 2.5 mg / mL lysozyme solution as the mobile phase and a flow rate of 1.5 mL / min until the spectrum line was flat. The time at 10% penetration was used to calculate the DBC (Dynamic binding capacity), that is, the time t corresponding to 10% of the maximum absorbance value was used to calculate the amount of absorbed protein), and finally 40 mM PB + 1 M NaCl (pH = 5.8) eluent was used as the mobile phase to elute the adsorbed protein until the spectrum line was flat and the next test was performed.

[0152] DBC=(v*t*C) / V(mg / mL)

[0153] Wherein, v: sample loading flow rate, 1.5 mL / min; V: chromatographic filler volume, 0.581 mL; t: time corresponding to 10% of the maximum absorbance value, min; C: lysozyme concentration in the loading solution, 2.5 mg / mL.

[0154] (7) Hemoglobin purity test method:

[0155] Test conditions:

[0156] Solution A: 15 mM phosphate buffer, pH 5.6;

[0157] Solution B: 80 mM phosphate buffer, pH 6.5;

[0158] Flow rate: 1.50 mL / min;

[0159] Running gradient: 0-0.58min: 100% A, 0.58-6.58min: 100% D, 6.58-10.0min: 100% A;

[0160] Detection wavelength: 415nm;

[0161] Column temperature: 25°C;

[0162] Cation exchange chromatography column: XT-5um-4.6*35mm (H100Plus high performance liquid chromatography supporting column);

[0163] Instrument: Agilent 1260Bio Infinity II.

[0164] Example 1-1

[0165] This embodiment is a porous polymer microsphere, and the preparation method is as follows:

[0166] (1) Preparation of PST seed particles

[0167] Add 250g of ethanol and 10.0g of PVPK30 to a 500mL beaker. After complete dissolution, add 4.85g of sodium diisooctyl sulfosuccinate and 50mL of water. Stir and dissolve at room temperature to obtain solution 1. Stir and dissolve 1.53g of AIBN and 100g of St (styrene) to obtain solution 2. Pass nitrogen into a 2000mL three-necked flask, add solution 2 to solution 1 and continue stirring and mixing, and add the evenly mixed solution to the reaction bottle. Under the conditions of stirring speed of 250rpm and nitrogen protection, heat to 65℃ and continue to keep warm for 6h. After the reaction is completed, pour out the reaction solution, centrifuge at a speed of 5000rpm, and the centrifugation time is about 10min. After discarding the upper layer of reaction solution, wash with 200mL of ethanol 3 times; continue to wash with 200mL of ultrapure water 6 times, centrifuge at a speed of 5000rpm, and obtain PST seeds. The particle size D50 is 2.95μm tested by scanning electron microscopy (SEM). Figure 1 , Figure 2 shown.

[0168] (2) Preparation of porous microspheres P(GMA-DVB)

[0169] Dissolve 1.8g of sodium dodecylbenzene sulfonate (SDBS) in 600g of pure water, and stir to dissolve completely to obtain mixed phase 1 (water phase). In a 500mL beaker, weigh 18.75g of glycidyl methacrylate, 131.25g of divinylbenzene (DVB) and 150.0g of toluene as porogens, add 3.75g of dioctanoyl peroxide as an initiator, and stir magnetically to dissolve completely to obtain mixed phase 2 (oil phase). Add mixed phase 1 and mixed phase 2 into a beaker and ultrasonically emulsify them into an emulsion of about 0.5μm to 1μm. Add 4.1g of PST seeds obtained in step (1) and the emulsion made of mixed phase 1 (water phase) and mixed phase 2 (oil phase) into a beaker, put it into a magnetic beaker and stir it with a magnetic stirrer to mix, and then swell at room temperature, control the stirring speed: 100rpm, and the swelling time: 24h. After the swelling was completed, 243 g of 2% PVA aqueous solution was added, the stirring speed was 150 rpm, and the stirring was carried out for 30 min. Under the protection of N2, the temperature was raised to 75°C and the reaction was kept warm for 12 h. After the reaction was completed, the reaction liquid was filtered through a Buchner funnel with filter paper, and then washed three times with 500 mL of deionized water, three times with 300 mL of ethanol, and three times with 300 mL of acetone to obtain porous microspheres P (GMA-DVB) with a crosslinking degree of 70%. The average particle size D50 was 13.44 μm as measured by scanning electron microscopy (SEM). Figure 3 , Figure 4 The epoxy value of the microspheres is 5.2 mmol / g. The average pore size measured by the BET method is The specific surface area is 456m 2 It can be understood that the particle size, pore size and specific surface area of ​​the porous microspheres P(GMA-DVB) are equivalent to those of the final product microspheres.

[0170] Example 1-2

[0171] This embodiment is a porous polymer microsphere, and its preparation method is the same as that of Example 1-1, with the main difference being that the type and amount of the porogen in step (2) are different, thereby making the average pore size and specific surface area of ​​the porous microsphere P (GMA-DVB) different.

[0172] The specific preparation method is as follows:

[0173] (1) Preparation of PST seed particles: same as in Example 1-1.

[0174] (2) Preparation of porous microspheres P(GMA-DVB):

[0175] Dissolve 1.8g of sodium dodecylbenzene sulfonate (SDBS) in 600g of pure water, and stir to dissolve completely to obtain mixed phase 1 (water phase). In a 500mL beaker, weigh 18.75g of glycidyl methacrylate, 131.25g of divinylbenzene (DVB), 160.0g of toluene, and 40.0g of n-hexanol as porogens, add 3.75g of dioctanoyl peroxide as an initiator, and stir magnetically to dissolve completely to obtain mixed phase 2 (oil phase). Add mixed phase 1 and mixed phase 2 into a beaker and ultrasonically emulsify them into an emulsion of about 0.5μm to 1μm. Add 3.0g of the PST seeds obtained in step (1) and the emulsion made of mixed phase 1 (water phase) and mixed phase 2 (oil phase) into a beaker, put it into a magnetic beaker and stir it with a magnetic stirrer to mix, and swell at room temperature, control the stirring speed: 100rpm, and the swelling time: 24h. After the swelling is completed, add 243g of 2% PVA solution, stir at a speed of 150rpm, stir for 30min, raise the temperature to 75°C under N2 protection, and keep the reaction warm for 12h. After the reaction is completed, use qualitative filter paper to filter the reaction liquid on a Buchner funnel, then wash three times with 500mL of deionized water, three times with 300mL of ethanol, and three times with 300mL of acetone to obtain porous microspheres P (GMA-DVB) with a crosslinking degree of 70%. The average particle size D50 tested by scanning electron microscopy (SEM) is 14.5μm. Epoxy value of microspheres: 3.8mmol / g. The average pore size measured by the BET method is The specific surface area is 275m 2 / g.

