Platelet separation and purification method and application of platelet obtained through separation and purification in PRP or PRF

Abstract

The invention provides a separation and purification method of platelets and application of the platelets obtained through separation and purification in PRP or PRF. The method comprises the following steps: carrying out chemical cross-linking on an anti-erythrocyte antibody and an anti-leukocyte antibody to prepare an antibody compound, incubating the antibody compound and a blood sample to obtain a mixed solution, centrifuging, collecting supernate, and removing parenchyma cells and agglomerates to obtain the platelets. The platelet in the blood sample is separated and purified by using the method provided by the invention, the recovery rate of the platelet is greater than or equal to 92%, the activity is improved by more than 30% compared with that of a centrifugal method, the residual leukocyte is less than 1 * 10 < 6 > / mL, the residual erythrocyte is less than 5 * 10 < 7 > / mL, the clinical infusion standard is reached, the separation and purification operation steps are simple, the whole process time is less than or equal to 20 minutes, the platelet separation and purification efficiency is improved, and the cost is reduced. And the cost is reduced by more than 50%. The PRP or PRF prepared from the platelets separated and purified by the method provided by the invention can be applied to the fields of department of stomatology, maxillofacial surgery, orthopedics, plastic surgery and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and blood component separation technology, specifically to a method for separating and purifying platelets and the application of the separated and purified platelets in PRP or PRF. Background Technology

[0002] Platelets are tiny but vital components of blood, formed from anucleate cell fragments produced by the division of mature megakaryocytes. They play an indispensable role in maintaining blood clotting, physiological hemostasis, promoting vascular repair during wound healing, and ensuring the integrity of vascular endothelial cells. Platelet transfusion is an important treatment for hemorrhagic diseases caused by thrombocytopenia or thrombocytopenia, and obtaining high-purity, highly active platelets is a prerequisite for related in vitro biological research. Therefore, developing efficient and mild platelet separation and purification techniques has significant clinical and research value.

[0003] The most commonly used platelet separation methods currently available include density gradient centrifugation and biomagnetic bead sorting. Density gradient centrifugation separates platelets mainly by utilizing the differences in density and sedimentation velocity among platelets, erythrocytes, and leukocytes, achieving preliminary separation through differential centrifugation. However, this method often requires multiple centrifugation steps, and the resulting platelets have limited purity and low recovery rates (<70%). The obtained platelet products often contain a significant number of leukocytes and erythrocytes. These residual cells are not only potential sources of immunogens but may also affect the storage quality and transfusion safety of platelets. Residual leukocytes (>5%) may lead to inflammatory responses. Biomagnetic bead sorting technology is a more efficient and convenient method for platelet separation and purification. It mainly utilizes platelet surface marker proteins for separation. Specifically, different monoclonal antibody proteins are coated onto magnetic beads. Then, the monoclonal antibody-coated magnetic beads specifically bind to platelets. The magnetic beads carry the bound platelets and adsorb onto the separation column. No negative cells bound to the magnetic beads flow out, thus achieving platelet separation. This biomagnetic bead sorting requires complex coupling processes and specialized magnetic separation equipment, which is costly and can easily cause mechanical damage to platelets. Furthermore, this biomagnetic bead sorting technology uses single-target antibody magnetic beads, which can only remove a single cell type. It requires multiple steps in combination, resulting in low platelet separation and purification efficiency. Summary of the Invention

[0004] To address the problems of low platelet recovery rate and purity in existing platelet separation and purification methods using density gradient centrifugation, and the complex processes, high costs, and low platelet separation and purification efficiency in platelet separation and purification using biomagnetic bead sorting technology, this invention provides a platelet separation and purification method and the application of the purified platelets in PRP or PRF.

[0005] According to a first aspect of the present invention, a method for isolating and purifying platelets is provided, comprising the following steps: S1. Chemically cross-link anti-erythrocyte antibodies and anti-leukocyte antibodies to obtain antibody complexes; S2. The antibody complex is mixed with the blood sample and then incubated to obtain a mixture; S3. After centrifuging the mixture, collect the supernatant, remove the impurities and clots in the supernatant, and obtain platelets.

[0006] In the platelet separation and purification method provided by this invention, anti-erythrocyte antibodies and anti-leukocyte antibodies are cross-linked across cells through chemical cross-linking to construct an antibody complex that can simultaneously target the surface antigens of erythrocytes and leukocytes. This antibody complex is mixed with a blood sample and incubated. Large agglomerates are formed through a "erythrocyte-antibody complex-leukocyte" bridging cross-linking network mediated by the antibody complex. Centrifugation of the resulting mixture causes these large agglomerates to settle to the lower layer, simultaneously removing both erythrocytes and leukocytes. The supernatant is collected, and impurities and agglomerates are removed, thereby achieving the separation and purification of platelets from the blood sample, ultimately yielding platelets. Using the method provided by this invention to separate and purify platelets from blood samples, the platelet recovery rate is ≥92%, and the activity (assessed by thromboxane B2 release) is more than 30% higher than that of the centrifugation method. The residual leukocytes in the purified platelets are <1×10⁻⁶. 6 Red blood cells / mL, residual red blood cells <5×10 7 The platelet count / mL meets clinical transfusion standards, and the entire separation and purification process is simple to operate and takes less time (≤20 minutes), which greatly improves the platelet separation and purification efficiency. No magnetic separation equipment is required, and the cost is reduced by more than 50%.

[0007] Preferably, in S1, the anti-erythrocyte antibody and the anti-leukocyte antibody are chemically cross-linked through a bifunctional PEG linker, which is selected from one of N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide (NHS-PEG-NHS), polyethylene glycol diamine (NH2-PEG-NH2), aminooxy-PEG-NHS, acylhydrazide-PEG-NHS, and maleimide-PEG-NHS.

[0008] The aforementioned bifunctional PEG linker is used to chemically cross-link anti-erythrocyte antibodies and anti-leukocyte antibodies to obtain antibody complexes. The two ends of the antibody complexes target the CD235a protein (glycoprotein A) on the surface of erythrocytes and the CD45 protein (leukocyte common antigen) on the surface of leukocytes, respectively. This enables simultaneous high-affinity binding across cell types, allowing the antibody complexes to bind to both leukocytes and erythrocytes in the blood sample to form large particle aggregates. These aggregates can quickly settle to the lower layer under low-speed centrifugation, while platelets remain suspended in the supernatant. This method can simultaneously remove both erythrocytes and leukocytes, achieving efficient platelet separation.

[0009] Preferably, in S1, the anti-erythrocyte antibody is an anti-CD235a monoclonal antibody, and the anti-leukocyte antibody is an anti-CD45 monoclonal antibody.

[0010] Preferably, the above antibody complex is prepared by the following steps: a. Pretreatment with PBS buffer or HEPES buffer to obtain the first antibody solution and pretreatment with PBS buffer or HEPES buffer to obtain the second antibody solution. b. Add the first antibody solution and the second antibody solution to N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide (NHS-PEG-NHS, also known as bis-NHS-PEG) and react at 20~25℃ in the dark for 15~60 min. Then add Tris-HCl or glycine to the reaction system and react for 10~20 min to obtain the antibody complex.

