A citrate dialysis concentrate and its preparation method

Abstract

The present invention relates to the technical field of hemodialysis, and more specifically, to a citrate dialysis concentrate and a preparation method thereof. The present invention for the first time provides an anticoagulant factor X antibody and applies it to the preparation of a citrate dialysis concentrate. Through result analysis, a synergistic effect is achieved for the anticoagulation effect on peripheral blood after the combination of the two, and the anticoagulation effect is extremely significant, which can significantly improve the application prospect of the citrate dialysis solution.

Description

Technical Field

[0001] The present invention relates to the technical field of hemodialysis, and more specifically, to a citric acid dialysis concentrate and a preparation method thereof. Background Art

[0002] Hemodialysis (HD) is one of the renal replacement therapies for patients with acute and chronic renal failure. It involves draining the blood in the body to the outside, passing it through a dialyzer composed of countless hollow fibers. Inside and outside each hollow fiber, the blood and an electrolyte solution (dialysate) with a concentration similar to that of the body carry out mass exchange through the principles of diffusion, ultrafiltration, adsorption, and convection to remove metabolic wastes in the body, maintain electrolyte and acid-base balance; at the same time, remove excess water in the body, and the whole process of returning the purified blood is called hemodialysis.

[0003] Citrate anticoagulation is an extracorporeal anticoagulation technique. Calcium ions are a very important coagulation factor in normal human blood and participate in all aspects of blood coagulation. If the blood calcium ion concentration drops to a certain level, the coagulation process of blood circulation will be severely inhibited. During hemodialysis, if citrate is pumped into the extracorporeal circulation, citrate will form calcium citrate with calcium ions in the blood, thereby reducing the blood calcium ion concentration to a very low level, thus achieving the anticoagulation effect.

[0004] However, in the prior art, the anticoagulation effect of the disclosed citric acid dialysis concentrate is poor, and thrombus formation occurs in some patients during hemodialysis, resulting in a decline in the hemodialysis effect. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the present invention provides a citric acid dialysis concentrate and a preparation method thereof. The present invention first provides an anticoagulation factor X antibody and applies it to the preparation of a citric acid dialysis concentrate. Through result analysis, the combination of the two has a synergistic effect on the anticoagulation effect of peripheral blood, and the anticoagulation effect is extremely significant, which can significantly improve the application prospect of the citric acid dialysate.

[0006] Specifically, the present invention first provides a citric acid dialysis concentrate, which is composed of 1.1 - 1.5 M sodium chloride, 10 - 50 mM potassium chloride, 5 - 20 mM calcium chloride, 15 - 45 mM magnesium chloride, 100 - 400 g / L glucose, 1 - 10 mM citric acid, and 100 - 200 g / L anticoagulation factor X antibody in every 1 L of injection water.

[0007] Preferably, the citric acid dialysis concentrate is composed of 1.1 M sodium chloride, 10 mM potassium chloride, 5 mM calcium chloride, 15 mM magnesium chloride, 100 g / L glucose, 1 mM citric acid, and 100 g / L anticoagulation factor X antibody in every 1 L of injection water.

[0008] Preferably, the citrate dialysis concentrate consists of 1.2 M sodium chloride, 25 mM potassium chloride, 15 mM calcium chloride, 30 mM magnesium chloride, 250 g / L glucose, 5 mM citric acid and 150 g / L anticoagulant factor X antibody in each 1 L of water for injection.

[0009] Preferably, the citrate dialysis concentrate consists of 1.5 M sodium chloride, 50 mM potassium chloride, 20 mM calcium chloride, 45 mM magnesium chloride, 400 g / L glucose, 10 mM citric acid and 200 g / L anticoagulant factor X antibody in each 1 L of water for injection.

[0010] More preferably, the anticoagulant factor X antibody is obtained by immunizing BALB / c mice with coagulation factor X as an antigen using hybridoma technology, separating mouse spleen cells, fusing them with SP2 / 0 myeloma cells, and then screening for the anticoagulant factor X antibody.

[0011] More preferably, the heavy chain variable region sequence of the anticoagulant factor X antibody is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

[0012] More preferably, the heavy chain variable region of the anticoagulant factor X antibody includes CDR1, CDR2 and CDR3, and their sequences correspond to those shown in SEQ ID NOs.3 - 5 respectively. The light chain variable region of the anticoagulant factor X antibody includes CDR4, CDR5 and CDR6, and their sequences correspond to those shown in SEQ ID NOs.6 - 8 respectively.

[0013] Another aspect of the present invention is also to provide a method for preparing a citrate dialysis concentrate, which includes the following steps:

[0014] 4) After repeatedly rinsing the clean plastic bucket 3 - 5 times, disinfect it with 75 - 100% ethanol 2 - 5 times, and then wash it with water for injection 3 - 5 times and set it aside for use;

[0015] 5) Accurately calculate the dosage of each component according to the above concentrate culture, weigh them one by one and put them into the plastic bucket in step 1);

[0016] 6) Add an appropriate amount of water for injection, stir it mechanically to dissolve it fully, filter it through a filter screen, and then add the remaining water for injection to make up the full volume.