[0176] Examples 1-3

[0177] This embodiment is a porous polymer microsphere, and its preparation method is the same as that of Example 1-1, with the main difference being that the amount of porogen used in step (2) is different, thereby making the average pore size and specific surface area of ​​the porous microsphere P (GMA-DVB) different.

[0178] The specific preparation method is as follows:

[0179] (1) Preparation of PST seed particles: same as in Example 1-1.

[0180] (2) Preparation of porous microspheres P(GMA-DVB):

[0181] Dissolve 1.8g of sodium dodecylbenzene sulfonate (SDBS) in 600g of pure water, and stir to dissolve completely to obtain mixed phase 1 (water phase). In a 500mL beaker, weigh 18.75g of glycidyl methacrylate, 131.25g of divinylbenzene (DVB) and 100.0g of toluene as porogens, add 3.75g of dioctanoyl peroxide as an initiator, and stir magnetically to dissolve completely to obtain mixed phase 2 (oil phase). Add mixed phase 1 and mixed phase 2 into a beaker and ultrasonically emulsify them into an emulsion of about 0.5μm to 1μm. Add 3.0g of the PST seeds obtained in step (1) and the emulsion made of mixed phase 1 (water phase) and mixed phase 2 (oil phase) into a beaker, put it into a magnetic beaker and stir it with a magnetic stirrer to mix, and then swell at room temperature, control the stirring speed: 100rpm, and the swelling time: 24h. After the swelling is completed, add 243g of 2% PVA solution, stir at 150rpm, stir for 30min, raise the temperature to 75°C under N2 protection, and keep the reaction for 12h. After the reaction is completed, use qualitative filter paper to filter the reaction liquid on a Buchner funnel, then wash three times with 500mL of deionized water, three times with 300mL of ethanol, and three times with 300mL of acetone to obtain porous microspheres P (GMA-DVB) with a crosslinking degree of 70%. The average particle size D50 tested by scanning electron microscopy (SEM) is 12.9μm, and the epoxy value of the microspheres is 3.8mmol / g. The average pore size is measured by the BET method. The specific surface area is 680m 2 / g.

[0182] Examples 1-4

[0183] This embodiment is porous polymer microspheres, and its preparation method is the same as that of Example 1-1, with the main difference being that the amount of porogen used in step (2) is different, thereby making the average pore size and specific surface area of ​​the porous microspheres P (GMA-DVB) different.

[0184] The specific preparation method is as follows:

[0185] (1) Preparation of PST seed particles: same as in Example 1-1.

[0186] (2) Preparation of porous microspheres P(GMA-DVB):

[0187] Dissolve 1.8g of sodium dodecylbenzene sulfonate (SDBS) in 600g of pure water, and stir to dissolve completely to obtain mixed phase 1 (water phase). In a 500mL beaker, weigh 18.75g of glycidyl methacrylate, 131.25g of divinylbenzene (DVB) and 75.0g of toluene as porogens, add 3.75g of dioctanoyl peroxide as an initiator, and stir magnetically to dissolve completely to obtain mixed phase 2 (oil phase). Add mixed phase 1 and mixed phase 2 into a beaker and ultrasonically emulsify them into an emulsion of about 0.5μm to 1μm. Add 8.0g of the PST seeds obtained in step (1) and the emulsion made of mixed phase 1 (water phase) and mixed phase 2 (oil phase) into a beaker, put it into a magnetic beaker and stir it with a magnetic stirrer to mix, and then swell at room temperature, control the stirring speed: 100rpm, and the swelling time: 24h. After the swelling is completed, add 243g of 2% PVA solution, stir at 150rpm, stir for 30min, raise the temperature to 75°C under N2 protection, and keep the reaction for 12h. After the reaction is completed, use qualitative filter paper to filter the reaction liquid on a Buchner funnel, then wash three times with 500mL of deionized water, three times with 300mL of ethanol, and three times with 300mL of acetone to obtain porous microspheres P (GMA-DVB) with a crosslinking degree of 70%. The particle size D50 tested by scanning electron microscopy (SEM) is 10.2μm, and the epoxy value of the microspheres is 3.8mmol / g. The average pore size is measured by the BET method. The specific surface area is 810m 2 / g.

[0188] Examples 1-5

[0189] This embodiment is a porous polymer microsphere, and its preparation method is the same as that of Example 1-1, with the main difference being that the type and amount of the porogen in step (2) are different, thereby making the average pore size and specific surface area of ​​the porous microsphere P (GMA-DVB) different.

[0190] The specific preparation method is as follows:

[0191] (1) Preparation of PST seed particles: same as in Example 1-1.

[0192] (2) Preparation of porous microspheres P(GMA-DVB):

[0193] Dissolve 1.8g of sodium dodecylbenzene sulfonate (SDBS) in 600g of pure water, and stir to dissolve completely to obtain mixed phase 1 (water phase). In a 500mL beaker, weigh 18.75g of glycidyl methacrylate, 131.25g of divinylbenzene (DVB), 200.0g of toluene, and 100.0g of n-hexanol as porogens, add 3.75g of dioctanoyl peroxide as an initiator, and stir magnetically to dissolve completely to obtain mixed phase 2 (oil phase). Add mixed phase 1 and mixed phase 2 into a beaker and ultrasonically emulsify them into an emulsion of about 0.5μm to 1μm. Add 3.0g of the PST seeds obtained in step (1) and the emulsion made of mixed phase 1 (water phase) and mixed phase 2 (oil phase) into a beaker, put it into a magnetic beaker, stir it magnetically, and swell it at room temperature. Control the stirring speed: 100rpm, and the swelling time: 24h. After the swelling is completed, add 243g of 2% PVA solution, stir at 150rpm, stir for 30min, raise the temperature to 75°C under N2 protection, and keep the reaction for 12h. After the reaction is completed, use qualitative filter paper to filter the reaction liquid on a Buchner funnel, then wash three times with 500mL of deionized water, three times with 300mL of ethanol, and three times with 300mL of acetone to obtain porous microspheres P (GMA-DVB) with a crosslinking degree of 70%. The particle size D50 tested by scanning electron microscopy (SEM) is 15.5μm, and the epoxy value of the microspheres is 4.8mmol / g. The average pore size is measured by the BET method. The specific surface area is 102m 2 / g.

[0194] Examples 1-6

[0195] This embodiment is a cation exchange polymer porous microsphere, and the specific preparation method is as follows:

[0196] (1) Preparation of PST seed particles: same as in Example 1-1.