[0011] Preferably, in step a, both the phosphate-buffered saline (PBS) buffer and the 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer contain NaCl, and the concentration of NaCl in both the PBS buffer and the HEPES buffer is 0.1~0.2 M. The concentration of the PBS buffer is 20~50 mM and the pH is 7.8~8.2, and the concentration of the HEPES buffer is 20~50 mM and the pH is 7.8~8.2.

[0012] The antibody complex involved in this method is prepared in an amine-free buffer system (PBS buffer or HEPES buffer) at pH 7.8–8.2. Under these conditions, the active groups (bis-NHS esters) at both ends of the NHS-activated PEG (NHS-PEG-NHS, also known as bis-NHS-PEG) can undergo amidation coupling with the lysine primary amine on the surface of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody via amide bonds. After the reaction, the residual NHS ester is blocked with Tris-HCl or glycine to obtain a bispecific antibody complex that can simultaneously bridge erythrocytes and leukocytes. Applying the prepared antibody complex to the separation and purification of platelets can improve the efficiency of platelet separation and purification.

[0013] Preferably, in step a, the protein concentration in both the first antibody solution and the second antibody solution is 1~5 mg / mL.

[0014] Preferably, in the preparation step of the antibody complex, the amount of anti-CD235a monoclonal antibody is n1, the amount of anti-CD45 monoclonal antibody is n2, and the amount of N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide (NHS-PEG-NHS) is n3, and n1, n2, and n3 satisfy n1:n2=(1~2):(1~2) and n3:(n1+n2)=(0.2~1):1.

[0015] Preferably, in step b, the final concentration of Tris-HCl in the reaction system after adding Tris-HCl is 8~12 mM.

[0016] Preferably, in step b, the final concentration of glycine in the reaction system after adding glycine is 45-55 mM.

[0017] Adding Tris-HCl or glycine to terminate the coupling reaction after conjugating anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody with N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide can quench residual active esters in the reaction system and reduce the risk of over-polyethylene glycolation or conjugation of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody.

[0018] Preferably, in step b, the number-average molecular weight of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide is 5000~6000 g / mol (1 kDa=1000 g / mol, equivalent to 5~6 kDa).

[0019] During the construction of the antibody complex, the number-average molecular weight of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide, which is used to conjugate anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody, is controlled within the above-mentioned range. This helps to reduce steric hindrance and improve the conjugation efficiency between the two antibodies, thereby further improving the efficiency of platelet separation and purification using this antibody complex.

[0020] Preferably, the above antibody complex is prepared by the following steps: a. Prepare an antibody mixture using anti-CD235a monoclonal antibody, anti-CD45 monoclonal antibody, and 2-morpholinoethanesulfonic acid (MES) buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the antibody mixture and react in the dark at 0-25°C for 10-30 min to obtain the reaction solution. b. Add PBS buffer or HEPES buffer to the reaction solution, then add polyethylene glycol diamine (NH2-PEG-NH2) and react at 20~25℃ for 15~60 min. Then add Tris-HCl or glycine to the reaction system and react for 10~20 min to obtain the antibody complex.

[0021] Preferably, in step a, the concentration of 2-morpholinoethanesulfonic acid (MES) buffer is 40-60 mM and the pH is 5.2-6.0.

[0022] Preferably, in step b, both the PBS buffer and the HEPES buffer contain NaCl, and the concentration of NaCl in both the PBS buffer and the HEPES buffer is 0.1~0.2 M. The concentration of the PBS buffer is 20~50 mM and the pH is 7.8~8.2. The concentration of the HEPES buffer is 20~50 mM and the pH is 7.8~8.2.

[0023] The Fc segment (fragment crystallizable) of an antibody (immunoglobulin) is the part at the bottom of the "Y"-shaped structure of the antibody molecule. It is part of the antibody constant region and is essential for the antibody to perform its effector function.

[0024] The antibody complexes involved in this protocol were constructed using an acidic MES buffer system at pH 5.2–6.0. ​​First, the Fc fragments of the anti-CD235a and anti-CD45 monoclonal antibodies used for constructing the antibody complexes were activated using EDC and NHS. During this process, EDC activated the accessible carboxyl groups (glutamic acid Glu / aspartic acid Asp side chains or C-terminal carboxyl groups -COOH) on the Fc regions of the anti-CD235a and anti-CD45 monoclonal antibodies, generating a highly reactive O-acylisourea intermediate. This intermediate is extremely unstable and will react in aqueous solution. Due to rapid hydrolysis and failure, the crosslinking efficiency is low. The added NHS can react with the above-mentioned O-acylisourea intermediate and convert it into a more stable and more specific NHS ester (succinimide ester). Subsequently, under neutral to slightly alkaline conditions, the O-acylisourea intermediate can undergo nucleophilic attack with the primary amine group in the terminal amine PEG, i.e., polyethylene glycol diamine (NH2-PEG-NH2), and finally couple to form a stable amide bond (-CO-NH-), which is blocked with Tris-HCl or glycine. In summary, this scheme utilizes EDC and NHS to activate the Fc fragments of the anti-CD235a and anti-CD45 monoclonal antibodies used to construct antibody complexes. This creates conditions for their subsequent amide bond coupling with primary amine-containing double-ended active PEG, i.e., polyethylene glycol diamine (NH2-PEG-NH2). This improves the coupling efficiency of NH2-PEG-NH2 with both anti-CD235a and anti-CD45 monoclonal antibodies, thereby enhancing the platelet separation and purification efficiency using the constructed antibody complexes.

[0025] Preferably, in step a, after adding 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the antibody mixture, the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the system is 4~6 mM and the final concentration of N-hydroxysuccinimide is 2~5 mM.

[0026] Preferably, in step b, the molecular weight of polyethylene glycol (PEG) in polyethylene glycol diamine (NH2-PEG-NH2) is 5~6 kDa (1 kDa = 1000 g / mol, equivalent to 5000~6000 g / mol).

[0027] Preferably, in the preparation step of the antibody complex, the amount of anti-CD235a monoclonal antibody is n1, the amount of anti-CD45 monoclonal antibody is n2, and the amount of polyethylene glycol diamine (NH2-PEG-NH2) is n3, and n1, n2, and n3 satisfy n1:n2=(1~2):(1~2) and n3:(n1+n2)=(2~4):1.

[0028] Preferably, in step a, the protein concentration in the antibody mixture is 1-3 mg / mL.

[0029] Preferably, in step b, the final concentration of Tris-HCl in the reaction system after adding Tris-HCl is 8~12 mM.

[0030] Preferably, in step b, the final concentration of glycine in the reaction system after adding glycine is 45-55 mM.

[0031] Preferably, the above antibody complex is prepared by the following steps: a. Prepare an anti-CD235a monoclonal antibody solution using anti-CD235a monoclonal antibody and sodium acetate buffer. Add sodium periodate (NaIO4) to the anti-CD235a monoclonal antibody solution and incubate at 0-4°C in the dark for 25-35 min. Then add ethylene glycol to the system and incubate at 0-4°C for 2-5 min to obtain aldehyde-containing antibody. b. Mix the aldehyde-containing antibody with aminooxy-PEG-NHS or acylhydrazide-PEG-NHS, add it to sodium acetate buffer, and incubate at 20-25°C for 10-20 min. Then add aniline to the system and react at 0-4°C for 2-4 h to obtain the first intermediate. c. Mix the first intermediate with the anti-CD45 monoclonal antibody and add it to PBS buffer or HEPES buffer. React at 20-25°C for 15-45 min. Then add Tris-HCl to the system and react for 10-20 min to obtain the antibody complex.

