Citric acid dialysis concentrated solution and preparation method thereof

Abstract

The invention relates to the technical field of hemodialysis, in particular to a citric acid dialysis concentrated solution and a preparation method thereof. The anticoagulant factor X antibody is provided for the first time and is applied to preparation of the citric acid dialysis concentrated solution, result analysis shows that after the anticoagulant factor X antibody and the citric acid dialysis concentrated solution are combined, a synergistic effect is achieved on the anticoagulant effect of peripheral blood, the anticoagulant effect is extremely remarkable, and the application prospect of the citric acid dialysis solution can be remarkably improved.

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 drains blood from the body to the outside of the body and passes through a dialyzer composed of countless hollow fibers. The blood and the electrolyte solution (dialysate) containing a concentration similar to that of the body are exchanged inside and outside the hollow fibers through diffusion, ultrafiltration, adsorption and convection principles to remove metabolic waste from the body, maintain electrolyte and acid-base balance, remove excess water from the body, and return the purified blood. The whole process is called hemodialysis.

[0003] Citrate anticoagulation is an extracorporeal anticoagulation technology. Calcium ions are a very important coagulation factor in normal human blood and are involved in all aspects of 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 citric acid is pumped into the extracorporeal circulation, citric acid will form calcium citrate with calcium ions in the blood, thereby reducing the calcium ion concentration in the blood to a very low level, thereby achieving the anticoagulation effect.

[0004] However, the prior art discloses that the anticoagulant effect of the citric acid dialysis concentrate is relatively poor, and some patients may develop thrombus formation during hemodialysis, resulting in a decrease in the hemodialysis effect. Summary of the invention

[0005] In view of 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 provides an anti-coagulation factor X antibody for the first time and applies it to the preparation of citric acid dialysis concentrate. The result analysis shows that 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 citric acid dialysis fluid.

[0006] Specifically, the present invention first provides a citric acid dialysis concentrate, wherein each 1L of water for injection comprises 1.1-1.5M sodium chloride, 10-50mM potassium chloride, 5-20mM calcium chloride, 15-45mM magnesium chloride, 100-400g / L glucose, 1-10mM citric acid and 100-200g / L anti-coagulation factor X antibody.

[0007] Preferably, the citric acid dialysis concentrate 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 anti-factor X antibody per 1 L of water for injection.

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

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

[0010] More preferably, the anti-factor X antibody is prepared by immunizing BALB / c mice with factor X as an antigen using hybridoma technology, separating mouse spleen cells, and fusing them with SP2 / 0 myeloma cells to screen the anti-factor X antibody.

[0011] More preferably, the heavy chain variable region sequence of the anti-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] Further preferably, the anti-coagulation factor X antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively, and the anti-coagulation factor X antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.

[0013] Another aspect of the present invention is to provide a method for preparing a citric acid dialysis concentrate, comprising the following steps:

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

[0015] 5) Accurately calculate the amount of each component according to the above concentrated solution 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 and stir mechanically to fully dissolve it. After filtering through a filter, add the remaining water for injection to the full amount.

[0017] Another aspect of the present invention is to provide use of anti-factor X antibody in preparing citrate dialysis concentrate.

[0018] Preferably, the heavy chain variable region sequence of the anti-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 anti-coagulation factor X antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively, and the anti-coagulation factor X antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.

[0020] The advantages of the present invention are as follows: the present invention provides an anti-coagulation factor X antibody for the first time, and applies the antibody to prepare a citrate dialysis concentrate. The results show that 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 the citrate dialysis concentrate. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments 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 intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0023] In the examples 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 examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0024] Example 1

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

[0026] The anti-coagulation factor X antibody is prepared by using hybridoma technology to immunize BALB / c mice with coagulation factor X as an antigen, and then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells to screen the anti-coagulation factor X antibody.

[0027] The results showed that the EC of anti-factor X antibody binding to factor X 50 The value was 0.86 nM, which confirmed that it has a strong and specific binding with coagulation factor X and has a good anticoagulant effect.