[0197] (2) Preparation of porous microspheres P(GMA-DVB): same as Example 1-1.

[0198] (3) Hydrolysis of porous microspheres P(GMA-DVB)

[0199] In a 1000mL three-necked flask, 50mL of the porous microspheres P(GMA-DVB) prepared in step (2) were dispersed in 500ml of 0.5M sulfuric acid aqueous solution and stirred evenly, and the temperature was raised to 40°C for hydrolysis reaction for 4h. After the reaction solution was cooled to room temperature, it was washed and filtered with deionized water and anhydrous ethanol (water washing to pH = 7.0) to obtain porous P(GMA-DVB)-OH microspheres, and the residual epoxy value of the dried microspheres was ≤0.20mmol / g.

[0200] (4) Hydrophilic spacer arm of porous P(GMA-DVB)-OH microspheres:

[0201] In a 500mL three-necked flask, 50mL of the hydroxylated porous microspheres P(GMA-DVB)-OH prepared in step (3) were dispersed in 200mL of dioxane solvent, and then 30mL of 1,4-butanediol and 30g of epichlorohydrin were added to the flask. After stirring evenly, 2mL of boron trifluoride ether complex was added. The reaction solution was heated to 75°C under nitrogen protection, the stirring speed was controlled to 150rpm, and the reaction was continued for 8h. After the reaction solution was cooled to room temperature, it was washed with deionized water to pH = 7.0 to obtain hydrophilic spacer-armed epoxy porous P(GMA-DVB)-EPOXY microspheres, and the epoxy value of the microspheres was 3.5mmol / g.

[0202] (5) Synthesis of carboxylated porous microspheres P(GMA-DVB)-COOH:

[0203] 30mL of the microspheres in step (4) and 30mL of ultrapure water are transferred into a 250mL three-necked round-bottom flask. A solution consisting of 70mL of H2O and 25g of 3-chloropropionic acid is prepared and added to the above-mentioned 250mL three-necked round-bottom flask, heated with a water bath under water cooling of a condenser tube until the temperature rises to 60-65°C, 36g of a 48% sodium hydroxide aqueous solution is added dropwise, and the mixture is kept at 65°C for 16h before being cooled to room temperature, then filtered on a suction funnel, washed once with 30mL of a 10% Na2CO3 aqueous solution, and then washed 10 times with ultrapure water until neutral (pH=7.0) (30mL each time), to obtain carboxylated microspheres. The microspheres with a carboxyl structure are washed 3 times with ethanol, and finally drained to obtain carboxylated weak cation exchange microspheres (P(GMA-DVB)-COOH). The ion exchange capacity of the dried microspheres was 0.55 mmol / L, and the lysozyme loading was 32 mg / mL.

[0204] Standard protein performance test method test spectrum such as Figure 5 As shown, it can be seen that the prepared weak cation exchange polymer porous microspheres can be used as fillers to effectively separate three proteins: ribonuclease A, cytochrome C and lysozyme.

[0205] Examples 1-7

[0206] This embodiment is a cation exchange polymer porous microsphere, the main difference is: the type of cationic active group used is different.

[0207] (1) Preparation of PST seed particles: same as in Examples 1-6.

[0208] (2) Preparation of porous microspheres P(GMA-DVB): same as in Examples 1-6.

[0209] (3) Hydrolysis of porous P(GMA-DVB)-OH microspheres: Same as Example 1-6.

[0210] (4) Hydrophilic spacer arming of porous P(GMA-DVB)-OH microspheres: Same as in Example 1-6.

[0211] (5) Synthesis of sulfonated porous microspheres P(GMA-DVB)-SO3:

[0212] Transfer 30 mL of the microspheres from step (4) and 30 mL of ultrapure water into a 250 mL three-necked round-bottom flask. Prepare a solution consisting of 15.2 mL of H2O and 11.5 g of sodium sulfite, add it to the above 250 mL three-necked round-bottom flask, heat it in a water bath under water cooling with a condenser, raise the temperature to 65°C, keep it warm for 16 hours and then cool it to room temperature, then filter it on a suction funnel and wash it 10 times with ultrapure water until it is neutral (pH = 7.0) (30 mL each time) to obtain sulfonated microspheres. Wash the microspheres with sulfonic acid structure with ethanol 3 times and finally drain them to obtain sulfonic acid strong cation exchange microspheres (P(GMA-DVB)-SO3H). The ion exchange capacity of the dried microspheres is 0.53 mmol / L, and the lysozyme loading is 68 mg / mL.

[0213] Standard protein performance test method test spectrum such as Figure 6 It can be seen that the prepared strong cation exchange polymer porous microspheres can be used as fillers to effectively separate three proteins: ribonuclease A, cytochrome C and lysozyme, and the peak time and separation degree are significantly different from those in Examples 1-6.

[0214] Examples 1-8

[0215] This embodiment is a cation exchange polymer porous microsphere, and its preparation method is the same as that of Embodiments 1-6, with the main difference being that the pore size of the microsphere used in step (2) is different.

[0216] The specific preparation method is as follows:

[0217] (1) Preparation of PST seed particles: same as in Example 1-1.

[0218] (2) Preparation of porous microspheres P(GMA-DVB): same as in Examples 1-4.

[0219] (3) Hydrolysis of porous P(GMA-DVB)-OH microspheres: Same as Example 1-6.

[0220] (4) Hydrophilic spacer arming of porous P(GMA-DVB)-OH microspheres: Same as in Example 1-6.

[0221] (5) Synthesis of carboxylated porous microspheres P(GMA-DVB)-COOH: Same as Example 1-6.

[0222] The obtained porous microspheres (P(GMA-DVB)-COOH) with weak cation exchange were hydroxypropylated. The ion exchange capacity of the dried microspheres was 1.03 mmol / L, and the lysozyme loading was 9.5 mg / mL.

[0223] Standard protein performance test method test spectrum such as Figure 7 As shown. It can be seen that the pore size of the microspheres has a great influence on the separation effect of the ion exchange polymer porous microspheres as fillers. The steric hindrance effect of the pores on the surface that are too small on the protein leads to insufficient ion exchange. The retention time of the three proteins ribonuclease A, cytochrome C and lysozyme is very short. The two protein standards ribonuclease A and cytochrome C are not separated and peak at 0.911min at the same time.

[0224] Examples 1-9

[0225] This embodiment is a cation exchange polymer porous microsphere, and its preparation method is the same as that of Embodiments 1-6, with the main difference being that the pore size of the microsphere used in step (2) is different.

[0226] The specific preparation method is as follows:

[0227] (1) Preparation of PST seed particles: same as in Example 1-1.

[0228] (2) Preparation of porous microspheres P(GMA-DVB): same as Example 1-2.