[0032] This method involves mildly oxidizing the N-glycan chain in the Fc region of the anti-CD235a monoclonal antibody with sodium periodate (NaIO4) to generate an aldehyde group. Subsequently, the anti-CD45 monoclonal antibody is linked to the aldehyde group via an oxime bond or a hydrazone bond using aminooxy-PEG-NHS or hydrazide-PEG-NHS, thereby achieving the conjugation of the two antibodies, anti-CD235a and anti-CD45. After the reaction, the antibody complex is blocked with Tris-HCl. Applying the prepared antibody complex to the separation and purification of platelets can improve the efficiency of platelet separation and purification.

[0033] Preferably, the sodium acetate buffer solution has a concentration of 40-60 mM and a pH of 5.2-5.5.

[0034] Preferably, in step a, the protein concentration in the anti-CD235a monoclonal antibody solution is 1~3 mg / mL.

[0035] Preferably, in step a, the final concentration of NaIO4 in the system after adding NaIO4 to the anti-CD235a monoclonal antibody solution is 0.5~3.5 mM.

[0036] Preferably, in step a, the final concentration of ethylene glycol in the system after adding ethylene glycol is 8-12 mM.

[0037] Preferably, in step b, the molar ratio of aldehyde antibody to ammonia-PEG-NHS is (1~1.5):1.

[0038] Preferably, in step b, the molar ratio of the aldehyde antibody to the hydrazide-PEG-NHS is (1~1.5):1.

[0039] Preferably, in step b, the molar ratio of the first intermediate to the anti-CD45 monoclonal antibody is (0.3~1):1.

[0040] Preferably, in step b, the final concentration of aniline in the system after adding aniline is 10~50 mM.

[0041] Preferably, in step c, both the PBS buffer and the HEPES buffer contain NaCl, and the concentration of NaCl in both the PBS buffer and the HEPES buffer is 0.15 M. The concentration of the PBS buffer is 20-50 mM and the pH is 7.8-8.2. The concentration of the HEPES buffer is 20-50 mM and the pH is 7.8-8.2.

[0042] Preferably, in step c, the final concentration of Tris-HCl in the reaction system after adding Tris-HCl is 8~12 mM.

[0043] Preferably, the above antibody complex is prepared by the following steps: a. Prepare an anti-CD235a monoclonal antibody solution by mixing anti-CD235a monoclonal antibody with the first buffer. Add a thiol reducing agent (TCEP) or dithiothreitol (DTT) to the anti-CD235a monoclonal antibody solution and incubate at 20-25°C for 15-30 min to obtain the first mixture. b. Add maleimide PEG active ester (Maleimide-PEG-NHS) to the first mixture and mix well. Then add HEPES buffer or PBS buffer to the system and incubate at 20-25°C in the dark for 15-45 min to obtain the second intermediate. c. After mixing the second intermediate with the anti-CD45 monoclonal antibody, add HEPES buffer or PBS buffer to the system and react at 20-25℃ for 15-45 min. Then add Tris-HCl to the system and react for 10-20 min to obtain the antibody complex.

[0044] This method involves mildly reducing the hinge disulfide bonds of the anti-CD235a monoclonal antibody to generate free thiol groups, which then undergo thiol-maleimide addition with the maleimide terminus of Maleimide-PEG-NHS. The resulting intermediate is coupled to the lysine residue on the surface of the anti-CD45 monoclonal antibody to construct an antibody complex, which is then blocked with Tris-HCl or glycine. Applying the prepared antibody complex to the separation and purification of platelets can improve the efficiency of platelet separation and purification.

[0045] Preferably, the pH of the first buffer solution is 6.8 to 7.2, and the first buffer solution contains the following components: 15 to 25 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 0.1 to 0.2 M NaCl, and 0.5 to 2 mM ethylenediaminetetraacetic acid (EDTA).

[0046] Preferably, in step a, the protein concentration in the anti-CD235a monoclonal antibody solution is 1~3 mg / mL.

[0047] Preferably, in step a, the final concentration of TCEP in the system after adding TCEP to the anti-CD235a monoclonal antibody solution is 0.2~0.5 mM, or the final concentration of DTT in the system after adding DTT to the anti-CD235a monoclonal antibody solution is 0.5~1 mM.

[0048] Preferably, in step b, the molar ratio of Maleimide-PEG-NHS to the anti-CD235a monoclonal antibody in the first mixture is (1~1.5):1.

[0049] Preferably, in step b, the pH of the HEPES buffer is 6.5-7.0, and the pH of the PBS buffer is 6.5-7.0.

[0050] Preferably, in step c, the pH value of the HEPES buffer is 7.8~8.2, and the pH value of the PBS buffer is 7.8~8.2.

[0051] Preferably, in step c, the final concentration of Tris-HCl in the reaction system after adding Tris-HCl is 8~12 mM.

[0052] Preferably, in S2, the blood sample includes heparin-anticoagulated whole blood.

[0053] The platelet separation and purification method provided by this invention can efficiently separate and purify platelets from blood samples such as heparin-anticoagulated whole blood.

[0054] Preferably, in step S2, after mixing the antibody complex with the blood sample, the process further includes adding PEG 6000 to the system and bringing the final concentration of PEG 6000 in the system to 2-4 wt%.

[0055] After the antibody complex is mixed with the blood sample, the high molecular weight polymer PEG 6000 is added to the system and the final concentration of PEG 6000 in the system is controlled within the above range. This can promote the accelerated sedimentation of the agglomerate and obtain high-purity, high-functioning platelets.

[0056] Preferably, S3 includes the following operation: centrifuging the mixture at 250~350 g for 5~10 minutes and collecting the supernatant, filtering the supernatant using a microporous membrane with a pore size of 5 μm to remove impurities and clots in the supernatant, and obtaining platelets.

[0057] The mixture obtained by mixing and incubating the antibody complex with the blood sample can quickly settle to the lower layer under low-speed centrifugation, while the platelets remain suspended in the supernatant. This process can simultaneously remove both red blood cells and white blood cells, efficiently separating platelets. The entire process avoids high-speed centrifugation (>1000 g) and repeated centrifugation operations. The low shear stress during the centrifugation process can maximize the preservation of the integrity of platelet membrane glycoproteins (GPIIb / IIIa, i.e., integrin αIIbβ3), the functional activity of particle contents (ADP, thromboxane A2 precursor), and mitochondria, which is beneficial for separating and purifying highly active platelets.

[0058] According to a second aspect of the present invention, a platelet is provided, which is obtained by the platelet separation and purification method described above.

[0059] According to a third aspect of the invention, the use of the above-described platelets in the preparation of platelet-rich plasma or platelet-rich fibrin is provided.

