Preparation method of human activated C1 esterase

Abstract

The invention relates to the field of blood products, in particular to a preparation method of human activated C1 esterase. The preparation method comprises the following steps: carrying out contact adsorption on cryoprecipitate-removed human plasma and anion exchange resin, washing and eluting to obtain an eluent containing C1 proesterase, carrying out membrane filtration, and carrying out ultrafiltration desalination treatment; adding an anionic substance into the eluent, incubating in vitro to effectively activate the C1 esterase, and filtering by a membrane to obtain a feed liquid containing the activated C1 esterase; and carrying out purification treatment on the feed liquid to obtain the human activated C1 esterase, and carrying out cryopreservation. According to the method, an anion substance is introduced into an eluent system obtained through anion exchange, mild and effective activation of the C1 proesterase is achieved under the condition of not depending on foreign proteolytic enzyme, the risk of non-specific hydrolysis and self-cutting inactivation is remarkably reduced, good synergy is formed with a follow-up purification process, and the method is suitable for industrial production. The human activated C1 esterase product with relatively high activity and good stability is obtained.

Description

Technical Field

[0001] This invention relates to the field of blood products, and more particularly to a method for preparing human activated C1 esterase. Background Technology

[0002] The complement system is an important component of the body's innate immunity, with the classical complement pathway playing a crucial role in antibody-mediated immune responses. Complement esterases (C1s) are the core functional enzymes in the classical complement pathway, belonging to the serine esterase family. Upon activation, they specifically cleave complement proteins C4 and C2, thereby initiating subsequent complement cascade reactions. Therefore, activating C1 esterases has significant application value in complement function research, elucidation of immune regulatory mechanisms, and screening of relevant inhibitors.

[0003] In existing technologies, the activation of C1 esteraseogen is generally considered to be a process dependent on the direct action of the "activating enzyme-substrate" interaction. Therefore, the in vitro activation of C1 esteraseogen mainly relies on proteolysis, that is, the targeted cleavage of C1 esteraseogen by C1r or other proteases, transforming it from a single-chain structure to a double-chain activated structure. However, this type of activation generally suffers from the problem of high controllability. On the one hand, the proteolysis reaction itself is difficult to precisely restrict to a single activation site, easily triggering non-specific cleavage or autocleavage reactions, resulting in enzyme structure damage or reduced activity. On the other hand, the activation process is highly sensitive to reaction time, enzyme dosage, and system conditions, exhibiting poor reproducibility and stability, which is not conducive to the development of a controllable and reliable in vitro activation method. Summary of the Invention

[0004] To address the problems of uncontrollable activation, easy inactivation, and difficult purification of C1 esterase in existing technologies, this invention provides a method for preparing human activated C1 esterase. This method involves introducing anionic substances into the eluent obtained through anion exchange to promote the effective activation of C1 esterase precursor under in vitro conditions. Combined with multi-stage chromatography, this achieves efficient separation and purification of the activated C1 esterase, thereby obtaining a structurally intact and stably active human activated C1 esterase product. The specific technical solution is as follows: A method for preparing human activated C1 esterase includes the following steps: (1) Human plasma with cryoprecipitate removed was contacted with anion exchange resin for adsorption, and then washed and eluted to obtain an eluent containing C1 esterase progenitourinus. After membrane filtration, it was subjected to ultrafiltration desalting treatment. (2) Add anionic substances to the eluent obtained in step (1), incubate in vitro to effectively activate C1 esterase, and obtain a feed solution containing activated C1 esterase by membrane filtration. (3) The liquid obtained in step (2) is purified to obtain human activated C1 esterase and then frozen.

[0005] By introducing anionic substances during the in vitro activation stage, effective activation of C1 esteraseogen was achieved under mild conditions, avoiding reliance on complex cascade reactions or non-specific protein hydrolysis processes, thus significantly improving the controllability and stability of the activation process. The activated C1 esterase obtained by this method exhibits high activity and low levels of contaminating proteins, making it suitable for subsequent purification and separation.

[0006] Furthermore, in step (2), the anionic substance is one or both of perlite and diatomaceous earth. Perlite and diatomaceous earth are loose and porous, which increases the contact area between the liquid and the anions, and their activation efficiency for C1 esterase is significantly better than that of other materials.

[0007] Furthermore, in step (2), the amount of anionic substance added is 0.5% to 5% of the mass of the eluent, the incubation temperature is 2 to 20°C, the incubation time is 1 to 15 hours, and the pore size of the filter membrane used is 0.1 to 1 μm.

