A kilogram-level automatic production device and method for high-concentration active heterophilic antibody blockers

Abstract

The present invention discloses a kilogram-level automatic production device and method for high-concentration active heterophilic antibody blockers, the device comprising a liquid preparation tank, a treatment tank, a liquid storage tank, a sample loading tank, an automatic chromatography device, a first ultrafiltration membrane package equipment circulation tank, a second ultrafiltration membrane package equipment circulation tank, a centrifugal device, a filtering device, and pipelines and valves connected to each tank body; the present invention adopts octanoic acid precipitation + HP ion exchange for production and purification. In view of the small single batch in the current stage, the method of a 500L liquid preparation tank is now used for liquid preparation, and the single batch liquid preparation volume is large, which meets the downstream purification requirements. Fully automatic purification equipment is used for purification, and the purification efficiency is high. Ultrafiltration membrane package equipment is used for automatic liquid exchange processing, the dialysis efficiency is high, and it also has the function of concentration, which meets the concentration requirements of high-concentration products.

Description

Technical Field

[0001] The invention belongs to the technical field of blocker production, and specifically relates to a kilogram-level automatic production device and a preparation method for a high-concentration active heterophilic antibody blocker. Background Art

[0002] Blockers are a general term for materials that can be used in the development of in vitro diagnostic reagents to reduce interference from proteins in patient samples or other factors in the test process, which may cause false positive or false negative test results.

[0003] In the early stages of the development of my country's in vitro diagnostic reagent industry, various in vitro diagnostic reagent manufacturers did not fully realize the importance of blocking agents. Most in vitro diagnostic reagent companies generally simply formulated them according to production needs, and the repeatability, stability, and use effects were uneven. As a result, the clinical application performance of domestically developed and produced in vitro diagnostic reagents was somewhat different from that of foreign imported brands, and false positive and false negative results were difficult to eliminate.

[0004] Blockers are key excipients in diagnostic reagent raw materials, and are widely used and used in large quantities. With market changes, there is a high demand for high-concentration and large-volume blockers. And production costs need to be lowered.

[0005] Currently, membrane packages are used for liquid exchange and concentration. The membrane packages have a high degree of clarity for the operating liquid, and filtration treatment is required before use. The production scale is small. Dialysis bags are commonly used for liquid exchange in this field. This process is inefficient and requires the dialysate to be replaced at a fixed time period. The batch size processed is small, and the loss during the dialysis process is large.

[0006] There are many types of blockers, and immune blockers can be divided into active blockers and passive blockers. Active blockers are a type of reagent that has a high affinity for interfering antibodies. They bind to interfering antibodies with high affinity to form a steric effect, thereby preventing the binding of interfering antibodies to detection antibodies or capture antibodies, reducing interference and false positive results in the test, such as RF blockers, HAMA blockers, etc. Passive blockers use non-specific substances, such as mouse IgG, mouse serum, non-specific monoclonal antibodies, polymerized IgG, etc. Their working principle is to provide optional binding sites for detection antibodies through the binding of interfering antibodies and capture substances.

[0007] The purification methods currently used for immunoblockers mainly include the following two methods:

[0008] 1. Affinity chromatography: such as G column affinity chromatography, which uses the specific affinity between antigen and antibody for purification. When the sample containing the blocker passes through the G column, the antibody component in the blocker will specifically bind to the column, while other impurities will be eluted. Then, by changing the elution conditions, such as adjusting the pH value or ionic strength of the buffer, the bound blocker will be eluted to obtain a purified blocker. For example, when preparing a monoclonal antibody blocker, a Protein G column can be used for affinity chromatography to obtain a high-purity antibody blocker.

[0009] 2. Ion exchange chromatography: purification is achieved through the ion interaction between ion exchange resin and blocker. For example, in blue gel + Q column ion exchange chromatography, the sample is first loaded onto the ion exchange column, and the blocker will adsorb to the ion exchange resin according to its surface charge, and then gradient elution is performed with salt solutions of different concentrations. According to the charge difference between the blocker and the impurities, they are eluted in turn at different salt concentrations, thereby achieving the purpose of separation and purification.

[0010] At present, the general concentration of active blocking agents is 20mg / ml. The dosage added during use is relatively large. The market has a demand for high-concentration samples (50mg / ml). At present, samples are separated and purified by affinity chromatography. The dissociation conditions are highly acidic, which can easily damage antibodies. Under acidic conditions, IgG is prone to form aggregates, which are unstable and easy to precipitate during freeze-thaw. The single batch size is 100g, which is relatively small. There are batch differences between different batches of products. Different batches need to be verified during downstream production, which has a greater impact on downstream production. Summary of the invention

[0011] In order to overcome the defects of small production volume and large batch-to-batch differences of existing active blockers, the present invention provides a kilogram-level automatic production device and method for high-concentration active heterophilic antibody blockers.