[0229] (3) Hydrolysis of porous P(GMA-DVB)-OH microspheres: Same as Example 1-6.

[0230] (4) Hydrophilic spacer arming of porous P(GMA-DVB)-OH microspheres: Same as in Example 1-6.

[0231] (5) Synthesis of carboxylated porous microspheres P(GMA-DVB)-COOH: Same as Example 1-6.

[0232] The porous microspheres (P(GMA-DVB)-COOH) with carboxypropylated weak cation exchange were obtained. The ion exchange capacity of the dried microspheres was 0.41mmol / L, and the lysozyme loading capacity could not be completed. The main reason was that the particle size was about 15um and the average pore size was about The pressure resistance of the polymer porous microsphere ion exchange filler is poor. Under the conditions of this standard protein performance test method, the chromatographic column caused the instrument pressure alarm to sound, reaching the upper limit of the instrument, and the standard protein separation performance test could not be completed.

[0233] Example 2-1

[0234] This embodiment is a method for preparing hemoglobin biological components.

[0235] (1) Samples: Whole blood red blood cell lysate from patients with β-thalassemia was used as raw material. HbA1c (<6.0%), HbF (>6.0%) and HbA2 (>4.0%) were required. All samples were free of abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0236] 2. The cation exchange polymer porous microspheres prepared in Examples 1-6 were used as ion exchange fillers to fill a 10 mm×150 mm preparative chromatography column, and high-purity HbF, HbA1c, HbA0 and HbA2 were prepared on a preparative chromatograph.

[0237] Specifically, the chromatographic conditions are as follows:

[0238] 2.1. Preparative chromatography instrument: Hitachi chromatograph;

[0239] 2.2. Preparation of chromatographic column: cation exchange chromatographic column, 10 mm × 150 mm;

[0240] 2.3. The flow rate of the chromatography system ranges from 0 to 10 ml / min;

[0241] 2.4. The pressure range is 0~40MPa;

[0242] 2.5. Column temperature: room temperature (20℃~25℃);

[0243] 2.6. Detector wavelength: 415nm;

[0244] 2.7. Manual injection valve: Rheodyne7725;

[0245] 2.8. Flow rate: 2.5 mL / min;

[0246] 2.9. Mobile phase: Buffer No. 1 is 15 mM phosphate buffer, pH 5.6, and buffer No. 2 is 80 mM phosphate buffer, pH 6.5.

[0247] 2.10. Gradient:

[0248] Step 1: Pump buffer #1 for 20 minutes, at which time HbA1a, HbA1b, HbF, LA1c, and HbA1c have all peaked;

[0249] Step 2: Pump with buffer #1 to buffer #2 for 20 minutes, 0-100% buffer #2, at which time the impurity peaks P3 and P4 in front of hemoglobin, HbA1c, and A0 have all been completely discharged;

[0250] Step 3: Buffer #2 was pumped for 20 minutes, at which time both HbA0 and HbA2 were completely discharged;

[0251] Step 4: Pump with buffer #1 for 20 minutes to equilibrate the baseline;

[0252] Next injection.

[0253] Injection volume: 0.5 mL (sample concentration: 15-fold dilution of red blood cell lysis buffer).

[0254] The chromatogram of the separation process is as follows Figure 8 The A, B, C, and D segments were collected and concentrated through a 30KD nanofiltration membrane to prepare samples of HbF, HbA1c, HbA0, and HbA2. The yield and purity of each component are shown in Table 2 below. The overlay of the thalassemia sample and the purified sample is shown in Fig. 9 shown.

[0255] Table 2

[0256] Serial number Purified sample name Purity of target peak (%) Target peak yield (%) A HkDJ 87.13 58.5 B HbA1C 81.58 72.6 C HbA0 100.00 79.2 D HbA2 95.74 65.4

[0257] Example 2-2

[0258] This embodiment is a method for preparing hemoglobin biological components.

[0259] (1) The red blood cell lysate of diabetic blood samples is used as the raw material. Human blood samples (high value HbA1C>9.0%) are required. All samples are free of abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0260] 2. The cation exchange polymer porous microspheres prepared in Examples 1-6 were used as ion exchange fillers to fill a 10 mm×150 mm preparative chromatography column, and high-purity HbA1c and HbA0 were prepared on a preparative chromatograph. Specifically, the chromatographic conditions were as follows:

[0261] 2.1. Preparative chromatography instrument: Hitachi chromatograph;

[0262] 2.2. Preparation of chromatographic column: cation exchange chromatographic column, 10 mm × 150 mm;

[0263] 2.3. The flow rate of the chromatography system ranges from 0 to 10 ml / min;

[0264] 2.4. The pressure range is 0~40MPa;

[0265] 2.5. Column temperature: room temperature (20℃~25℃);

[0266] 2.6. Detector wavelength: 415nm;

[0267] 2.7. Manual injection valve: Rheodyne7725;

[0268] 2.8. Flow rate: 2.5 mL / min;

[0269] 2.9. Mobile phase: Buffer No. 1 is 15 mM phosphate buffer, pH 5.6, and buffer No. 2 is 80 mM phosphate buffer, pH 6.5.

[0270] 2.10. Gradient:

[0271] Step 1: Pump buffer #1 for 20 minutes, at which time HbA1a, HbA1b, HbF, LA1c, and HbA1c have all peaked;

[0272] Step 2: Buffer #2 was pumped for 40 minutes, at which time the impurity peaks P3, P4, and HbA0 in front of hemoglobin A0 had all been completely eliminated;

[0273] Step 3: Pump with buffer #1 for 20 minutes to equilibrate the baseline;

[0274] Next injection.

[0275] Injection volume: 0.5 mL (sample concentration: 15-fold dilution of red blood cell lysis buffer).

[0276] The chromatogram of the separation process is as follows Fig.10 The A and B segments were collected and concentrated by 30KD nanofiltration membrane to prepare purified samples of HbA1c and HbA0. The yield of HbA1c was 81.6% and the purity was 100%. The yield of HbA0 was 89.0% and the purity was 100%.

[0277] Example 2-3

[0278] This embodiment is a method for preparing hemoglobin biological components.

[0279] (1) Samples: Blood samples from patients with β-thalassemia, with HbF (>10.0%) and HbA2 (2.0%~3.5%) required. All samples should be free of abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0280] 2. Ordinary human red blood cell lysate was used as raw material, and the cation exchange polymer porous microspheres prepared in Example 1-6 were used as ion exchange fillers to fill a 10 mm×150 mm chromatography preparation column, and high-purity HbF was prepared on a preparative chromatograph.