[0008] Furthermore, in step (1), the method for preparing human plasma with removed cryoprecipitate is as follows: fresh frozen human plasma is dissolved, mixed and centrifuged to obtain supernatant, and then filtered through a membrane to obtain human plasma with removed cryoprecipitate; the centrifugation temperature is 0~4℃ and the pore size of the filter membrane is 0.2~0.45μm.

[0009] Furthermore, the anion exchange resin in step (1) is one or a combination of DEAE Sephadex A50, DEAE Sepharose FF, Q Sepharose FF, Q Sepharose XL, Capto DEAE, Capto Q; Eshmuno Q, Fractogel EMDTMAE, and Fractogel EMD DEAE.

[0010] Furthermore, in step (1), the solution used for washing and elution is one or more of sodium citrate solution, sodium citrate solution, and sodium chloride solution, with a solution pH of 6.5 to 7.3.

[0011] Furthermore, the concentration of the sodium citrate solution is 0.01~0.05M, the concentration of the sodium citrate solution is 0.01~0.05M, and the concentration of the sodium chloride solution can be 0.1~0.2M.

[0012] Furthermore, in step (3), the purification process includes one or more chromatographic methods selected from anion exchange chromatography, affinity chromatography, gel filtration chromatography, and hydrophobic chromatography.

[0013] Furthermore, the affinity resin used in affinity chromatography is selected from one or more combinations of IgG Sepharose 6 Fast Flow, IgG Sepharose 4 Fast Flow, CNBr-activated Sepharose 4B, NHS-activated Sepharose 4 Fast Flow, NHS-activated Sepharose 6 Fast Flow, Epoxy-activated Sepharose 6B, Eupergit C, Eupergit C250L, ECR8204F, Affi-Gel 10, and Affi-Gel 15.

[0014] Furthermore, the anion exchange resin used in the anion exchange chromatography is selected from one or more combinations of DEAE Sephadex A50, DEAE Sepharose FF, Q Sepharose FF, Q Sepharose XL, Capto DEAE, Capto Q; Eshmuno Q, Fractogel EMD TMAE, and Fractogel EMD DEAE.

[0015] Furthermore, in step (4), the freezing temperature is -70~-20℃.

[0016] The beneficial effects of this invention are as follows: 1. The method for preparing human activated C1 esterase provided by this invention is based on previous mature blood product preparation process experience. Addressing the technical characteristics of human activated C1 esterase under in vitro conditions—namely, the difficulty in controlling activation, easy inactivation, and the unfavorable conditions for subsequent purification—this invention innovatively proposes a preparation method centered on high-efficiency activation. This method introduces anionic substances into the eluent system obtained through anion exchange, achieving mild and effective activation of C1 esterase progenitor without relying on exogenous proteolytic enzymes, significantly reducing the risk of non-specific hydrolysis and autoclast inactivation. Simultaneously, this activation method synergizes well with subsequent purification processes such as affinity chromatography and anion exchange chromatography, enabling the activated C1 esterase to maintain good structural stability and adaptability for separation and purification, thereby obtaining human activated C1 esterase products with high activity and good stability.

[0017] 2. The overall process route of this invention uses plasma as raw material. The process units employed are mature and reliable, with mild operating conditions, offering advantages such as high raw material utilization, high product yield, and low preparation cost. It is suitable for large-scale preparation and industrial promotion. The human activated C1 esterase prepared by the method of this invention not only expands the comprehensive utilization pathways of plasma resources and increases the added value of plasma products, but also fills the technological gap in the domestic field of human activated C1 esterase preparation, forming China's first independently developed human activated C1 esterase product. This has significant economic value and strategic significance for the comprehensive utilization of plasma, and plays an important role in enhancing my country's independent controllability in complement-related research and biological reagents. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Ultra-high performance liquid chromatogram of the sample prepared in Example 5 of this invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] Example 1 (1) Plasma pretreatment Fresh frozen human plasma was used as raw material, completely thawed at 0°C, and centrifuged at 4°C to remove cryoprecipitate from the plasma. The supernatant after centrifugation was filtered through a 0.22μm filter membrane to obtain plasma supernatant.