[0060] Blood samples are subjected to gradient centrifugation in a centrifuge. Based on the density differences of different blood components, the centrifugal force separates the blood into three layers: the bottom layer contains the densest red blood cells, the middle layer contains platelet-rich plasma (PRP) and white blood cells, and the top layer contains platelet-poor plasma (PPP). PRP is a platelet concentrate extracted from blood through centrifugation, and it is rich in platelets, a key component of blood responsible for clotting and tissue repair.

[0061] Platelet-Rich Fibrin (PRF) is a second-generation autologous platelet concentrate, also derived from blood. Rich in various growth factors, it can recruit stem cells to their homing sites, promoting stem cell migration, proliferation, and differentiation, thus facilitating wound healing and tissue regeneration. Simultaneously, PRF contains key immune cytokines, exerting local anti-inflammatory and anti-infective effects during the healing process of the damaged area. Furthermore, PRF possesses a three-dimensional network structure of fibrin gel, with a loose structure and large pores, serving as a natural scaffold material to provide a three-dimensional environment for cells. It is gradually degraded during tissue reconstruction and extracellular matrix formation. Therefore, PRF exhibits excellent biological properties for promoting periodontal ligament regeneration in replanted teeth.

[0062] Platelets separated and purified using the method provided by this invention can be used to prepare PRP or PRF. The prepared PRP or PRF can be widely used in fields such as dentistry, maxillofacial surgery, orthopedics, and plastic surgery. Attached Figure Description

[0063] Figure 1 The diagram illustrates the connection relationship between the anti-CD235a monoclonal antibody and the anti-CD45 monoclonal antibody in the antibody complex used in the platelet separation and purification method provided by this invention.

[0064] Figure 2 Microscopic images of platelet aggregation clumps at different magnifications (20×, 40×) during the process of separating and purifying platelets from blood samples using the method provided by this invention. Detailed Implementation

[0065] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] Amino PEG and PEG amine are a class of PEG binders containing amino groups. They can react with acids, succinimidyl-active esters (NHS esters), or pentafluorophenyl (PFP) and can be used for labeling, chemical modification, surface or particle modification.

[0067] In the preparation of the antibody complexes described in the following examples or comparative examples, the aminooxy-PEG-NHS (NHS-PEG-ONH2) used is prepared by reacting amino polyethylene glycol (Amino PEG) with NHS ester. Related product information can be found at https: / / broadpharm.com / product-categories / peg-linkers / amino-peg.

[0068] PEG hydrrazide linkers and hydrrazide reagents are a class of labeling reagents containing highly reactive hydrazide groups. They can react with aldehydes, ketones, and carboxyl groups and can be used in most protein labeling applications.

[0069] The hydrazide-PEG-NHS used in the preparation of the above antibody complex is prepared by reacting hydrazide-PEG linkers with NHS ester. Relevant product information can be found at https: / / broadpharm.com / product-categories / peg-linkers / peg-hydrazide.

[0070] Example 1 A method for isolating and purifying platelets, comprising the following steps: S1. Anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody are chemically cross-linked to obtain an antibody complex. The linking relationship between the anti-CD235a monoclonal antibody and the anti-CD45 monoclonal antibody in this antibody complex is as follows: Figure 1 As shown; The above antibody complex was prepared by the following steps: a. The anti-CD235a monoclonal antibody was pretreated with 30 mM PBS buffer (pH 8.0, containing 0.15 M NaCl) to obtain a first antibody solution with a protein concentration of 3 mg / mL. The anti-CD45 monoclonal antibody was pretreated with 30 mM PBS buffer (pH 8.0, containing 0.15 M NaCl) to obtain a second antibody solution with a protein concentration of 3 mg / mL. b. Add the first antibody solution and the second antibody solution to NHS-PEG-NHS (the number average molecular weight of PEG is 5000 g / mol) and react at 20°C in the dark for 45 min. Then, add Tris-HCl to the reaction system to make the final concentration in the reaction system 10 mM and react for 15 min to obtain the antibody complex. In this antibody complex, the anti-CD235a monoclonal antibody and the anti-CD45 monoclonal antibody are linked by a PEG linker (NHS-PEG-NHS). The molar ratio of anti-CD235a monoclonal antibody to anti-CD45 monoclonal antibody is 1:1, and the molar ratio of N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide (NHS-PEG-NHS) to the total antibody of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody is 0.5:1. S2. After mixing the above antibody complex with the blood sample (heparin-anticoagulated whole blood), add PEG 6000 to a final concentration of 3 wt%, gently mix at room temperature, and incubate for 15 minutes to obtain the mixture. S3. Centrifuge the above mixture at 300 g for 8 minutes, collect the supernatant, and filter the supernatant using a 5 μm microporous membrane to remove impurities and clots, thus obtaining platelets.

[0071] Example 2 This embodiment provides a method for separating and purifying platelets. Compared with Example 1, the difference is that the preparation method of the antibody complex in step S1 is different. The antibody complex used in this embodiment was prepared through the following steps: a. Prepare an antibody mixture with a protein concentration of 3 mg / mL using anti-CD235a monoclonal antibody, anti-CD45 monoclonal antibody and MES buffer with a concentration of 50 mM and a pH of 5.5. Add EDC and NHS to the antibody mixture and react at 20°C in the dark for 20 min to obtain the reaction solution. b. Add 30 mM HEPES buffer (containing 0.15 M NaCl) at pH 8.0 to the reaction solution, then add NH2-PEG-NH2 and react at 20°C for 45 min. Then add glycine to the reaction system to a final concentration of 10 mM and react for 10-20 min to obtain the antibody complex. The molar ratio of anti-CD235a monoclonal antibody to anti-CD45 monoclonal antibody is 1:1, and the molar ratio of NH2-PEG-NH2 to the total antibody of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody is 3:1.

[0072] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0073] Example 3 This embodiment provides a method for separating and purifying platelets. Compared with Example 1, the difference is that the preparation method of the antibody complex in step S1 is different. The antibody complex used in this embodiment was prepared through the following steps: a. An anti-CD235a monoclonal antibody solution with a protein concentration of 3 mg / mL was prepared by mixing anti-CD235a monoclonal antibody with sodium acetate buffer at a concentration of 50 mM and a pH of 5.5. Sodium periodate (NaIO4) was added to the anti-CD235a monoclonal antibody solution to bring the final concentration in the system to 2 mM, and the mixture was incubated at 4°C in the dark for 30 min. Then, ethylene glycol was added to the system to bring the final concentration in the system to 10 mM, and the mixture was incubated at 4°C for 4 min to obtain aldehyde-containing antibody. b. The aldehyde-containing antibody and aminooxy-PEG-NHS were mixed at a molar ratio of 1:1 and added to a 50 mM sodium acetate buffer solution with a pH of 5.5. The mixture was then incubated at 20°C for 15 min. Aniline was then added to the system and the mixture was reacted at 0-4°C for 2-4 h to obtain the first intermediate. c. The first intermediate and the anti-CD45 monoclonal antibody were mixed at a molar ratio of 1:1 and added to a 30 mM PBS buffer (containing 0.15 M NaCl) at pH 8.0. The mixture was reacted at 20°C for 30 min. Tris-HCl was then added to the system to make the final concentration in the reaction system 10 mM and the mixture was reacted for 15 min to obtain the antibody complex.