[0281] Specifically, the chromatographic conditions are as follows:

[0282] 2.1. Preparative chromatography instrument: Hitachi chromatograph;

[0283] 2.2. Preparation of chromatographic column: cation exchange chromatographic column, 10 mm × 150 mm;

[0284] 2.3. The flow rate of the chromatography system ranges from 0 to 10 ml / min;

[0285] 2.4. The pressure range is 0~40MPa;

[0286] 2.5. Column temperature: room temperature (20℃~25℃);

[0287] 2.6. Detector wavelength: 415nm;

[0288] 2.7. Manual injection valve: Rheodyne7725;

[0289] 2.8. Flow rate: 2.5 mL / min;

[0290] 2.9. Mobile phase: Buffer No. 1 is 15 mM phosphate buffer, pH 5.6, and buffer No. 2 is 80 mM phosphate buffer, pH 6.5.

[0291] 2.10. Gradient:

[0292] Step 1: Pump buffer #1 for 20 minutes, at which time HbA1a, HbA1b, HbF, LA1c, and HbA1c have all peaked;

[0293] Step 2: Buffer #2 was pumped for 40 minutes, at which time the impurity peaks P3, P4, and HbA0 in front of hemoglobin A0 had all been completely discharged;

[0294] Step 3: Pump with buffer #1 for 20 minutes to equilibrate the baseline;

[0295] Next injection.

[0296] Injection volume: 0.5 mL (sample concentration: 15-fold dilution of red blood cell lysis buffer).

[0297] The chromatogram of the separation process is as follows Fig.11 The HbF fraction was collected and concentrated by 30KD nanofiltration membrane to prepare a purified HbF sample with a yield of 74.2% and a purity of 93.18%.

[0298] Example 3-1

[0299] This example is the preparation of a high-value HbA1c quality control material (quality control material III (high level)), and the steps are as follows:

[0300] 1. First, ordinary people are screened, requiring that their HbA1c is <6.0%, and the measured values ​​of HbF (<3.0%) and HbA2 (2.0%-3.5%) are normal. All samples are free of common hemoglobin variants such as HbBart's, HbH, HbCS, HbE, HbD, HbS, and HbC.

[0301] 2. Take the HbA1c sample prepared in Example 2-1;

[0302] 3. The preparation process of high HbA1c quality control material is as follows:

[0303] Step 1: Take 100 mL of normal human whole blood, mix them together, and then centrifuge them at 5000 rpm / min for 30 minutes;

[0304] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0305] Step 3: adding physiological saline at a volume ratio of 10% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell lysate of normal concentration;

[0306] Step 4: Take a certain amount of purified HbA1c (i.e., the HbA1c sample prepared in Example 2-1 in step 2) and add it to the red blood cell lysate of normal concentration. After sufficient mixing, the mass percentage of HbA1c is determined by the H100Plus hemoglobin analyzer of Pumen Company, which is 11.06%, and the total area is 165,000 mOD, to obtain a high-concentration HbA1c quality control material, and then add 5% trehalose and 0.3% sodium azide. After packaging, the mixture is freeze-dried by vacuum freeze-drying technology to obtain a freeze-dried high-concentration HbA1c hemoglobin control material. The thalassemia pattern spectrum of the H100Plus high-performance liquid chromatograph is as follows: Fig.12 shown.

[0307] The preparation method of the above-mentioned quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use on glycosylated hemoglobin instruments. It can be used as a freeze-dried HbA1c quality control material for diabetes diagnosis, and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0308] Example 3-2

[0309] This example is the preparation of a low-value HbA1c quality control material for glycated hemoglobin (quality control material III (low level)), and the steps are as follows:

[0310] 1. First, ordinary people's samples are screened. The HbF (<3.0%) and HbA2 (2.0%-3.5%) values ​​of ordinary people are required to be normal, and all samples must not have abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0311] 2. Take the high-concentration HbA0 sample purified and prepared in Example 2-1.

[0312] 3. The preparation process of low-value glycated hemoglobin HbA1c quality control is as follows:

[0313] Step 1: Take 100 mL of normal human whole blood and mix them together, centrifuge them at 5000 rpm / min for 30 minutes;

[0314] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0315] Step 3: adding physiological saline at a volume ratio of 15% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell stock solution of normal concentration;

[0316] Step 4: Take a certain amount of high-concentration HbA0 sample (i.e., the high-concentration HbA0 sample prepared in step 2) and add it to the normal concentration of red blood cell lysate to adjust the mass percentage of HbA1c, and use Pumen's H100Plus hemoglobin analyzer to measure the mass percentage of HbA1c to be 5.29%, with a total area of ​​217,000 mOD, to obtain a low-concentration HbA1c quality control. If HbA1c is not between 4.0% and 6.0%, for samples with HbA1c>6.0%, purified HbA0 can be added for adjustment; then 5% trehalose and 0.3% sodium azide are added, and after packaging, vacuum freeze-drying technology is used for freeze-drying to obtain a freeze-dried low-concentration HbA1c quality control. The thalassemia pattern spectrum of the H100Plus high-performance liquid chromatograph is as follows: Fig.13 shown.

[0317] The preparation method of the above quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use in β-thalassemia screening instruments. It can be used as a quality control material for diabetes diagnosis and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0318] Example 3-3

[0319] This example is the preparation of HbA2 quality control material (high level quality control material I) for β-thalassemia screening, and the steps are as follows:

[0320] 1. First, ordinary people's samples are screened. The HbF (<3.0%) and HbA2 (2.0%-3.5%) values ​​of ordinary people are required to be normal, and all samples must not have abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0321] 2. Take the high-concentration HbA2 sample purified and prepared in Example 2-1;

[0322] 3. The preparation process of HbA2, the quality control material for β-thalassemia screening, is as follows:

[0323] Step 1: Take 100 mL of normal human whole blood, mix them together, and then centrifuge them at 5000 rpm / min for 30 minutes;

[0324] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0325] Step 3: adding physiological saline at a volume ratio of 15% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell lysate of normal concentration;

[0326] Step 4: Take a certain amount of purified HbA2 and add it to the normal concentration of red blood cell lysate. After fully mixing, use the H100Plus hemoglobin analyzer of Pumen Company to measure the mass percentage of HbA2 to be 5.81%, and the total area is 267,000 mOD to obtain a high-concentration HbA2 quality control material. Then add 5% trehalose and 0.3% sodium azide, and freeze-dry it using vacuum freeze-drying technology after packaging to obtain a freeze-dried hemoglobin control material containing high concentration HbA2; the thalassemia mode spectrum of H100Plus high-performance liquid chromatography is shown as follows: Fig.14 shown.