[0022] (2) Anion exchange adsorption and elution Adjust the pH of the plasma supernatant to 7.0, add DEAE Sephadex A-50 gel at a ratio of 1.5 g / kg, and adsorb for 30 min under stirring. Then wash with a buffer solution of 0.02 M sodium citrate, 0.2 M sodium chloride, and pH 7.0 to remove impurities and proteins, washing 3 times, each time for no less than 10 min. Then elute 3 times with a buffer solution of 0.02 M sodium citrate, 1 M sodium chloride, and pH 7.0. Collect the eluent, filter it through a 0.22 μm filter membrane, and then perform ultrafiltration desalting until the conductivity is lower than 15 mS / cm.

[0023] (3) Perlite-mediated activation treatment Add perlite at 5% of its mass to the eluent obtained in step (2), incubate at 4°C for 14 hours, and then filter with a 0.22 μm filter membrane to obtain the treated liquid.

[0024] (4) IgG Sepharose 6 Fast Flow affinity chromatography purification Affinity chromatography was performed using IgG Sepharose 6 Fast Flow as the chromatographic medium. Equilibration buffer was prepared with 0.15 M sodium chloride, 0.02 M Tris-HCl, pH 7.2. Washing was performed with a solution of 0.15 M sodium chloride, 0.02 M Tris-HCl, 0.025 M Tween-80, pH 7.2, followed by elution with a solution of 0.5 M sodium chloride, 0.02 M Tris-HCl, 0.025 M Tween-80, pH 7.2. The eluent was collected.

[0025] (5) Capto Q anion exchange chromatography purification Purification was performed using Capto Q as the anion exchange chromatography medium. An equilibration solution of 0.07 M sodium chloride, 0.02 M disodium hydrogen phosphate, 0.01 M sodium citrate, and pH 7.2 was used. The mixture was washed with a solution of 0.09 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2, followed by elution with a solution of 0.2 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2. The eluent was collected to obtain human activated C1 esterase, which was then stored at -70°C.

[0026] Example 2 (1) Plasma pretreatment Fresh frozen human plasma was used as raw material, completely thawed at 0°C, and centrifuged at 4°C to remove cryoprecipitate from the plasma; the supernatant after centrifugation was filtered through a 0.22μm filter membrane to obtain plasma supernatant.

[0027] (2) DEAE Sephadex A-50 adsorption and elution Adjust the pH of the plasma supernatant to 7.0, add DEAE Sephadex A-50 gel at a ratio of 1.5 g / kg, and adsorb for 30 min under stirring. Then wash the impurities three times with a buffer of 0.02 M sodium citrate, 0.2 M sodium chloride, and pH 7.0, with each wash lasting at least 10 min. Elute three times with a buffer of 0.02 M sodium citrate, 1 M sodium chloride, and pH 7.0, and collect the eluent. Filter the eluent through a 0.22 μm filter membrane and then perform ultrafiltration desalting until the conductivity is below 15 mS / cm.

[0028] (3) Diatomaceous earth-mediated activation treatment Add 0.5% of the mass of diatomaceous earth to the eluent obtained in step (2) and incubate at 4°C for 14 hours. After incubation, filter the solution using a 0.22μm filter membrane to obtain the activated solution.

[0029] (4) Capto Q anion exchange chromatography purification The solution obtained in step (3) was purified by anion exchange chromatography using Capto Q as the chromatographic medium. A solution of 0.07 M sodium chloride, 0.02 M disodium hydrogen phosphate, 0.01 M sodium citrate, and pH 7.2 was used as the equilibration solution. The solution was washed with a solution of 0.09 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2, and eluted with a solution of 0.2 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2. The eluent was collected.

[0030] (5) CNBr-activated Sepharose™ 4B affinity chromatography purification The CNBr-activated Sepharose™ 4B gel was pre-washed several times with 1mM HCl to remove the protective agent. Then, in alkaline coupling buffer (0.1M NaHCO3, 0.5M NaCl, pH 8.3), IgG (concentration ≥2mg / mL) was mixed with the gel at a 1:1 volume ratio, and the mixture was rotated at room temperature for 1.5 h to enhance coupling efficiency. After coupling, unreacted sites were blocked with 0.1M Tris-HCl (pH 8.0) for 1 h. Three washes were performed, alternating between 0.1M sodium acetate + 0.5M NaCl solution at pH 4.0 and 0.1M Tris-HCl + 0.5M NaCl solution at pH 8.0, to remove non-covalently bound proteins, with 3-5 column volumes per wash. Finally, the coupled IgG-Sepharose was equilibrated with the target chromatography buffer and stored in a preservation solution containing 20% ​​ethanol at 4°C for later use.