[0074] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0075] Example 4 This embodiment provides a method for separating and purifying platelets. Compared with Example 1, the difference is that the preparation method of the antibody complex in step S1 is different. The antibody complex used in this embodiment was prepared through the following steps: a. A solution of anti-CD235a monoclonal antibody with a protein concentration of 3 mg / mL was prepared by mixing anti-CD235a monoclonal antibody with a first buffer (pH 6.8-7.2, containing 20 mM HEPES, 0.15 M NaCl, and 1 mM EDTA). Thiol reducing agent (TCEP) was added to the anti-CD235a monoclonal antibody solution to make its final concentration in the system 0.3 mM, and the solution was incubated at 20°C for 20 min to obtain the first mixture. b. After adding Maleimide-PEG-NHS to the first mixture and mixing well, add HEPES buffer with a pH of 6.8 to the system and incubate at 20°C in the dark for 30 min to obtain the second intermediate; The molar ratio of Maleimide-PEG-NHS to the anti-CD235a monoclonal antibody in the first mixture is 1:1. c. After mixing the second intermediate with the anti-CD45 monoclonal antibody at a molar ratio of 1:1, HEPES buffer with a pH of 8.0 was added to the system and reacted at 20°C for 30 min. Then, Tris-HCl was added to the system to make the final concentration in the reaction system 10 mM and reacted for 15 min to obtain the antibody complex.

[0076] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0077] Example 5 This embodiment provides a method for separating and purifying platelets. Compared with Example 1, the difference is that in step b of preparing the antibody complex, the first antibody solution and the second antibody solution are added to NHS-PEG-NHS and reacted at 20°C in the dark for 45 min, and the step of adding Tris-HCl is omitted.

[0078] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0079] Example 6 This embodiment provides a method for separating and purifying platelets. Compared with Embodiment 1, the difference is that in step S2, after mixing the antibody complex with the blood sample (heparin-anticoagulated whole blood), the addition of PEG 6000 is omitted.

[0080] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0081] Comparative Example 1: Platelet separation by Ficoll gradient density centrifugation A method for isolating and purifying platelets includes the following steps: Take 10 mL of blood sample (heparin-anticoagulated whole blood), prepare Ficoll-Paque PLUS solutions of different concentrations (GE Healthcare) to form a density gradient, first slowly add Ficoll-Paque PLUS solution with a density of 1.077 g / mL to the bottom of a centrifuge tube, then add Ficoll-Paque PLUS solution with a density of 1.065 g / mL on top, avoiding stirring, then slowly add the blood sample to the top of the Ficoll-Paque PLUS solution gradient (density of 1.065 g / mL), centrifuge at 1500 g for 30 minutes (room temperature, slow start and stop), separate the platelet layer located at the Ficoll-Paque PLUS solution gradient interface, then transfer it to a new centrifuge tube, dilute with phosphate-buffered saline (PBS) buffer, centrifuge at 300 g for 10 minutes and wash, repeat the above operation twice to obtain platelets.

[0082] Comparative Example 2: Platelet separation using biomagnetic beads This comparative example refers to a method for separating and purifying platelets provided by Chinese invention patent CN 118685354 A, which includes the following steps: S1. Prepare the coupling buffer according to the following formula: 20 mM, pH 7.4 PBS buffer containing 0.1% (w / v) Tween-20, 0.05% (w / v) BSA, and 0.1% (w / v) Proclin 300; S2. Take 100 μL of Thermo Fisher Dynabeads with a concentration of 10 mg / mL. TM MyOne TM Streptavidin magnetic beads (1 μm in diameter) were washed with conjugation buffer (200 μL each time). The beads were then adsorbed using a magnetic rack for 2 min, and the supernatant was removed. This washing process was repeated 3 times to obtain pretreated magnetic beads. 200 μL of conjugation buffer was then added to the pretreated magnetic beads. 5 μg each of biotin-labeled anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody were added to the conjugation buffer containing the magnetic beads. The mixture was immediately vortexed and incubated at 25°C for 2 h for the conjugation reaction. After the conjugation reaction was completed, the beads were adsorbed using a magnetic rack for 2 min to obtain the magnetic bead-antibody complex. S3. Wash the obtained magnetic bead-antibody complex with conjugation buffer, using 200 μL of conjugation buffer each time. Adsorb using a magnetic rack for 2 min, remove the supernatant, and repeat the washing process 3 times to obtain the purified magnetic bead-antibody complex. Add 200 μL of conjugation buffer to the purified magnetic bead-antibody complex. Take 200 μL of blood sample (heparin-anticoagulated whole blood) and add it to the conjugation buffer containing the magnetic bead-antibody complex. Incubate at room temperature with shaking for 15 min, adsorb using a magnetic rack for 2 min to obtain the magnetic bead-antibody-marker protein complex. Wash the obtained magnetic bead-antibody-marker protein complex with conjugation buffer, using 500 μL of conjugation buffer each time. Adsorb using a magnetic rack for 2 min, remove the supernatant, and repeat the washing process 3 times to obtain the purified magnetic bead-antibody-marker protein complex. Then wash with elution buffer (elution buffer preparation: 20 mM, pH 7.4 PBS buffer, containing 1% (w / v) SDS, 0.1% (w / v) PBS). Eluting was performed using Tween-20 and 0.5% (w / v) biotin. The elution method was to add 500 μL of elution buffer, shake at room temperature (25°C) for 10 min, and then adsorb using a magnetic rack for 2 min. The supernatant was the purified platelet.

[0083] Comparative Example 3 This comparative example provides a method for separating and purifying platelets. Compared with Example 1, the difference in composition is that an equal amount of anti-CD235a monoclonal antibody is used instead of an equal amount of antibody complex.

[0084] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0085] Comparative Example 4 This comparative example provides a method for separating and purifying platelets. Compared with Example 1, the difference in composition is that the antibody complex is prepared by the following steps: anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody are directly physically mixed at a molar ratio of 1:1 (without chemical cross-linking) to obtain the antibody complex.

[0086] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0087] Test Example 1: Comparison of Platelet Separation and Purification Times This test case aims to compare the time required for the entire process of separating and purifying platelets from blood samples using the platelet separation and purification methods provided in Examples 1-6 and Comparative Examples 1-2. The results are shown in Table 1.

[0088] Table 1 Platelet isolation and purification time

[0089] As shown in Table 1, compared with the methods of separating and purifying platelets from blood samples using gradient density centrifugation in Comparative Example 1 and using biomagnetic beads in Comparative Example 2, the platelet separation and purification methods provided in Examples 1-6 have simpler operation steps and shorter overall time (≤20 minutes), which greatly improves the platelet separation and purification efficiency.

[0090] Test Example 2: Platelet recovery rate, platelet activity, and clearance efficiency of contaminating cells. This test case aims to investigate the recovery rate, purity, concentration, activity, and residual red blood cells and white blood cells of platelets during the separation and purification of platelets from blood samples using the methods provided in Examples 1-6 and Comparative Examples 1-4. The results are shown in Tables 2 and 3, where Table 2 shows the recovery rate, purity, concentration, and residual red blood cells and white blood cells of platelets, and Table 3 shows the platelet activity evaluation results.