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

[0029] Furthermore, the preparation method of the citric acid dialysis concentrate comprises the following steps:

[0030] 1) Rinse the clean plastic barrel repeatedly for 3 times, disinfect it with 75% ethanol for 2 times, and then clean it with injection water for 3 times before use;

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

[0032] 3) Add 1 / 2 of the water for injection and stir mechanically to fully dissolve it. After filtering through a filter, add the remaining water for injection to the full amount.

[0033] Example 2

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

[0035] The preparation steps are the same as those in Example 1.

[0036] Example 3

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

[0038] The preparation steps are the same as those in Example 1.

[0039] Comparative Example 1

[0040] A citric acid dialysis concentrate comprises 1.5M sodium chloride, 50mM potassium chloride, 20mM calcium chloride, 45mM magnesium chloride, 400g / L glucose and 10mM citric acid in each 1L of water for injection.

[0041] The preparation steps are the same as those in Example 1.

[0042] Comparative Example 2

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

[0044] The preparation steps are the same as those in Example 1.

[0045] Verification Example

[0046] APTT coagulation test

[0047] The peripheral blood samples of rabbits were collected and evenly divided into 5 parts, and then quickly mixed with the dialysis concentrate, with the volume ratio of the two being 9:1. Then, 0.1 mL of the mixed solution was taken out and 0.1 mL of APTT reagent preheated at 37°C for 10 min was added, and incubated at 37°C for 5 min. Then 0.1 mL of 0.025 M calcium chloride preheated at 37°C was added, and the stopwatch was immediately started to record the coagulation time.

[0048] TT experiment

[0049] The peripheral blood samples of rabbits were collected and evenly divided into 5 parts, and then quickly mixed with the dialysis concentrate, with the volume ratio of the two being 9:1. Subsequently, 0.2 mL of the mixed solution was taken out and 0.2 mL of TT reagent preheated at 37°C for 10 min was added. After mixing, the stopwatch was immediately started to record the coagulation time.

[0050] The results are shown in Table 1.

[0051] Table 1: Test results

[0052] APTT coagulation time (seconds) TT coagulation 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] From the results in Table 1, it can be seen that the anticoagulant effect on peripheral blood samples treated with the dialyzate concentrate described in the embodiment of the present invention is significantly better than that of the comparative example, further confirming that the combined effect of citric acid and anti-factor X antibody achieves a synergistic anticoagulant technical effect.

[0054] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution 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 modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A citric acid dialysis concentrate, characterized in that: Each 1L of water for injection includes 1.1-1.5M sodium chloride, 10-50mM potassium chloride, 5-20mM calcium chloride, 15-45mM magnesium chloride, 100-400g / L glucose, 1-10mM citric acid and 100-200g / L anti-coagulation factor X antibody.

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

3. The citric acid dialysis concentrate according to claim 1, characterized in that: Each 1L of water for injection includes 1.2M sodium chloride, 25mM potassium chloride, 15mM calcium chloride, 30mM magnesium chloride, 250g / L glucose, 5mM citric acid and 150g / L anti-factor X antibody.

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

5. The citric acid dialysis concentrate according to any one of claims 1 to 4, characterized in that: The anti-coagulation factor X antibody is prepared by using hybridoma technology to immunize BALB / c mice with coagulation factor X as an antigen, and then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells to screen the anti-coagulation factor X antibody.

6. The citric acid dialysis concentrate according to claim 5, characterized in that: The heavy chain variable region sequence of the anti-factor X antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

7. The citric acid dialysis concentrate according to claim 5, characterized in that: The anti-coagulation factor X antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively; the anti-coagulation factor X antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.

8. The method for preparing the citric acid dialysis concentrate according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: 1) Rinse the clean plastic barrel repeatedly 3-5 times, disinfect it with 75-100% ethanol 2-5 times, and then wash it with injection water 3-5 times for use; 2) Accurately calculate the amount 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 and stir to fully dissolve it. After filtering through a filter, add the remaining water for injection to the full amount.

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

2.

10. The use according to claim 9, characterized in that The anti-coagulation factor X antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively; the anti-coagulation factor X antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.