Glacial acetic acid dialysis concentrated solution and preparation method thereof

Abstract

The invention relates to the technical field of hemodialysis, in particular to a glacial acetic acid dialysis concentrated solution and a preparation method thereof. The invention provides the anticoagulant factor VIII antibody, the anticoagulant factor VIII antibody is applied to preparation of the glacial acetic acid dialysis concentrated solution, and result analysis shows that the blood coagulation phenomenon of the glacial acetic acid dialysis concentrated solution can be remarkably improved by introducing the anticoagulant factor VIII antibody, and the risk that coagulation and thrombosis are possibly increased by commercial glacial acetic acid dialysis solution is reduced. Meanwhile, the stability of the dialysate can be effectively improved, and the dialysate can be suitable for long-term storage and does not deteriorate. The application prospect of the glacial acetic acid dialysate is remarkably improved, and the glacial acetic acid dialysate has many advantages especially for hemodialysis of acute and chronic renal failure patients and drug poisoning patients.

Description

Technical Field

[0001] The present invention relates to the technical field of hemodialysis, and more specifically to a glacial acetic 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] Glacial acetic acid is usually used as a pH regulator, acidifier, and organic acid solvent in preparations. For example, injections and some eye drops whose solvents are hydrophilic solvents such as alcohols will choose glacial acetic acid with excellent water solubility as a pH regulator. Glacial acetic acid is also added to the hemodialysis fluid and hemodialysis dry powder A required for hemodialysis. The disadvantage of existing glacial acetic acid dialysis concentrates is that blood coagulation will occur when such products are used in conjunction with hemodialysis machines, especially for patients with acute and chronic renal failure and drug poisoning. It will cause adverse reactions to hemodialysis. In addition, the main problem facing commercial glacial acetic acid dialysis concentrates is how to improve their storage stability and reduce liquid deterioration and the generation of impurity molecules caused by long-term storage. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a glacial acetic acid dialysis concentrate and a preparation method thereof. The present invention provides an anti-coagulation factor VIII antibody for the first time, and applies it to the preparation of glacial acetic acid dialysis concentrate. According to the 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. At the same time, the stability of the dialysis fluid can be effectively improved, and it can be suitable for long-term storage without deterioration, which significantly improves the application prospect of glacial acetic acid dialysis fluid.

[0005] Specifically, the present invention first provides a glacial acetic acid dialysis concentrate, including anti-coagulation factor VIII antibodies.

[0006] Preferably, the anti-coagulation factor VIII antibody is prepared by using hybridoma technology to immunize BALB / c mice with coagulation factor X as an antigen, then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells to screen the anti-coagulation factor VIII antibody.

[0007] Preferably, the heavy chain variable region sequence of the anti-coagulation factor VIII antibody is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

[0008] Preferably, the heavy chain variable region of the anti-coagulation factor VIII antibody includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively, and the light chain variable region of the anti-coagulation factor VIII antibody includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

[0009] On the other hand, the present invention provides a low-calcium glacial acetic acid dialysis concentrate, characterized in that each 1L of water for injection comprises 1.1-1.5M sodium chloride, 10-25mM potassium chloride, 5-10mM calcium chloride, 15-30mM magnesium chloride, 10-25g / L glucose, 5-10g / L glacial acetic acid and 10-15g / L anti-coagulation factor VIII antibody.

[0010] Preferably, the low-calcium glacial acetic acid dialysis concentrate comprises 1.1M sodium chloride, 10mM potassium chloride, 5mM calcium chloride, 15mM magnesium chloride, 10g / L glucose, 5g / L glacial acetic acid and 10g / L anti-coagulation factor VIII antibody per 1L of water for injection.

[0011] Preferably, the low-calcium glacial acetic acid dialysis concentrate comprises 1.5M sodium chloride, 25mM potassium chloride, 10mM calcium chloride, 30mM magnesium chloride, 25g / L glucose, 10g / L glacial acetic acid and 15g / L anti-coagulation factor VIII antibody per 1L of water for injection.

[0012] On the other hand, the present invention provides a high calcium glacial acetic acid dialysis concentrate, characterized in that each 1L of water for injection comprises 1.5-2.0M sodium chloride, 50-60mM potassium chloride, 20-50mM calcium chloride, 45-60mM magnesium chloride, 40-55g / L glucose, 15-30g / L glacial acetic acid and 20-30g / L anti-coagulation factor VIII antibody.

[0013] Preferably, the high calcium glacial acetic acid dialysis concentrate comprises 1.5M sodium chloride, 50mM potassium chloride, 20mM calcium chloride, 45mM magnesium chloride, 40g / L glucose, 15g / L glacial acetic acid and 20g / L anti-coagulation factor VIII antibody per 1L of water for injection.