[0091] Platelet recovery rate, purity, concentration, activity, and residual red blood cells and white blood cells are tested or calculated using the following methods: 1. Platelet concentration, residual leukocytes, and residual leukocyte concentration Using a hematology analyzer (Sysmex XN-1000) and cell counting, along with staining with CD41a-FITC, CD42b-PE, CD45-APC, and CD235a-PerCP antibodies (BD Biosciences), and employing a CytoFLEX flow cytometer, the concentrations and proportions of platelets, residual leukocytes, and residual erythrocytes were determined. The residual leukocyte concentration was required to be <1×10⁻⁶. 6 The number of cells / mL and the concentration of residual red blood cells must be <5×10⁻⁶. 7 Only when the dose is 1 unit / mL can it meet the clinical infusion standard.

[0092] 2. Platelet recovery rate The total number of purified platelets and the total number of platelets in the original blood sample were obtained by using a blood cell analyzer (Sysmex XN-1000) and cell counting. The platelet recovery rate was calculated as follows: Recovery rate (%) = (Total number of purified platelets / Total number of platelets in the original blood) × 100%.

[0093] 3. Platelet purity Using a blood cell analyzer (Sysmex XN-1000), the total number of purified platelets, the number of residual white blood cells, and the number of residual red blood cells were obtained by cell counting. The purity of platelets was calculated as follows: Purity (%) = [Platelet count / (Platelet count + White blood cell count + Red blood cell count)] × 100%.

[0094] 4. Platelet activity evaluation (1) Morphological examination Platelets were stained with Wright-Gymsa and their morphology was observed under a microscope (1000×). The proportion of discoid platelets was counted. (2) Aggregation function Adenosine diphosphate (ADP) is an important energy transfer molecule in cells, and it plays a key role in the aggregation process of platelets.

[0095] Platelet aggregation function was determined by ADP (10 μM) and collagen (2 μg / mL) induced aggregation rate assay (transmissive method), and the results were expressed as ADP-induced aggregation rate. (3) Thromboside B2 (TXB2) release The concentration of TXB2 in platelet-containing supernatant after thrombin (1 U / mL) stimulation was determined using an ELISA method. In hematology and clinical testing, PLT is the standard abbreviation for platelet count, and the unit is ×10⁻¹⁰. 9 / L (meaning the number of billions of platelets per liter of blood sample). It represents the concentration of platelets in a blood sample. (4) Expression of α-granule membrane protein (CD62P / P-selectin) The basal P-selectin (CD62P) positivity rate of platelets before and after ADP stimulation was detected by flow cytometry.

[0096] Table 2. Platelet recovery rate, purity, concentration, and concentration of residual red blood cells and white blood cells.

[0097] Table 3. Results of platelet activity evaluation

[0098] In the platelet separation and purification methods provided in Examples 1-6, anti-erythrocyte antibodies and anti-leukocyte antibodies are cross-linked across cells via chemical cross-linking to construct antibody complexes that can simultaneously target the surface antigens of erythrocytes and leukocytes (a schematic diagram of the molecular mechanism of dual-antibody-mediated erythrocyte-leukocyte cross-linking is shown in Figure 1). Figure 1As shown in the figure, the antibody complex is mixed with a blood sample and incubated. Large agglomerates are formed through a "red blood cell-antibody complex-white blood cell" bridging network mediated by the antibody complex. Centrifugation of the resulting mixture causes these large agglomerates to settle to the lower layer, simultaneously removing both red blood cells and white blood cells. The supernatant is collected, and any remaining cells and agglomerates are removed (microscopic images of the agglomerates are shown in the figure). Figure 2 As shown, Figure 2 (AB are microscopic images of the agglomerate at different magnifications), thereby achieving the goal of separating and purifying platelets from blood samples, and finally obtaining platelets.

[0099] As shown in Tables 2 and 3, the platelet separation and purification methods provided in Examples 1-6 involve chemically cross-linking anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody using a bifunctional PEG linker. The resulting antibody complex can be used for efficient platelet separation, with platelet recovery rates ≥92% (of which, Examples 1-4 and Example 6 are all >93%), purity ≥98%, and residual red blood cells and residual white blood cells meeting the clinical platelet transfusion standards. Furthermore, the activity of the separated and purified platelets is significantly higher than that of Comparative Example 1 (platelet separation using the traditional density gradient centrifugation method).

[0100] Test Example 3 This test case aims to investigate the effects of reaction temperature and time (i.e., coupling temperature and time, with the coupling temperature ranging from 4 to 25°C and the coupling time ranging from 15 to 60 min) involved in the conjugation of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody during the construction of the antibody complex in step S1 of the platelet separation and purification method provided in Example 1) on the conjugation efficiency and the recovery rate and purity of platelets separated and purified from heparin-anticoagulated whole blood. The results are shown in Table 4.

[0101] Table 4. Effects of conjugation temperature and time on the conjugation efficiency of the two antibodies, as well as platelet recovery rate and purity.

[0102] Note: In Table 4, monomer retention rate refers to the proportion of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody that still exist in monomer form in the target antibody complex obtained after conjugation of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody; yield refers to the recovery rate of the target antibody complex obtained after conjugation and purification of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody.

[0103] As shown in Table 4, during the conjugation of activated anti-CD235a and anti-CD45 monoclonal antibodies using NHS-PEG-NHS, increasing the temperature significantly shortens the reaction time and improves the conjugation efficiency. However, exceeding 30 min leads to increased hydrolysis and non-specific cross-linking, resulting in a decrease in yield and monomer retention. Furthermore, the conjugation effect of the two antibodies is optimal at a conjugation temperature of 25℃ and a conjugation time of 30 min, with a conjugation efficiency ≥90%, monomer retention ≥92%, yield ≥90%, platelet recovery ≥95%, and platelet purity ≥98%. These optimal conjugation conditions are suitable for applications that are particularly sensitive to stability, achieving higher net yield and more controllable consistency with a shorter process time, while maximizing platelet recovery and purity.

[0104] Test Example 4 This test case aims to investigate the effect of the number-average molecular weight (2000, 5000, 6000, 10000 g / mol) of polyethylene glycol (PEG) in NHS-PEG-NHS used in the antibody complex construction process of step S1 of the platelet separation and purification method provided in Example 1 on the coupling efficiency between anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody. The results are shown in Table 5.

[0105] Table 5. Effects of the number-average molecular weight of PEG in NHS-PEG-NHS on the conjugation efficiency of the two antibodies and on platelet recovery, purity, and residual red blood cells and white blood cells.

[0106] Note: In Table 5, coupling efficiency, platelet recovery rate and platelet concentration are expressed as mean ± standard deviation (n=7).

