A glacial acetic acid dialysis concentrate and its preparation method

Abstract

The present invention relates to the field of hemodialysis technology, and more specifically, to a glacial acetic acid dialysis concentrate and a preparation method thereof. The present invention provides an anti-coagulation factor VIII antibody, and applies the antibody to the preparation of a glacial acetic acid dialysis concentrate. Analysis of the results shows that the introduction of the anti-coagulation factor VIII antibody significantly improves the blood coagulation phenomenon of the glacial acetic acid dialysis concentrate, and reduces 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 the dialysis fluid can be suitable for long-term storage without deterioration. The application prospects of glacial acetic acid dialysis fluid are significantly improved, especially for hemodialysis of patients with acute and chronic renal failure and patients with drug poisoning, which will bring many advantages.

Description

Technical Field

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

[0002] Hemodialysis (HD) is a renal replacement therapy for patients with acute and chronic renal failure. It involves draining blood from the body and passing it through a dialyzer composed of numerous hollow fibers. Blood and a solution containing electrolytes similar to those in the body (dialysate) exchange substances within and outside the fibers through diffusion, ultrafiltration, adsorption, and convection. This process removes metabolic waste, maintains electrolyte and acid-base balance, and simultaneously removes excess water. The purified blood is then returned to the body. This entire process is called hemodialysis.

[0003] Glacial acetic acid is commonly used in pharmaceutical preparations as a pH regulator, acidifier, and organic acid solvent. For example, injections and some eye drops using hydrophilic solvents such as alcohols will use glacial acetic acid, due to its excellent water solubility, as a pH regulator. Glacial acetic acid is also added to the hemodialysis fluid and agent A of the hemodialysis dry powder required for hemodialysis. A drawback of existing glacial acetic acid dialysis concentrates is that they can cause blood coagulation when used in conjunction with a hemodialysis machine, which can cause adverse reactions, especially for patients with acute and chronic renal failure and drug poisoning. Furthermore, the main problem facing commercial glacial acetic acid dialysis concentrates is how to improve their storage stability and reduce the risk of liquid deterioration and the generation of impurity molecules due to long-term storage. Summary of the Invention

[0004] To address the technical problems existing in the prior art, the present invention provides a glacial acetic acid dialysis concentrate and a method for preparing the same. This invention provides, for the first time, an anti-factor VIII antibody and applies it to the preparation of glacial acetic acid dialysis concentrate. Analysis of the results shows that the combination of the two achieves a synergistic and highly significant anticoagulant effect on peripheral blood. Furthermore, the concentrate effectively improves the stability of the dialysate, making it suitable for long-term storage without deterioration, significantly enhancing the application prospects of glacial acetic acid dialysate.

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

[0006] Preferably, the anti-factor VIII antibody is obtained 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 for anti-factor VIII antibodies.

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

[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-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 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.

[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-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 aside;

[0017] 2) Calculate the amount of each component, weigh it, and place it into the plastic bucket prepared 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-factor VIII antibody in the preparation of a glacial acetic acid dialysis concentrate.

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

[0021] Further 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.

[0022] The advantages of the present invention are as follows: The present invention provides an anti-factor VIII antibody and applies it to the preparation of glacial acetic acid dialysis concentrate. Analysis results show that the introduction of the anti-factor VIII antibody significantly improves the coagulation phenomenon of the glacial acetic acid dialysis concentrate, reducing the risk of coagulation and thrombosis that may be increased in commercial glacial acetic acid dialysis concentrate. Furthermore, the stability of the dialysis fluid is effectively improved, making it suitable for long-term storage without deterioration. This significantly enhances the application prospects of glacial acetic acid dialysis fluid, especially for hemodialysis of patients with acute and chronic renal failure and drug poisoning. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to 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 those of ordinary skill in the art without creative work are within the scope of protection 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 screened by using hybridoma technology to immunize BALB / c mice with coagulation factor VIII as antigen, separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells. The results showed that the EC binding of anti-coagulation factor VIII antibody to coagulation factor VIII was 50 The value was 0.78 nM, demonstrating its strong and specific binding to coagulation factor X.

[0029] The isolated antibodies were sequenced and identified. 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, whose sequences correspond to SEQ ID NOs. 3-5, respectively. The light chain variable region of the anti-coagulation factor VIII antibody includes CDR4, CDR5, and CDR6, whose sequences correspond to SEQ ID NOs. 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 three times, disinfect it twice with 75% ethanol, and then rinse it three times with water for injection before use;

[0032] 2) Accurately calculate the amount of each component according to the above concentrated solution culture, weigh them one by one and place 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 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 steps are the same as those in Example 1.

[0048] Verification Example 1

[0049] APTT coagulation test: Collect a rabbit peripheral blood sample and divide it into five equal portions. The sample is then quickly mixed with dialysis concentrate at a 9:1 volume ratio. Subsequently, 0.1 mL of the mixture is removed and added to 0.1 mL of APTT reagent pre-warmed at 37°C for 10 minutes. The mixture is incubated at 37°C for 5 minutes. Then, 0.1 mL of 0.025 M calcium chloride pre-warmed at 37°C is added. A stopwatch is immediately started, and the clotting time is recorded.

[0050] TT experiment: A rabbit peripheral blood sample was collected and evenly divided into five aliquots. The aliquots were then quickly mixed with dialysis concentrate at a volume ratio of 9:1. Subsequently, 0.2 mL of the mixed solution was added to 0.2 mL of TT reagent, pre-warmed at 37°C for 10 minutes. After mixing, a stopwatch was immediately started and the clotting time was recorded. The results are shown in Table 1.

[0051] Table 1: Test results

[0052] APTT clotting time (seconds) TT clotting 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] From the results in Table 1, it can be seen 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 example. 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) three months and long-term (25°C, 65% RH) six months conditions were selected to test the stability of the preparations.

[0056] Results As shown in the stability test results in Tables 2 and 3 below, the low-calcium and high-calcium hemodialysis concentrates of the present invention have superior stability in appearance, content uniformity, and pH compared to the comparative examples. They remain stable during the three-month accelerated storage period and the six-month long-term storage period, do not generate impurities, and do not cause reagent deterioration, 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 important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A glacial acetic acid dialysis concentrate, characterized in that: It comprises an anti-coagulation factor VIII antibody, wherein the heavy chain variable region sequence of the anti-coagulation factor VIII antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO.

2.

2. The glacial acetic acid dialysis concentrate according to claim 1, wherein The anti-coagulation factor VIII antibody is screened 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.

3. The glacial acetic acid dialysis concentrate according to claim 1, wherein 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.

4. 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, wherein 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 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, wherein 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.

6. The method for preparing the glacial acetic acid dialysis concentrate according to any one of claims 1 to 5, 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 aside; 2) Calculate the amount of each component, weigh it, and place it into the plastic bucket prepared in step 1); 3) Add 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.

7. Use of an anti-coagulation factor VIII antibody in the preparation of a 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.

8. The use according to claim 7, wherein The heavy chain variable region of the anti-factor VIII antibody includes CDR1, CDR2 and CDR3, and the sequences thereof correspond to SEQ ID NOs. 3-5, respectively.

9. The use according to claim 7, wherein 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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