[0014] Preferably, the high calcium glacial acetic acid dialysis concentrate comprises 2.0 M sodium chloride, 60 mM potassium chloride, 50 mM calcium chloride, 60 mM magnesium chloride, 55 g / L glucose, 30 g / L glacial acetic acid and 30 g / L anti-coagulation factor VIII antibody per 1 L of water for injection.

[0015] Another aspect of the present invention is to provide a method for preparing a glacial acetic acid dialysis concentrate, comprising the following steps:

[0016] 1) Rinse and disinfect the plastic barrel, then clean it with water for injection and set it aside;

[0017] 2) Calculate the amount of each component, weigh it and put it into the plastic bucket in step 1);

[0018] 3) Add an appropriate amount of water for injection and stir to fully dissolve it, then filter it and add the remaining water for injection to the full amount.

[0019] Another aspect of the present invention is to provide use of an anti-blood coagulation factor VIII antibody in the preparation of glacial acetic acid dialysis concentrate.

[0020] Preferably, the heavy chain variable region sequence of the anti-coagulation factor VIII antibody is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

[0021] Further preferably, the anti-coagulation factor VIII antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively, and the anti-coagulation factor VIII antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

[0022] The advantages of the present invention are as follows: The present invention provides an anti-coagulation factor VIII antibody, and applies it to the preparation of glacial acetic acid dialysis concentrate. According to the analysis results, the introduction of the anti-coagulation factor VIII antibody can significantly improve the blood coagulation phenomenon of the glacial acetic acid dialysis concentrate, and reduce the risk of increased coagulation and thrombosis in commercial glacial acetic acid dialysis fluid. At the same time, the stability of the dialysis fluid can be effectively improved, and it can be suitable for long-term storage without deterioration. The application prospects of glacial acetic acid dialysis fluid are significantly improved, especially for the hemodialysis of patients with acute and chronic renal failure and drug poisoning patients, which will bring many advantages. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Example 1

[0027] A low-calcium glacial acetic acid dialysis concentrate comprises 1.1M sodium chloride, 10mM potassium chloride, 5mM calcium chloride, 15mM magnesium chloride, 10g / L glucose, 5g / L glacial acetic acid and 10g / L anti-coagulation factor VIII antibody in each 1L of water for injection.

[0028] The anti-coagulation factor VIII antibody was prepared by using hybridoma technology to immunize BALB / c mice with coagulation factor VIII as an antigen, and then the mouse spleen cells were separated and fused with SP2 / 0 myeloma cells to screen the anti-coagulation factor VIII antibody. 50 The value was 0.78 nM, confirming that it binds strongly and specifically to coagulation factor X.

[0029] The isolated antibodies were sequenced and identified, and the heavy chain variable region sequence of the anti-coagulation factor VIII 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 VIII antibody includes CDR1, CDR2 and CDR3, and their sequences correspond to SEQ ID NO. 3-5, respectively, and the light chain variable region of the anti-coagulation factor VIII antibody includes CDR4, CDR5 and CDR6, and their sequences correspond to SEQ ID NO. 6-8, respectively.

[0030] Furthermore, the preparation method of the glacial acetic acid dialysis concentrate comprises the following steps:

[0031] 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;

[0032] 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);

[0033] 3) 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.

[0034] Example 2

[0035] A low-calcium glacial acetic acid dialysis concentrate comprises 1.5M sodium chloride, 25mM potassium chloride, 10mM calcium chloride, 30mM magnesium chloride, 25g / L glucose, 10g / L glacial acetic acid and 15g / L anti-coagulation factor VIII antibody in each 1L of water for injection.

[0036] The preparation steps are the same as those in Example 1.

[0037] Example 3

[0038] A high calcium glacial acetic acid dialysis concentrate comprises 1.5M sodium chloride, 50mM potassium chloride, 20mM calcium chloride, 45mM magnesium chloride, 40g / L glucose, 15g / L glacial acetic acid and 20g / L anti-coagulation factor VIII antibody in each 1L of water for injection.

[0039] Example 4

[0040] A high calcium glacial acetic acid dialysis concentrate comprises 2.0 M sodium chloride, 60 mM potassium chloride, 50 mM calcium chloride, 60 mM magnesium chloride, 55 g / L glucose, 30 g / L glacial acetic acid and 30 g / L anti-coagulation factor VIII antibody in each 1 L of water for injection.

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

[0042] Comparative Example 1

[0043] A low-calcium glacial acetic acid dialysis concentrate comprises 1.5M sodium chloride, 50mM potassium chloride, 10mM calcium chloride, 45mM magnesium chloride, 40g / L glucose and 15g / L glacial acetic acid in each 1L of water for injection.

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

[0045] Comparative Example 2

[0046] A high calcium hemodialysis concentrate comprises 2.0 M sodium chloride, 60 mM potassium chloride, 50 mM calcium chloride, 60 mM magnesium chloride, 55 g / L glucose and 20 g / L anti-coagulation factor VIII antibody in each 1 L of water for injection.