[0012] The technical solution adopted by the present invention is:

[0013] A kilogram-level automatic production device for high-concentration active heterophilic antibody blockers, the device comprises a liquid preparation tank, a treatment tank, a liquid storage tank, a sample loading tank, a deep filtration system, an automatic chromatography device, a first ultrafiltration membrane package equipment circulation tank, a second ultrafiltration membrane package equipment circulation tank, a centrifugal device, a filtering device, and pipelines and valves connected to each tank body; wherein the liquid preparation tank is connected to the liquid storage tank, the liquid storage tank is connected to the treatment tank and the automatic chromatography device, the deep filtration system is connected to the treatment tank, the treatment tank is connected to the centrifugal device, the centrifugal device is connected to the first ultrafiltration membrane package equipment circulation tank, the first ultrafiltration membrane package equipment circulation tank is connected to the filtering device, the filtering device is connected to the sample loading tank, the sample loading tank is connected to the automatic chromatography device, and the automatic chromatography device is connected to the second ultrafiltration membrane package equipment circulation tank.

[0014] The liquid preparation tank is a 500L stainless steel tank, which is automatically controlled by a PLC control system for water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time, and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank;

[0015] The liquid storage tank is a 500L stainless steel tank used for storing the solution.

[0016] The treatment tank is a 500L stainless steel tank, and the solution stored in the liquid storage tank is discharged into the treatment tank for octanoic acid precipitation;

[0017] The centrifugal device is a continuous flow disc centrifuge;

[0018] The sample is centrifuged in a continuous flow disc centrifuge, and the supernatant is collected and placed in the circulation tank of the first ultrafiltration membrane package equipment, and then enters the filtration device after the liquid is replaced and dialyzed;

[0019] The filtering device is a capsule filter; the filtered sample is transferred to a sample loading tank; an automated chromatography device is used to load the sample and collect the flow-through liquid; the collected sample is passed through a second ultrafiltration membrane bag device circulation tank for liquid exchange and concentration to 50 mg / ml, and is sterilized and filtered at 0.22 μm before being packaged.

[0020] The method for producing kilogram-level high-concentration active heterophilic antibody blocking agent using the above device comprises the following steps:

[0021] 1) Liquid preparation: A 500L stainless steel tank is used as the liquid preparation tank. The PLC control system can automatically control water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time, and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank;

[0022] 2) 100 L of mouse ascites obtained from immunized mice was initially filtered through filter cloth, and then the sample was deep filtered through 4-18 μm to clarify;

[0023] 3) Transfer the treated clarified raw material to a 500L treatment tank, add 40mM NaAc (pH4.0) diluent 4 times the raw material volume, and stir evenly; add 10% n-octanoic acid according to the raw material volume, add slowly, and stir for 4 hours;

[0024] 4) The stirred sample is centrifuged in a continuous flow disc centrifuge, and the supernatant is collected and placed in the first ultrafiltration membrane package equipment circulation tank for 2 hours, and the pH of the sample is adjusted to 7.2;

[0025] 5) The pH-adjusted sample was dialyzed in an ultrafiltration membrane pack device using 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol solution with a salt exchange ratio of 1:10;

[0026] 6) Filter the liquid replacement sample through a 0.45 μm capsule filter and transfer the filtered sample to a sample loading tank;

[0027] 7) Prepare 200 L of HP weak anion exchange medium and fill it into the chromatography column; use equilibration buffer 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol to equilibrate the HP DEAE column for 5 times column volume. After the column is equilibrated, adjust the protein UV detector display value to 0 as the baseline;

[0028] 8) Load the sample at a loading capacity of 10 mg / ml using an automated chromatography device at 2 L / min, collect the flow-through, and start collecting protein when the protein UV detector displays a value greater than 0.2; balance with 10 mmPB + 0.3 M NaCl (pH 7.4) + 5% glycerol, and stop collecting protein when the protein UV detector displays a value less than 0.2; 9) Replace the collected sample with a second ultrafiltration membrane package (10 mmPB + 0.3 M NaCl (pH 7.4) + 2% Tween), and concentrate to 50 mg / ml, sterilize and filter the sample at 0.22 μm, and then package; at this time, the product batch is 1000 g.

[0029] The mouse ascites described in the present invention is the ascites formed by inoculating cells (such as hybridoma cells) producing heterophilic antibody blockers into the peritoneal cavity of mice, growing and multiplying in the peritoneal environment, and secreting heterophilic antibody blockers, such as active blockers such as HBR-4, HBR-5, and HBR-5 / S.