[0017] Another aspect of the present invention is also to provide the use of the anticoagulant factor X antibody in the preparation of a citrate dialysis concentrate.

[0018] Preferably, the heavy chain variable region sequence of the anticoagulant factor X antibody is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

[0019] Further preferably, the heavy chain variable region of the anti - factor X antibody comprises CDR1, CDR2 and CDR3, the sequences of which correspond to those shown in SEQ ID NO.3 - 5 respectively, and the light chain variable region of the anti - factor X antibody comprises CDR4, CDR5 and CDR6, the sequences of which correspond to those shown in SEQ ID NO.6 - 8 respectively.

[0020] The advantages of the present invention are as follows: The present invention provides an anti - factor X antibody for the first time and applies it to the preparation of citrate dialysis concentrate. Through result analysis, the combination of the two has a synergistic effect on the anticoagulant effect of peripheral blood, and the anticoagulant effect is extremely significant, which can significantly improve the application prospect of citrate dialysis solution. Specific embodiments

[0021] The following specific embodiments are used to further illustrate the present invention in detail, so that those skilled in the art can understand the present invention more clearly.

[0022] The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0023] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well - known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well - known to those skilled in the art.

[0024] Example 1

[0025] A citrate dialysis concentrate, which comprises 1.1 M sodium chloride, 10 mM potassium chloride, 5 mM calcium chloride, 15 mM magnesium chloride, 100 g / L glucose, 1 mM citrate and 100 g / L anti - factor X antibody in every 1 L of injection water.

[0026] Among them, the anti - factor X antibody is obtained by immunizing BALB / c mice with factor X as an antigen using the hybridoma technology, separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells, and then screening the anti - factor X antibody.

[0027] The results show that: the EC 50 value of the anti - factor X antibody binding to factor X is 0.86 nM, which confirms its strong and specific binding to factor X and has a good anticoagulant effect.

[0028] The isolated antibody was sequenced and identified. The heavy chain variable region sequence of the anti - coagulation factor X antibody of the present invention is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2. The heavy chain variable region of the anti - coagulation factor X antibody includes CDR1, CDR2 and CDR3, and their sequences correspond to those shown in SEQ ID NOs.3 - 5 respectively. The light chain variable region of the anti - coagulation factor X antibody includes CDR4, CDR5 and CDR6, and their sequences correspond to those shown in SEQ ID NOs.6 - 8 respectively.

[0029] Furthermore, the preparation method of the citrate dialysis concentrate includes the following steps:

[0030] 1) After repeatedly rinsing the clean plastic bucket 3 times, disinfect it with 75% ethanol 2 times, and then wash it with injection water 3 times and set it aside for use;

[0031] 2) Accurately calculate the dosage of each component according to the above - mentioned concentrate culture, weigh them one by one and put them into the plastic bucket in step 1);

[0032] 3) Add 1 / 2 of the injection water, fully dissolve it by mechanical stirring, filter it through a filter screen, and then add the remaining injection water to make up the full volume.

[0033] Example 2

[0034] A citrate dialysis concentrate, which consists of 1.2 M sodium chloride, 25 mM potassium chloride, 15 mM calcium chloride, 30 mM magnesium chloride, 250 g / L glucose, 5 mM citrate and 150 g / L anti - coagulation factor X antibody in every 1 L of injection water.

[0035] Among them, the preparation method steps are the same as those in Example 1.

[0036] Example 3

[0037] A citrate dialysis concentrate, which consists of 1.5 M sodium chloride, 50 mM potassium chloride, 20 mM calcium chloride, 45 mM magnesium chloride, 400 g / L glucose, 10 mM citrate and 200 g / L anti - coagulation factor X antibody in every 1 L of injection water.

[0038] Among them, the preparation method steps are the same as those in Example 1.

[0039] Comparative Example 1

[0040] A citrate dialysis concentrate, which consists of 1.5 M sodium chloride, 50 mM potassium chloride, 20 mM calcium chloride, 45 mM magnesium chloride, 400 g / L glucose and 10 mM citrate in every 1 L of injection water.

[0041] Among them, the preparation method steps are the same as those in Example 1.

[0042] Comparative Example 2

[0043] A hemodialysis concentrate, which comprises 1.5 M sodium chloride, 50 mM potassium chloride, 20 mM calcium chloride, 45 mM magnesium chloride, 400 g / L glucose, and 200 g / L anticoagulant factor X antibody in every 1 L of injection water.

[0044] Among them, the preparation method steps are the same as those in Example 1.

[0045] Verification Example

[0046] APTT Coagulation Experiment

[0047] Collect rabbit peripheral blood samples, evenly divide them into 5 parts, and quickly mix them evenly with the dialysis concentrate. The volume ratio of the two is 9:1. Subsequently, take out 0.1 mL of the mixed solution and add 0.1 mL of APTT reagent pre-warmed at 37°C for 10 min, incubate at 37°C for 5 min. Then add 0.1 mL of 0.025 M calcium chloride pre-warmed at 37°C, immediately start the stopwatch, and record the coagulation time.