[0047] The preparation method and steps are the same as those in Example 1.

[0048] Verification Example 1

[0049] APTT coagulation test: Collect peripheral blood samples from rabbits and divide them into 5 parts, and quickly mix them with dialysis concentrate, with the volume ratio of the two being 9:1. Then, take out 0.1mL of the mixed solution and add 0.1mL of APTT reagent preheated at 37℃ for 10min, and incubate at 37℃ for 5min. Then add 0.1mL of 0.025M calcium chloride preheated at 37℃, start the stopwatch immediately, and record the coagulation time.

[0050] TT experiment: peripheral blood samples of rabbits were collected and divided into 5 parts, and then quickly mixed with 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. The results are shown in Table 1.

[0051] Table 1: Test results

[0052] APTT coagulation time (seconds) TT coagulation time (seconds) Example 1 38.7 48.9 Example 2 47.2 60.2 Example 3 68.9 81.3 Example 4 82.6 95.8 Comparative Example 1 25.7 30.5 Comparative Example 2 16.8 25.6

[0053] It can be seen from the results in Table 1 that the anticoagulation time of peripheral blood samples treated with the dialysis concentrate described in the embodiment of the present invention is significantly better than that of the control group. At the same time, the high calcium formula has a stronger anticoagulant effect than the low calcium formula, which can further reduce the risk of increased coagulation and thrombosis caused by commercial glacial acetic acid dialysis fluid.

[0054] Verification Example 2

[0055] With reference to the requirements and specifications for long-term testing of pharmaceutical preparations in the Chinese Pharmacopoeia and the Guiding Principles for Stability Testing of Raw Drugs and Preparations, the stability of the low-calcium and high-calcium hemodialysis concentrates described in the present invention was analyzed, specifically, the changes under accelerated (40°C) for three months and long-term (25°C, 65% RH) conditions for six months were selected to test the stability of the preparations.

[0056] Results As shown in the stability test results in Tables 2 and 3 below, it can be seen that compared with the comparative example, the low calcium and high calcium hemodialysis concentrates of the present invention have superior stability in appearance, content uniformity and pH, and remain stable during the accelerated three-month period and the long-term six-month storage period, without generating impurities and causing the reagents to deteriorate, which is conducive to commercial long-term storage and application.

[0057] Table 2 Accelerated three-month test results

[0058]

[0059]

[0060] Table 3 Long-term six-month test results

[0061]

[0062]

[0063] 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 glacial acetic acid dialysis concentrate, characterized in that: These include anti-factor VIII antibodies.

2. The glacial acetic acid dialysis concentrate according to claim 1, characterized in that The anti-coagulation factor VIII 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 VIII antibody.

3. The glacial acetic acid dialysis concentrate according to claim 2, characterized in that: The heavy chain variable region sequence of the anti-coagulation factor VIII antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

4. The glacial acetic acid dialysis concentrate according to claim 3, characterized in that: The heavy chain variable region of the anti-coagulation factor VIII antibody includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5 respectively, and the light chain variable region of the anti-coagulation factor VIII antibody includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8 respectively.

5. A low-calcium glacial acetic acid dialysis concentrate, characterized in that: Each 1L of water for injection includes 1.1-1.5M sodium chloride, 10-25mM potassium chloride, 5-10mM calcium chloride, 15-30mM magnesium chloride, 10-25g / L glucose, 5-10g / L glacial acetic acid and 10-15g / L anti-coagulation factor VIII antibody.

6. A high calcium glacial acetic acid dialysis concentrate, characterized in that: Each 1L of water for injection includes 1.5-2.0M sodium chloride, 50-60mM potassium chloride, 20-50mM calcium chloride, 45-60mM magnesium chloride, 40-55g / L glucose, 15-30g / L glacial acetic acid and 20-30g / L anti-coagulation factor VIII antibody.

7. The method for preparing the glacial acetic acid dialysis concentrate according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: 1) Rinse and disinfect the plastic barrel, then clean it with water for injection and set it aside; 2) Calculate the amount of each component, weigh it and put it into the plastic bucket in step 1); 3) Add an appropriate amount of water for injection and stir to fully dissolve it, then filter it and add the remaining water for injection to the full amount.

8. Use of an anti-coagulation factor VIII antibody in the preparation of glacial acetic acid dialysis concentrate, characterized in that: The heavy chain variable region sequence of the anti-coagulation factor VIII antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

9. The use according to claim 8, characterized in that The heavy chain variable region of the anti-coagulation factor VIII antibody includes CDR1, CDR2 and CDR3, and the sequences thereof correspond to those shown in SEQ ID NOs. 3-5, respectively.

10. The use according to claim 8, characterized in that The light chain variable region of the anti-coagulation factor VIII antibody includes CDR4, CDR5 and CDR6, and the sequences thereof correspond to SEQ ID NOs. 6-8, respectively.

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