[0327] The preparation method of the above quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use on β-thalassemia screening instruments. It can be used as a quality control material for β-thalassemia screening and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0328] Embodiment 3-4

[0329] This example is the preparation of the quality control material HbF (high level quality control material II) for β-thalassemia screening, and the steps are as follows:

[0330] 1. First, ordinary people's samples are screened. The HbF (<3.0%) and HbA2 (2.0%-3.5%) values ​​of ordinary people are required to be normal, and all samples must not have abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0331] 2. Take the high-concentration HbF sample purified and prepared in Example 2-1;

[0332] 3. The preparation process of HbF, the quality control material for β-thalassemia screening, is as follows:

[0333] Step 1: Take 100 mL of normal human whole blood, mix them together, and then centrifuge them at 5000 rpm / min for 30 minutes;

[0334] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0335] Step 3: adding physiological saline at a volume ratio of 15% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell lysate of normal concentration;

[0336] Step 4: The HbF sample prepared in Example 2-1 is added as a raw material to the red blood cell lysate of normal concentration. After being fully mixed, the HbF is measured by the H100Plus hemoglobin analyzer of Pumen Company to be between 15.52% and the total area is 239,000 mOD to obtain a high-concentration HbF quality control material, and then 5% trehalose and 0.3% sodium azide are added. After packaging, the vacuum freeze-drying technology is used for freeze-drying to obtain a freeze-dried high-concentration HbF hemoglobin control material for thalassemia screening; the thalassemia pattern spectrum of the H100Plus high-performance liquid chromatograph is as follows Fig.15 shown.

[0337] The preparation method of the above quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use on β-thalassemia screening instruments. It can be used as a quality control material for β-thalassemia screening and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0338] Embodiment 3-5

[0339] This example is the preparation of quality control materials (high-level quality control material IV and low-level quality control material IV) for β-thalassemia screening, and the steps are as follows:

[0340] 1. First, ordinary people's samples are screened. The HbF (<3.0%) and HbA2 (2.0%-3.5%) values ​​of ordinary people are required to be normal, and all samples must not have abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0341] 2. Take the high-concentration HbA2 sample and HbF sample purified and prepared in Example 2-1.

[0342] 3. The preparation process of quality control material for β-thalassemia screening is as follows:

[0343] Step 1: Take 100 mL of normal human whole blood, mix them together, and centrifuge them in a centrifuge at 5000 rpm / min for 30 minutes;

[0344] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0345] Step 3: adding physiological saline at a volume ratio of 15% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell lysate of normal concentration;

[0346] Step 4: Take a certain amount of purified HbA2 and HbF and add them to the normal concentration of red blood cell lysate. After fully mixing, use the H100Plus hemoglobin analyzer of Pumen Company to measure HbA2 at 5.86%, HbF at 5.05%, and the total area at 176,000 mOD to obtain high-concentration HbA2 and HbF quality control materials, then add 5% trehalose and 0.3% sodium azide, and freeze-dry them using vacuum freeze-drying technology after packaging to obtain freeze-dried high-concentration HbA2 and HbF hemoglobin control materials. The thalassemia pattern spectrum of H100Plus high-performance liquid chromatography is shown as follows Fig.16 As shown;

[0347] Step 5: Take a certain amount of normal concentration of red blood cell lysate and use Pumen's H100Plus hemoglobin analyzer to measure HbA2 at 2.67%, HbF at 2.13%, and the total area at 275,000 mOD to obtain low concentration HbA2 and HbF quality control materials. If HbA2 is not between 2.0% and 3.5%, for samples with HbA2>3.5%, purified HbA0 can be added for adjustment; for samples with HbA2<2.0%, purified HbA2 can be added for adjustment so that the HbA2 of the sample is in the range of 2.0% to 3.5%; if HbF is not between 1.0% and 3.0%, purified HbA0 can be added for adjustment for samples with HbF>3.0%; for samples with HbF<1.0%, purified HbF can be added for adjustment so that the HbF of the sample is in the range of 1.0% to 3.0%; then 5% trehalose and 0.3% sodium azide are added, and after packaging, vacuum freeze-drying technology is used for freeze-drying to obtain freeze-dried low-concentration HbA2 and HbF quality control materials. The thalassemia mode spectrum of H100Plus high performance liquid chromatograph is as follows Fig.17 shown.

[0348] The preparation method of the above quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use on β-thalassemia screening instruments. It can be used as a quality control material for β-thalassemia screening and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0349] Embodiment 3-6

[0350] This example is the preparation of double quality control materials for screening of glycated hemoglobin and β-thalassemia (quality control material V of low concentration HbA1c, high concentration HbA2 and HbF, and quality control material V of high concentration HbA1c, low concentration HbA2 and HbF), and the steps are as follows:

[0351] 1. First, ordinary people's samples are screened. The HbF (<3.0%) and HbA2 (2.0%-3.5%) values ​​of ordinary people are required to be normal, and all samples must not have abnormal hemoglobin such as HbBart's, HbH, HbCS, HbE, HbD, HbS, HbC, etc.

[0352] 2. Take the HbA2 sample and HbF sample prepared in Example 2-1.

[0353] 3. The preparation process of double quality control materials for glycated hemoglobin and β-thalassemia screening is as follows:

[0354] Step 1: Take 100 mL of normal human whole blood, mix them together, and centrifuge them in a centrifuge at 5000 rpm / min for 30 minutes;

[0355] Step 2: Use a pipette to gently aspirate the plasma portion of the supernatant and retain the red blood cells in the precipitate;

[0356] Step 3: adding physiological saline at a volume ratio of 15% for washing, and repeating the washing for 3 times according to steps 1 and 2 to obtain a red blood cell lysate of normal concentration;

[0357] Step 4: Take a certain amount of purified HbA2 and HbF and add them to the normal concentration of red blood cell lysate. After sufficient mixing, use Pumen's H100Plus hemoglobin analyzer to measure HbA1c at 4.84%, HbA2 at 5.37%, HbF at 6.07%, and the total area at 138,000 mOD to obtain high-concentration HbA2 and HbF quality control materials. If HbA1c is not between 4.0% and 6.0%, for samples with HbA1c>6.0%, purified HbA0 can be added for adjustment; for samples with HbA1c<4.0%, purified HbA1c can be added for adjustment so that the HbA1c of the sample is within the range of 4.0% to 6.0%; then 5% trehalose and 0.3% sodium azide are added, and after subpackaging, vacuum freeze-drying technology is used for freeze-drying to obtain freeze-dried low-concentration HbA1c, high-concentration HbA2 and HbF hemoglobin control substances. The thalassemia mode spectrum of H100Plus high-performance liquid chromatography is shown as follows Fig.18 As shown;