[0048] TT Experiment

[0049] Collect rabbit peripheral blood samples, evenly divide them into 5 parts, and quickly mix them evenly with the dialysis concentrate. The volume ratio of the two is 9:1. Subsequently, take out 0.2 mL of the mixed solution and add 0.2 mL of TT reagent pre-warmed at 37°C for 10 min, mix well, immediately start the stopwatch, and record the coagulation time.

[0050] The results are shown in Table 1.

[0051] Table 1: Test Results

[0052] APTT clotting time (seconds) TT clotting time (seconds) Example 1 35.6 42.7 Example 2 45.5 58.6 Example 3 65.7 75.7 Comparative Example 1 24.5 28.9 Comparative Example 2 15.8 20.8

[0053] It can be seen from the results in Table 1 that under the treatment of the dialysis concentrate described in the embodiment of the present invention, the anticoagulant effect on peripheral blood samples is significantly better than that of the comparative example, further confirming that the combined effect of citric acid and anticoagulant factor X antibody has achieved a synergistic anticoagulant technical effect.

[0054] It is necessary to point out here that the above embodiments are only for further elaborating and explaining the technical solutions of the present invention, and are not further limitations on the technical solutions of the present invention. The method of the present invention is only a preferred implementation scheme and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A citrate dialysis concentrate, characterized in that, Each 1 L of water for injection comprises 1.1 - 1.5 M sodium chloride, 10 - 50 mM potassium chloride, 5 - 20 mM calcium chloride, 15 - 45 mM magnesium chloride, 100 - 400 g / L glucose, 1 - 10 mM citric acid and 100 - 200 g / L anticoagulant factor X antibody; the heavy chain variable region of the anticoagulant factor X antibody comprises CDR1, CDR2 and CDR3, and their sequences respectively correspond to those shown in SEQ ID NO.3 - 5, and the light chain variable region of the anticoagulant factor X antibody comprises CDR4, CDR5 and CDR6, and their sequences respectively correspond to those shown in SEQ ID NO.6 - 8.

2. The citrate dialysis concentrate according to claim 1, characterized in that, Each 1 L of water for injection comprises 1.1 M sodium chloride, 10 mM potassium chloride, 5 mM calcium chloride, 15 mM magnesium chloride, 100 g / L glucose, 1 mM citric acid and 100 g / L anticoagulant factor X antibody.

3. The citrate dialysis concentrate according to claim 1, wherein, Each 1 L of water for injection comprises 1.2 M sodium chloride, 25 mM potassium chloride, 15 mM calcium chloride, 30 mM magnesium chloride, 250 g / L glucose, 5 mM citric acid and 150 g / L anticoagulant factor X antibody.

4. The citrate dialysis concentrate according to claim 1, characterized in that, Each 1 L of water for injection comprises 1.5 M sodium chloride, 50 mM potassium chloride, 20 mM calcium chloride, 45 mM magnesium chloride, 400 g / L glucose, 10 mM citric acid and 200 g / L anticoagulant factor X antibody.

5. The citrate dialysis concentrate according to any one of claims 1-4, characterized in that, The anticoagulant factor X antibody is obtained by immunizing BALB / c mice with coagulation factor X as an antigen using hybridoma technology, separating mouse spleen cells, fusing them with SP2 / 0 myeloma cells, and then screening for the anticoagulant factor X antibody.

6. The citrate dialysis concentrate according to claim 5, wherein, The sequence of the heavy chain variable region of the anticoagulant factor X antibody is as shown in SEQ ID NO.1, and the sequence of the light chain variable region is as shown in SEQ ID NO.

2.

7. The preparation method of the citrate dialysis concentrate according to any one of claims 1-6, characterized in that, The method comprises the following steps: 1) After repeatedly rinsing a clean plastic bucket 3 - 5 times, disinfecting it with 75 - 100% ethanol 2 - 5 times, and then washing it with water for injection 3 - 5 times, it is set aside for use; 2) Accurately calculate the dosage of each component according to the concentrated solution culture, weigh them one by one and put them into the plastic bucket in step 1); 3) Add an appropriate amount of water for injection, stir to dissolve it fully, filter it through a filter screen, and then add the remaining water for injection to make up the full volume.

8. Use of an anticoagulant factor X antibody in the preparation of a citrate dialysis concentrate, characterized in that, The sequence of the heavy chain variable region of the anticoagulant factor X antibody is as shown in SEQ ID NO.1, and the sequence of the light chain variable region is as shown in SEQ ID NO.

2.

9. The use according to claim 8, characterized in that, The heavy chain variable region of the anticoagulant factor X antibody comprises CDR1, CDR2 and CDR3, and their sequences respectively correspond to those shown in SEQ ID NO.3 - 5, and the light chain variable region of the anticoagulant factor X antibody comprises CDR4, CDR5 and CDR6, and their sequences respectively correspond to those shown in SEQ ID NO.6 - 8.