[0107] As shown in Table 5, in step S1 of the platelet separation and purification method provided in Example 1, the PEG in the NHS-PEG-NHS used in the construction of the antibody complex is between 2 and 10 kDa. The coupling efficiency of the two antibodies, anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody, shows an inverted U-shaped trend, that is, as the number-average molecular weight of PEG increases, the coupling efficiency of the two antibodies first increases and then decreases. Medium molecular weight (5-6 kDa) PEG has a better balance between spatial configuration and effective collision frequency, and the coupling efficiency and functional performance (platelet recovery rate / purity) are the best. PEG chains that are too short (2 kDa) are easily affected by steric hindrance and span limitation, thus affecting the coupling efficiency. PEG chains that are too long (10 kDa) are also affected by steric hindrance and span limitation, thus affecting the coupling efficiency. When the number average molecular weight (NMR) of PEG in NHS-PEG-NHS is too high or too low, it will affect the conjugation efficiency of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody. This will result in a decrease in the recovery rate and purity of platelets extracted from heparin-anticoagulated whole blood using the antibody complex obtained by conjugation. Furthermore, a NMR of PEG in NHS-PEG-NHS between 5 and 6 kDa can provide a more stable transcellular link, which optimizes the conjugation efficiency of anti-CD235a monoclonal antibody and anti-CD45 monoclonal antibody, thereby improving the recovery rate and purity of platelets extracted from heparin-anticoagulated whole blood using the antibody complex obtained by conjugation.

[0108] Test Example 5 This test case aims to utilize the effects of the final concentration (0-5 wt%) of PEG6000 added in step S2 of the platelet separation and purification method provided in Example 1 and the sedimentation time (i.e., static incubation time) on the particle size of the agglomerate and the recovery rate, purity, and activity of platelets separated and purified from heparin-anticoagulated whole blood. The results are shown in Table 6.

[0109] Table 6. Effects of final PEG 6000 concentration and sedimentation time on particle size of agglomerates and on platelet recovery, purity, and activity.

[0110] As shown in Table 6, when the final concentration of PEG 6000 is 3 wt%, the exclusion force reaches the optimal level, the particle size of the aggregates increases to 62 μm, and the sedimentation rate is significantly improved. When the concentration is too high (>5%), the solution viscosity is too high (relative viscosity >2.5), which hinders the sedimentation of impurity cells and may slightly affect the fluidity of platelet membranes, resulting in a slight decrease in activity.

[0111] Test Example 6 This test case aims to utilize the centrifugation conditions in step S3 of the platelet separation and purification method provided in Example 1 to affect the recovery rate, purity, activity, and residual red blood cells and residual white blood cells of platelets separated and purified from heparin-anticoagulated whole blood. The results are shown in Table 7.

[0112] Table 7. Effects of centrifugation conditions on platelet recovery, purity, activity, and residual red blood cells and white blood cells.

[0113] As shown in Table 7, in the process of separating and purifying platelets from heparin-anticoagulated whole blood using the method provided in Example 1, the optimal centrifugation conditions for the mixture in step S3 are 300 g centrifugation force and 8 minutes centrifugation time. Under these conditions, impurity cells are fully settled while platelet loss and decreased activity are avoided.

[0114] Test Example 7 This test case aims to apply the platelets isolated and purified from blood samples using the methods provided in Examples 1-4 and Comparative Example 1 to the preparation of platelet-rich plasma (PRP), and to evaluate the feasibility of its clinical application by applying the prepared PRP to in vitro cell experiments. The specific experimental procedures are as follows: 1. Preparation of PRP Take 8.2 mL of platelet suspension obtained from Examples 1-4 and Comparative Example 1, and add autologous plasma (prepared by centrifugation at 3000 g × 15 min) until the platelet concentration reaches (1.0~1.5) × 10⁻⁶. 6 Mix each sample at a density of 1 μL gently to obtain approximately 10 mL of PRP.

[0115] 2. ELISA method for detecting growth factor content The contents of three growth factors—human platelet-derived growth factor (PDGF-AB), transforming growth factor-β1 protein (TGF-β1), and vascular endothelial growth factor (VEGF)—in PRP prepared from platelets isolated and purified using Examples 1-4 and Comparative Example 1 were detected by ELISA. The results are shown in Table 8.

[0116] Table 8 Results of growth factor content detection in PRP

[0117] As shown in Table 8, the contents of the three growth factors PDGF-AB, TGF-β1 and VEGF in PRP obtained from platelets isolated and purified using the methods provided in Examples 1 to 4 are all higher than those in Comparative Example 1.

[0118] 3. Clinical applications (in vitro cell experiments) (1) Cell proliferation and differentiation experiments were conducted using human fibroblasts (HFF-1) and human mesenchymal stem cells (hMSC); (2) Seed cells in 96-well plates (5 × 10⁶ cells / well). 3 Cells / wells were each supplemented with 10% PRP, and 10% fetal bovine serum (FBS) was added as a negative control group. (3) After 72 hours of culture, the proliferation of HFF-1 cells was detected by CCK-8 assay, and the expression of osteogenic differentiation marker genes (RUNX2, ALP, OCN) of hMSCs was detected by qRT-PCR to assess the osteogenic differentiation of hMSCs. The results are shown in Table 9. In the results of osteogenic differentiation marker gene expression detection, the osteogenic differentiation marker gene expression level after culturing hMSCs with PRP prepared from platelets isolated and purified from Comparative Example 1 was used as the baseline (denoted as c1). The osteogenic differentiation marker gene expression level after culturing hMSCs with PRP prepared from platelets isolated and purified from Examples 1 to 4 was denoted as c2. The final result of osteogenic differentiation marker gene expression level after culturing hMSCs with PRP prepared from platelets isolated and purified from Examples 1 to 4 was expressed as c2 / c1, that is, how many times the osteogenic differentiation marker gene expression level of Comparative Example 1 was compared with the baseline (i.e., Comparative Example 1) (i.e., relative expression level).

[0119] Table 9. HFF-1 cell proliferation and osteogenic differentiation of hMSC cells

[0120] As shown in Table 9, the PRP prepared from platelets isolated and purified using the methods provided in Examples 1-4 exhibited a higher proliferation rate in HFF-1 cells compared to Comparative Example 1. Furthermore, the relative expression levels of osteogenic differentiation marker genes in hMSC cells prepared from PRP prepared from platelets isolated and purified using the methods provided in Examples 1-4 were 1.7-1.9 times higher than those in Comparative Example 1. These findings regarding HFF-1 cell proliferation and hMSC cell osteogenic differentiation demonstrate that the highly active platelets prepared using the platelet isolation and purification method provided in this invention can be used to prepare high-quality PRP with high growth factor content and strong tissue regeneration promotion capacity, making it suitable for clinical applications in orthopedics, dentistry, and plastic surgery.

[0121] Test Example 8 This test case aims to utilize the method provided in Example 1 to isolate and purify platelets from blood samples for the preparation of platelet-rich fibrin (PRF), and then apply the prepared PRF to in vitro cell experiments to evaluate its clinical application feasibility. The specific experimental procedures are as follows: 1. Preparation of PRF Take 8.2 mL of platelet suspension obtained from Examples 1-4 and Comparative Example 1, add 100 μL of 10% CaCl2 solution (final concentration 10 mM) to activate coagulation, let stand at room temperature for 10 minutes to form PRF gel, centrifuge at 3000 g for 12 minutes, squeeze to remove serum, and obtain PRF membrane.