[0358] Step 5: Take a certain amount of the HbA1c quality control material of Example 3-1, adjust the concentration, and use the H100Plus hemoglobin analyzer of Pumen Company to measure HbA1c at 9.17%, HbA2 at 3.03%, HbF at 1.18%, and the total area at 242,000 mOD to obtain high-concentration HbA1c, low-concentration HbA2 and HbF quality control materials. The specific concentration adjustment operation is as follows: If HbA2 is not between 2.0% and 3.5%, for samples with HbA2>3.5%, purified HbA0 can be added for adjustment; for samples with HbA2<2.0%, purified HbA2 can be added for adjustment. Adjust the HbA2 of the sample to be in the range of 2.0% to 3.0%; if HbF is not between 1.0% and 3.0%, for samples with HbF>3.0%, purified HbA0 can be added for adjustment; for samples with HbF<1.0%, purified HbF can be added for adjustment to make the HbF of the sample in the range of 1.0% to 3.0%; then add 5% trehalose and 0.3% sodium azide, and freeze-dry by vacuum freeze-drying technology after packaging to obtain freeze-dried high-concentration HbA1c, low-concentration HbA2 and HbF quality control materials. The thalassemia mode spectrum of H100Plus high-performance liquid chromatography is shown as follows Fig.19 shown.

[0359] The preparation method of the above quality control material is simple, covers normal values ​​and medically determined levels, and has stable performance, making it more suitable for use in β-thalassemia screening instruments. It can be used as a dual quality control material for diabetes diagnosis and β-thalassemia screening, and is suitable for various clinical laboratories, testing departments and third-party laboratories.

[0360] The quality control materials of Examples 3-1 to 3-6 were tested:

[0361] In accordance with the requirements of "YYT 1789.1-2021 In vitro diagnostic test system performance evaluation method Part 1 Precision", excluding Saturdays and Sundays, the quality control materials for β-thalassemia screening prepared in Example 3-5 (high-level quality control material IV (Q1) and low-level quality control material IV (Q2)) were continuously measured for 20 days using the French Sebia electrophoresis Capillarys 2. Q1 and Q2 were measured twice a day. The total experimental period was one month, and 80 sets of high and low values ​​of HbF and HbA2 were obtained. The inter-day precision CV was required to be <4.0%. The specific measurement data are shown in Table 3 below.

[0362] At the same time, a new bottle of quality control material Q1 and Q2 was re-dissolved every month for 12 consecutive months, and each concentration was measured twice. The total experimental period was one year, and 24 sets of data for the high and low values ​​of the two items HbF and HbA2 were obtained. The precision CV was required to be <4.0%. The specific measurement data are shown in Table 4 below.

[0363] Table 3

[0364]

[0365] Table 4

[0366]

[0367] Conclusion: The prepared HbF and HbA2 quality control materials for β-thalassemia screening at high and low levels were continuously measured for 20 days and tested for 12 months after opening the bottle, and the precision CV was <4.0%, which met the clinical requirements for this quality control material. Therefore, the freeze-dried β-thalassemia screening quality control material developed by the present invention can be stably stored at 2-8°C for 1 year; after opening the bottle, re-dissolving and subpackaging, it can be stored at -20°C and can be stably stored for 30 days.

[0368] The remaining embodiments were tested in the same manner. The test results of the 20-day stability test of the quality control material of Example 3-1 are shown in Table 5 below. The inter-day precision meets CV < 4%, the test is qualified, and the stability meets the design requirements.

[0369] Table 5

[0370] Quality Control Testing days MEAN Standard Deviation SD Inter-day precision CV Control III (High Level HbA1c (%)) 20 days 11.06 0.2423 2.19％

[0371] The test results of the quality control material of Example 3-2 for 20 days are shown in Table 6 below. The inter-day precision meets CV<4%.

[0372] Table 6

[0373] Quality Control Testing days MEAN Standard Deviation SD Inter-day precision CV Control III (Low Level HbA1c (%)) 20 days 5.29 0.1063 2.01％

[0374] The test results of the 20-day stability test of the quality control material of Example 3-3 are shown in Table 7 below. The inter-day precision meets CV<4%, the test is qualified, and the stability meets the design requirements.

[0375] Table 7

[0376] Quality Control Testing days MEAN Standard Deviation SD Inter-day precision CV Control Material I (High Level HbA2 (%)) 20 days 5.81 0.1633 2.81％

[0377] The test results of the 20-day stability test of the quality control material of Example 3-4 are shown in Table 8 below. The inter-day precision meets CV<4%, the test is qualified, and the stability meets the design requirements.

[0378] Table 8

[0379] Quality Control Testing days MEAN Standard Deviation SD Inter-day precision CV Control Material II (High Level HbF (%)) 20 days 15.52 0.3302 2.13％

[0380] The test results of the 20-day stability test of the quality control material of Example 3-6 are shown in Table 9 below. The inter-day precision meets CV<4%, the test is qualified, and the stability meets the design requirements.

[0381] Table 9

[0382]

[0383] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0384] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the contents of the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A cation exchange polymer porous microsphere, characterized in that: include: A vinyl monomer-polyvinyl crosslinking agent copolymer mother ball, wherein the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball is modified with a hydroxyl group; The cation exchange active groups of the vinyl monomer-polyvinyl crosslinker copolymer mother ball are grafted via the hydroxyl group; The vinyl monomer-multi-vinyl crosslinking agent copolymer mother ball comprises: Vinyl monomer-polyvinyl crosslinker copolymer porous particles, wherein the vinyl monomer-polyvinyl crosslinker copolymer porous particles are modified with epoxy groups; the vinyl monomer-polyvinyl crosslinker copolymer porous particles are formed by crosslinking polystyrene seed microspheres and vinyl monomers with a polyvinyl crosslinker in the presence of a porogen; the vinyl monomer contains an epoxy group and / or the epoxy group is hydrolyzed and then grafted with a compound containing an epoxy group to obtain the epoxy group; A hydroxy compound grafted to the porous particles of the vinyl monomer-multi-vinyl crosslinker copolymer via the epoxy group, the hydroxy compound comprising the hydroxyl group; The average pore size of the cation exchange polymer porous microspheres is The average particle size D50 of the cation exchange polymer porous microspheres is 11 μm to 15 μm.

2. The cation exchange polymer porous microspheres according to claim 1, characterized in that: The average pore size of the cation exchange polymer porous microspheres is 3. The cation exchange polymer porous microspheres according to claim 1, characterized in that: The specific surface area of ​​the cation exchange polymer porous microspheres is 10 m 2 / g~1000m 2 / g.