[0030] The invention has expanded the original small-scale production by more than 10 times. Different from the small-scale production, large-scale production adds different protective agents and neutralizing agents during sample recovery to avoid the coagulation of the target protein after process amplification. The volume of the medium used for purification is different. Glycerol is added as a protective agent during large-scale medium recovery, and NaCl is added to the neutralizing solution to protect the protein.

[0031] The molecular weight cut-off of the ultrafiltration membrane package is 30 kd.

[0032] HP weak anion exchange medium is HPDEAE-30.

[0033] Beneficial effects:

[0034] The present invention adopts octanoic acid precipitation + HP ion exchange for production and purification. In view of the small single batch in the current stage, a 500L liquid preparation tank method is now used for liquid preparation, and the single batch liquid preparation volume is large, which meets the downstream purification requirements. Fully automatic purification equipment is used for purification, and the purification efficiency is high. Ultrafiltration membrane bag equipment is used for automatic liquid exchange processing, the dialysis efficiency is high, and it also has the effect of concentration, which meets the concentration requirements of high-concentration products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the automatic production device of the present invention; wherein: 1-liquid preparation tank, 2-treatment tank, 3-liquid storage tank, 4-sampling tank, 5-automatic chromatography device, 6-first ultrafiltration membrane package equipment circulation tank, 7-second ultrafiltration membrane package equipment circulation tank, 8-centrifugal equipment, 9-filtration equipment, 10-deep filtration system.

[0036] Figure 2 The turbidity of products produced by different production processes. On the left: caprylic acid + ion exchange process, concentration 50 mg / ml, turbidity 10 NTU (clear appearance). On the right: affinity chromatography process, concentration 50 mg / ml, turbidity 107 NTU (with flocculent precipitation).

[0037] Figure 3 The product purity of different production processes is 83% for affinity chromatography and 91% for caprylic acid + ion exchange. DETAILED DESCRIPTION

[0038] The ultrafiltration membrane package used in the following examples has a molecular weight cutoff of 30 kd.

[0039] HP weak anion exchange medium is HPDEAE-30

[0040] Example 1

[0041] A kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, the device comprising a liquid preparation tank (1), a treatment tank (2), a liquid storage tank (3), a sample loading tank (4), an automatic chromatography device (5), a first ultrafiltration membrane package device circulation tank (6), a second ultrafiltration membrane package device circulation tank (7), a centrifugal device (8), a filtering device (9), a deep filtration system (10), and pipelines and valves connected to each tank body; wherein the liquid preparation tank is connected to the liquid storage tank, the liquid storage tank is connected to the treatment tank and the automatic chromatography device, the deep filtration system is connected to the treatment tank, the treatment tank is connected to the centrifugal device, the centrifugal device is connected to the first ultrafiltration membrane package device circulation tank, the first ultrafiltration membrane package device circulation tank is connected to the filtering device, the filtering device is connected to the sample loading tank, the sample loading tank is connected to the automatic chromatography device, and the automatic chromatography device is connected to the second ultrafiltration membrane package device circulation tank.

[0042] The liquid preparation tank is a 500L stainless steel tank, which is automatically controlled by a PLC control system for water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time, and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank;

[0043] The liquid storage tank is a 500L stainless steel tank used for storing heterophilic antibody blocking agent solution.

[0044] The treatment tank is a 500L stainless steel tank, and the heterophilic antibody blocking agent solution stored in the storage tank is discharged into the treatment tank for caprylic acid precipitation;

[0045] The centrifugal device is a continuous flow disc centrifuge;

[0046] The sample is centrifuged in a continuous flow disc centrifuge, and the supernatant is collected and placed in the circulation tank of the first ultrafiltration membrane package equipment, and then enters the filtration device after the liquid is replaced and dialyzed;

[0047] The filtering device is a capsule filter; the filtered sample is transferred to a sample loading tank; an automated chromatography device is used to load the sample and collect the flow-through liquid; the collected sample is passed through a second ultrafiltration membrane bag device circulation tank for liquid exchange and concentration to 50 mg / ml, and is sterilized and filtered at 0.22 μm before being packaged.

[0048] The method for producing kilogram-level high-concentration active heterophilic antibody blocking agent using the above device comprises the following steps:

[0049] 1) Liquid preparation: A 500L stainless steel tank is used as the liquid preparation tank. The PLC control system can automatically control water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time, and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank;

[0050] 2) 100L of mouse ascites (active blocker produced) obtained from immunized mice was initially filtered through filter cloth, and then the sample was deep filtered through 4-18 μm to clarify;

[0051] Mouse ascites acquisition process:

[0052] 1. Select healthy mice over 50 days old as inoculation subjects;

[0053] Aspirate the liquid paraffin / ascites adjuvant, pierce the lower abdominal cavity of the mouse about 2-3 mm, and then push 0.5 ml / mouse of the liquid paraffin / ascites adjuvant 1 cm into the abdominal cavity.