[0031] The above-mentioned IgG-Sepharose was used as the affinity chromatography medium to purify the eluent obtained in step (4) by affinity chromatography. A solution of 0.15M sodium chloride, 0.02M Tris-HCl, and pH 7.2 was used as the affinity equilibration solution. The solution was washed with a solution of 0.15M sodium chloride, 0.02M Tris-HCl, 0.025M Tween-80, and pH 7.2, and then eluted with a solution of 0.5M sodium chloride, 0.02M Tris-HCl, 0.025M Tween-80, and pH 7.2. The eluent was collected.

[0032] (6) Fractogel EMD DEAE anion exchange chromatography purification The eluent obtained in step (5) was purified by anion exchange chromatography using Fractogel EMDDEAE as the chromatographic medium. An equilibration solution of 0.07 M sodium chloride, 0.02 M disodium hydrogen phosphate, 0.01 M sodium citrate, and pH 7.2 was used. The eluent was washed with a solution of 0.09 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2, and eluted with a solution of 0.2 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2. The eluent was collected to obtain human activated C1 esterase, which was then frozen at -20°C.

[0033] Example 3 (1) Plasma pretreatment Fresh frozen human plasma was used as raw material, completely thawed at 0°C, and centrifuged at 4°C to remove cryoprecipitate from the plasma; the plasma supernatant after centrifugation was filtered through a 0.22μm filter membrane to obtain plasma supernatant.

[0034] (2) DEAE Sephadex A-50 adsorption and elution Adjust the pH of the plasma supernatant to 7.0, add DEAE Sephadex A-50 gel at a ratio of 1.5 g / kg, and adsorb for 30 min under stirring. Then wash the impurities three times with a buffer of 0.02 M sodium citrate, 0.2 M sodium chloride, and pH 7.0, with each wash lasting at least 10 min. Elute three times with a buffer of 0.02 M sodium citrate, 1 M sodium chloride, and pH 7.0. Collect the eluent, filter it through a 0.22 μm filter membrane, and then perform ultrafiltration desalting until the conductivity is below 15 mS / cm.

[0035] (3) Perlite / diatomite-mediated activation treatment Add 1% perlite and 2% diatomaceous earth by mass of the eluent obtained in step (2) to the eluent and incubate at 4°C for 14 hours. After incubation, filter the solution using a 0.22 μm filter membrane to obtain the activated solution.

[0036] (4) IgG Sepharose 6 Fast Flow affinity chromatography purification The solution obtained in step (3) was purified by affinity chromatography using IgG Sepharose 6 Fast Flow as the affinity chromatography medium. The solution was equilibrated with a solution of 0.1 M sodium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M Tris-HCl, and pH 7.2. It was washed with a solution of 0.15 M sodium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M Tris-HCl, 0.025 M Tween-80, and pH 7.2. The solution was eluted with a solution of 0.5 M sodium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M Tris-HCl, 0.025 M Tween-80, and pH 7.2, and the eluent was collected.

[0037] (5) Fractogel EMD TMAE anion exchange chromatography purification The eluent obtained in step (4) was purified by anion exchange chromatography using Fractogel EMDTMAE as the chromatographic medium. A solution of 0.07 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2 was used as the equilibration solution. The eluent was washed with a solution of 0.09 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2, followed by washing with a solution of 0.2 M sodium chloride, 0.02 M disodium hydrogen phosphate, and pH 7.2. The eluent was collected to obtain human activated C1 esterase, which was then frozen at -50°C.

[0038] Example 4: Detection of activated C1 esterase titer To quantitatively determine the titer of C1 esterase in the eluent obtained from chromatography, a standard curve of C1 esterase activity versus concentration based on reaction rate (A405 / min) was established. First, a series of gradient solutions of C1 esterase standards with concentrations of 20, 25, 30, 35, 40, 60, and 80 μg / mL were prepared. Under fixed reaction conditions, a chromogenic substrate C1E5603 (CH3OCLys(Cbo)-Gly-Arg-pNA·AcOH) with a final concentration of 1.5 μmol / mL was added to each standard solution to initiate the enzymatic reaction. The absorbance change at 405 nm was continuously monitored using a microplate reader, and the reaction rate (A405 / min) corresponding to each concentration was calculated. A standard curve was plotted with C1 esterase concentration on the x-axis and reaction rate (A405 / min) on the y-axis, and linear regression was performed to obtain the regression equation between C1 esterase activity and concentration. Subsequently, the flow-through or eluted samples obtained by chromatography were measured under the same substrate concentration and reaction conditions as the standard to obtain the corresponding A405 / min value. This value was then substituted into the above regression equation for calculation to determine the potency of activated C1 esterase in the sample.