[0122] 2. Morphological examination The PRF membrane structure was observed using scanning electron microscopy (SEM). The results showed that the fibrin network in the PRF membrane prepared from platelets isolated and purified in Examples 1-4 was dense, with pore sizes between 2 and 5 μm. Platelets were uniformly distributed within the fibrin network, with a density of approximately (3.5 ± 0.4) × 10⁻⁶. 5 / mm 2 In contrast, the PRF membrane prepared from platelets purified using Comparative Example 1 had a sparse fibrin network with pore sizes between 5 and 10 μm and a platelet density of only (1.8 ± 0.3) × 10⁻⁶. 5 / mm 2 .

[0123] 3. Growth factor release kinetics PRF membranes were immersed in PBS and stored at 4°C. Samples were taken periodically to detect the concentrations of two growth factors, platelet-derived growth factor (PDGF-AB) and transforming growth factor-β1 (TGF-β1). The results are shown in Table 10.

[0124] Table 10 Results of growth factor concentration detection in PRF membrane

[0125] As shown in Table 10, the concentrations of PDGF-AB and TGF-β1 in the PRF membranes prepared from platelets isolated and purified using the methods provided in Examples 1-4 are higher than those in Comparative Example 1. Furthermore, during storage at 4°C, both PDGF-AB and TGF-β1 in the PRF membranes prepared from platelets isolated and purified using the methods provided in Examples 1-4 exhibit a typical "slow-release" pattern. The concentrations of PDGF-AB and TGF-β1 reach a peak on day 3 and then slowly decrease, with a sustained release time exceeding 14 days, meeting the long-term requirements for tissue regeneration. These results demonstrate that the highly active platelets prepared using the platelet isolation and purification method provided by this invention can be used to prepare high-quality PRF with high growth factor content, strong tissue regeneration promotion ability, and slow-release growth factors, making it suitable for the long-term needs of tissue regeneration and applicable in orthopedics, dentistry, and plastic surgery.

[0126] The above results demonstrate that the highly active platelets prepared using the platelet separation and purification method provided by this invention can be used to prepare high-quality PRF, which has high platelet density and sustained release of growth factors, making it suitable for clinical applications such as dental implants, periodontal regeneration, and bone defect repair.

[0127] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A method for separating and purifying platelets, characterized by, The method comprises the following steps: S1. chemically cross-linking an anti-red blood cell antibody and an anti-white blood cell antibody to obtain an antibody complex; S2. incubating the antibody complex after mixing with a blood sample to obtain a mixed solution; S3. collecting supernatant after centrifuging the mixed solution, removing the mixed cells and coagulation clots in the supernatant, and obtaining platelets.

2. The method for separating and purifying platelets according to claim 1, wherein: the blood sample is centrifuged at 1,000 to 2,000 rpm for 10 to 20 minutes. In the S1, the anti-red blood cell antibody and the anti-white blood cell antibody are chemically cross-linked by a bifunctional PEG linker selected from one of N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide, polyethylene glycol diamine, aminooxy-PEG-NHS, hydrazine-PEG-NHS, and maleimide-PEG-NHS; In the S1, the anti-red blood cell antibody is an anti-CD235a monoclonal antibody, and the anti-white blood cell antibody is an anti-CD45 monoclonal antibody.

3. The method for separating and purifying platelets according to claim 2, wherein The antibody complex is prepared by the following steps: a. pretreating the anti-CD235a monoclonal antibody with a PBS buffer or a HEPES buffer to obtain a first antibody solution, and pretreating the anti-CD45 monoclonal antibody with a PBS buffer or a HEPES buffer to obtain a second antibody solution; b. adding the first antibody solution and the second antibody solution into N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide, and reacting at 20-25 DEG C in the dark for 15-60 min, then adding Tris-HCl or glycine into the reaction system and reacting for 10-20 min to prepare the antibody complex.

4. The method for separating and purifying platelets according to claim 3, wherein the blood sample is centrifuged at 1,000 to 2,000 rpm for 10 to 20 minutes. In the b, the number average molecular weight of polyethylene glycol in the N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide is 5000-6000 g / mol.

5. The method for separating and purifying platelets according to claim 2, wherein The antibody complex is prepared by the following steps: a. preparing an antibody mixture by using the anti-CD235a monoclonal antibody, the anti-CD45 monoclonal antibody, and 2-morpholinoethanesulfonic acid buffer, adding 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide into the antibody mixture, and reacting at 0-25 DEG C in the dark for 10-30 min to obtain a reaction solution; b. adding a PBS buffer or a HEPES buffer into the reaction solution, then adding polyethylene glycol diamine and reacting at 20-25 DEG C for 15-60 min, then adding Tris-HCl or glycine into the reaction system and reacting for 10-20 min to prepare the antibody complex.

6. The method for separating and purifying platelets according to claim 2, wherein The antibody complex is prepared by the following steps: a. preparing an anti-CD235a monoclonal antibody solution by using the anti-CD235a monoclonal antibody and sodium acetate buffer, adding NaIO4 into the anti-CD235a monoclonal antibody solution and mixing and incubating at 0-4 DEG C in the dark for 25-35 min, then adding ethylene glycol into the system and incubating at 0-4 DEG C for 2-5 min to obtain an aldehyde-bearing antibody; b. After mixing the aldehyde-bearing antibody with aminooxy-PEG-NHS or hydrazide-PEG-NHS, the mixture is added to a sodium acetate buffer and incubated at 20-25℃ for 10-20 min, then aniline is added to the system and reacted at 0-4℃ for 2-4 h to obtain a first intermediate; c. After mixing the first intermediate with the anti-CD45 monoclonal antibody, the mixture is added to a PBS buffer or a HEPES buffer and reacted at 20-25℃ for 15-45 min, then Tris-HCl is added to the system and reacted for 10-20 min to obtain the antibody complex.

7. The method for separating and purifying platelets according to claim 2, wherein The antibody complex is prepared by the following steps: a. An anti-CD235a monoclonal antibody solution is prepared by using the anti-CD235a monoclonal antibody and a first buffer, TCEP or DTT is added to the anti-CD235a monoclonal antibody solution and incubated at 20-25℃ for 15-30 min to obtain a first mixture; b. After adding maleimide PEG-NHS to the first mixture, HEPES buffer or PBS buffer is added to the system and incubated at 20-25℃ in the dark for 15-45 min to obtain a second intermediate; c. After mixing the second intermediate with the anti-CD45 monoclonal antibody, HEPES buffer or PBS buffer is added to the system and reacted at 20-25℃ for 15-45 min, then Tris-HCl is added to the system and reacted for 10-20 min to obtain the antibody complex.

8. The method for separating and purifying platelets according to claim 1, wherein: the blood sample is collected from a patient suffering from a disease selected from the group consisting of leukemia, lymphoma, and myeloma. In the S2, after mixing the antibody complex with the blood sample, PEG 6000 is further added to the system so that the final concentration of PEG 6000 in the system is 2-4 wt%.

9. A platelet, characterized by: The platelets are isolated and purified by the method for isolating and purifying platelets according to any one of claims 1-8.

10. The platelets of claim 9 are used in the preparation of platelet-rich plasma or platelet-rich fibrin.

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