4. The cation exchange polymer porous microspheres according to claim 1, characterized in that: The specific surface area of ​​the cation exchange polymer porous microspheres is 100 m 2 / g~800m 2 / g.

5. The cation exchange polymer porous microspheres according to claim 1, characterized in that: The cation exchange active group is one or both of a carboxyl functional group and a sulfonic acid functional group.

6. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The crosslinking degree of the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball is 20% to 80%.

7. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether, 4-vinylbenzyl glycidyl ether and methacrylic acid glycidyl ether.

8. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The epoxy group-containing compound is selected from at least one of epichlorohydrin, glycidyl ether and ethylene glycol diglycidyl ether.

9. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The average particle size D50 of the polystyrene seed microspheres is 1 μm to 10 μm; And / or, the hydroxy compound is at least one selected from ethylene glycol, 1,3-propylene glycol and 1,4-butanediol; And / or, the polyvinyl crosslinking agent is at least one selected from divinylbenzene, divinyltoluene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trivinylbenzene, divinylnaphthalene and trivinylnaphthalene.

10. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The amount of the vinyl monomer used is 10% to 80% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent.

11. The porous cation exchange polymer microspheres according to any one of claims 1 to 5, characterized in that: The porogen is one or more of toluene, xylene, diethylbenzene, dodecylbenzene, hexane, heptane, isopentanol, cyclohexanol, n-hexanol, octanol, diethyl phthalate and n-butyl phthalate; And / or, the amount of the porogen is 50% to 300% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent.

12. The method for preparing the porous microspheres of cation exchange polymers according to any one of claims 1 to 11, characterized in that: The steps include: preparing the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball; Grafting the cation exchange active groups onto the vinyl monomer-polyvinyl crosslinker copolymer mother ball via the hydroxyl reaction; The method for preparing the vinyl monomer-polyvinyl crosslinker copolymer mother ball comprises the following steps: Preparation of porous particles of vinyl monomer-polyvinyl crosslinker copolymer, wherein the porous particles of the vinyl monomer-polyvinyl crosslinker copolymer are modified with epoxy groups, and the preparation steps include: using polystyrene seed microspheres as seeds, emulsifying together with vinyl monomer and polyvinyl crosslinker in the presence of porogen, stabilizer and emulsifier, and then polymerizing in a medium by seed swelling method; the vinyl monomer contains epoxy groups and / or the epoxy groups are hydrolyzed and then grafted with epoxy-containing compounds to obtain the epoxy groups; The hydroxyl compound is grafted onto the porous particles of the vinyl monomer-multi-vinyl crosslinking agent copolymer through the epoxy reaction, and the hydroxyl compound comprises the hydroxyl group.

13. The method for preparing porous cation exchange polymer microspheres according to claim 12, characterized in that: The steps for preparing the porous particles of the vinyl monomer-multi-vinyl crosslinking agent copolymer have at least one of the following characteristics: (1) The initiator used in the polymerization is an oily initiator; (2) The amount of the initiator used in the polymerization is 0.1% to 10% by weight of the vinyl monomer; (3) The stabilizer is a water-soluble polymer; (4) The amount of the stabilizer is 0.5% to 15% by weight of the vinyl monomer; (5) The emulsifier is one or more of a cationic surfactant, a nonionic surfactant, an amphoteric surfactant and an anionic surfactant; (6) The amount of the emulsifier used is 0.05% to 10% by weight of the vinyl monomer.

14. Use of the cation exchange polymer porous microspheres according to any one of claims 1 to 11 in chromatographic fillers.

15. Use of the porous microspheres of cation exchange polymers according to any one of claims 1 to 11 in the preparation of biological components.

16. A method for preparing a hemoglobin biological component, characterized in that: The steps include: Obtaining a first red blood cell lysate; Performing chromatography on the first red blood cell lysate, collecting eluates of each hemoglobin biological component respectively, and preparing the hemoglobin biological component; The filler of the chromatography column used in the chromatography treatment comprises the cation exchange polymer porous microspheres according to any one of claims 1 to 11.

17. The method for preparing a hemoglobin biological component according to claim 16, characterized in that: The hemoglobin biological component is one or more of HbA2, HbF, HbA1c and HbA0.

18. The method for preparing a hemoglobin biological component according to claim 16 or 17, characterized in that: The mobile phases used in the chromatography treatment include mobile phase A and mobile phase B; the mobile phase A includes 1mM-20mM phosphate buffer with a pH of 5.3-6; the mobile phase B includes 30mM-100mM phosphate buffer with a pH of 6-7.

2.

19. The method for preparing a hemoglobin biological component according to claim 18, characterized in that: The chromatography treatment uses mobile phase A and mobile phase B in sequence for isocratic elution.

20. The method for preparing a hemoglobin biological component according to claim 16 or 17, characterized in that: The chromatography process has at least one of the following characteristics: (1) The chromatography column used in the chromatography treatment has an inner diameter of 10 mm to 50 mm and a length of 50 mm to 500 mm; (2) The flow rate of the mobile phase used in the chromatography treatment is 1 mL / min to 30 mL / min; (3) The chromatography treatment is carried out using a high performance liquid preparative chromatography system.

21. A quality control material, characterized in that: It comprises a hemoglobin biological component prepared by the preparation method according to any one of claims 16 to 20, and a second red blood cell lysate; The hemoglobin biological components include at least one or two of HbA2 and HbF; The purity of HbA2 and HbF are both ≥80%.

22. The quality control material according to claim 21, characterized in that: Lyoprotectants and preservatives are also included.

23. The quality control material according to claim 21 or 22, characterized in that: The quality control material is a quality control material for screening β-thalassemia, and the quality control material includes quality control material I and / or quality control material II; the quality control material I includes HbA2 prepared by the preparation method of any one of claims 16 to 20, and red blood cell lysate derived from normal whole blood; the quality control material II includes HbF prepared by the preparation method of any one of claims 16 to 20 or red blood cell lysate derived from newborn whole blood, and red blood cell lysate derived from normal whole blood.

24. The quality control material according to claim 21 or 22, characterized in that: The quality control material is quality control material IV for β-thalassemia screening, and the quality control material IV comprises HbA2 and HbF prepared by the preparation method of any one of claims 16 to 20, and red blood cell lysate derived from normal whole blood.

25. The quality control material according to claim 21 or 22, characterized in that: The quality control material is a dual quality control material V for diabetes diagnosis and β-thalassemia screening, and the quality control material V comprises HbA1c, HbA2, HbF prepared by the preparation method of any one of claims 16 to 20, and red blood cell lysate derived from normal whole blood.

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