[0039] Samples from one batch of Example 1, one batch of Example 2, and three batches of Example 3 were selected, and their activated C1 esterase titers were determined. The results are shown in Table 1.

[0040] Table 1. Activated C1 esterase titer of different batches of products

[0041] Example 5: HPLC Purity Detection The purity of the sample obtained in Example 1 was determined by ultra-high performance liquid chromatography (UPLC / HPLC). In the chromatographic analysis, the mobile phase was a buffer solution prepared from sodium phosphate, sodium chloride, and acetonitrile. A UV detector was used, and the detection wavelength was set to 280 nm. The chromatographic system employed a single-pump system for isocratic elution at a flow rate of 0.25 mL / min. A TOSOH TSKgel UP-SW Aggregate size exclusion column was used, with an injection volume of 10 μL and a single run time of 25 min. The detection results were calculated using the area normalization method, and each chromatographic peak was automatically integrated by the system. The purity of the sample obtained in Example 1 was 77.8%, and its UPLC chromatogram is shown below. Figure 1 As shown.

[0042] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for preparing human activated C1 esterase, characterized in that, Includes the following steps: (1) Human plasma with cryoprecipitate removed was contacted with anion exchange resin for adsorption, and then washed and eluted to obtain an eluent containing C1 esterase progenitourinus. After membrane filtration, it was subjected to ultrafiltration desalting treatment. (2) Add anionic substances to the eluent obtained in step (1), incubate in vitro to effectively activate C1 esterase, and obtain a feed solution containing activated C1 esterase by membrane filtration. (3) The liquid obtained in step (2) is purified to obtain human activated C1 esterase and then frozen.

2. The preparation method according to claim 1, characterized in that, In step (2), the anionic substance is one or both of perlite and diatomite.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the amount of anionic substance added is 0.5% to 5% of the mass of the eluent, the incubation temperature is 2 to 20°C, the incubation time is 1 to 15 hours, and the pore size of the filter membrane is 0.2 to 0.45 μm.

4. The preparation method according to claim 1, characterized in that, In step (1), the preparation method of human plasma with cryoprecipitate removed is as follows: fresh frozen human plasma is dissolved, mixed and centrifuged to obtain supernatant, and then filtered through a membrane to obtain human plasma with cryoprecipitate removed; the centrifugation temperature is 0~4℃ and the pore size of the filter membrane is 1~10μm.

5. The preparation method according to claim 1, characterized in that, The anion exchange resin in step (1) is one or a combination of DEAESephadex A50, DEAE Sepharose FF, Q Sepharose FF, Q Sepharose XL, Capto DEAE, CaptoQ; Eshmuno Q, Fractogel EMD TMAE, and Fractogel EMD DEAE.

6. The preparation method according to claim 1, characterized in that, In step (1), the washing and elution solutions used are one or more combinations of sodium citrate solution, sodium citrate solution, and sodium chloride solution, with a pH of 6.5 to 7.3; the concentration of sodium citrate solution is 0.01 to 0.05 M, the concentration of sodium citrate solution is 0.01 to 0.05 M, and the concentration of sodium chloride solution can be 0.1 to 0.2 M.

7. The preparation method according to claim 1, characterized in that, In step (3), the purification process includes one or more chromatographic methods, which are selected from one or more of anion exchange chromatography, affinity chromatography, gel filtration chromatography, and hydrophobic chromatography.

8. The preparation method according to claim 7, characterized in that, The affinity resin used in affinity chromatography is selected from one or more combinations of IgG Sepharose 6 Fast Flow, IgG Sepharose 4 Fast Flow, CNBr-activated Sepharose 4B, NHS-activated Sepharose 4 Fast Flow, NHS-activated Sepharose 6 Fast Flow, Epoxy-activated Sepharose 6B, Eupergit C, Eupergit C250L, ECR8204F, Affi-Gel 10, and Affi-Gel 15.

9. The preparation method according to claim 7, characterized in that, The anion exchange resin used in anion exchange chromatography is selected from one or more combinations of DEAE Sephadex A50, DEAE Sepharose FF, Q Sepharose FF, Q Sepharose XL, CaptoDEAE, Capto Q; Eshmuno Q, Fractogel EMD TMAE, and Fractogel EMD DEAE.

10. The preparation method according to claim 1, characterized in that, In step (4), the freezing temperature is -70~-20℃.

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