[0054] The inoculated cells should be complete in shape, uniform in size, with clear and smooth edges, strong in refractive index, densely grown, plump in cytoplasm, with few granules, no lipid droplets, and vacuoles in the cytoplasm; the cell survival rate should be >90% when counted under a microscope. The well-growing cells should be blown clean from the culture dish, mixed and collected in a 50ml sterile centrifuge tube, centrifuged at 1000rpm for 5 minutes, discarded the culture supernatant, and then added with sterile saline to blow the cells until uniform, centrifuged at 1000rpm for 5 minutes, discarded the saline supernatant, and washed once with sterile saline according to the above steps.

[0055] Discard the supernatant, add an appropriate amount of saline, and pipette evenly. The amount of saline added must ensure that the number of cells is greater than 1×10 6 / ml, take a drop and put it into the white blood cell counting plate, absorb the overflow outside the cover slip with filter paper, and count the number of cells in the four squares under a low-power microscope.

[0056] Count the total number of cells in the four grids of the counting plate, and only count the cells on the left and above the line. Then calculate according to the formula:

[0057] Total number of cells = total number of cells in four grids / 4×10 4 ×Multiple×Number of ml

[0058] According to the counting results, the number of cells required for immunization of mice was calculated, diluted to the required volume with 0.9% saline, and pipetted evenly.

[0059] Each mouse was injected intraperitoneally with 1×10 6 to 1.3×10 6 cells, the injection dose is 0.2ml / mouse;

[0060] Grab the mouse whose ascites is to be extracted and insert the needle tip into the mouse's lower abdominal cavity, vertically above the centrifuge tube, to allow the ascites to flow naturally into the collection tube.

[0061] 3) Transfer the treated clarified raw material to a 500L treatment tank, add 40mM NaAc (pH4.0) diluent 4 times the raw material volume, and stir evenly; add 10% n-octanoic acid according to the raw material volume, add slowly, and stir for 4 hours;

[0062] 4) The stirred sample is centrifuged in a continuous flow disc centrifuge, and the supernatant is collected and placed in the first ultrafiltration membrane package equipment circulation tank for 2 hours, and the pH of the sample is adjusted to 7.2;

[0063] 5) The pH-adjusted sample was dialyzed in an ultrafiltration membrane pack device using 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol solution with a salt exchange ratio of 1:10;

[0064] 6) Filter the liquid replacement sample through a 0.45 μm capsule filter and transfer the filtered sample to a sample loading tank;

[0065] 7) Prepare 200 L of HP weak anion exchange medium and fill it into the chromatography column; use equilibration buffer 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol to equilibrate the HP DEAE column for 5 times column volume. After the column is equilibrated, adjust the protein UV detector display value to 0 as the baseline;

[0066] 8) Load the sample at a loading capacity of 10 mg / ml using an automated chromatography device at 2 L / min, collect the flow-through, and start collecting protein when the protein UV detector displays a value greater than 0.2; balance with 10 mmPB + 0.3 M NaCl (pH 7.4) + 5% glycerol, and stop collecting protein when the protein UV detector displays a value less than 0.2; 9) Replace the collected sample with a second ultrafiltration membrane package (10 mmPB + 0.3 M NaCl (pH 7.4) + 2% Tween), and concentrate to 50 mg / ml, sterilize and filter the sample at 0.22 μm, and then package; at this time, the product batch is 1000 g.

[0067] The active heterophilic antibody blocker obtained by the device and process of the present invention has better freeze-thaw stability. Figure 2 As shown, the left figure is the caprylic acid + ion exchange process of the present invention, with a concentration of 50 mg / ml and a turbidity of 10 NTU (clear appearance), and the right figure is the affinity chromatography process, with a concentration of 50 mg / ml and a turbidity of 107 NTU (with flocculent precipitation).

[0068] The active heterophilic antibody blocker obtained by the device and process of the present invention has higher purity. Figure 3 As shown, the product purity obtained by the caprylic acid + ion exchange process of the present invention is 91%, while the purity of the affinity chromatography is 83%.

[0069] The active heterophilic antibody blocker obtained by using the device and process of the present invention has better activity. The results are shown in Table 1. The activity of the caprylic acid + ion exchange process of the present invention is increased by about 30% compared with the affinity chromatography process.

[0070] Table 1

[0071]

Claims

1. A kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, characterized in that: The device includes a liquid preparation tank, a treatment tank, a liquid storage tank, a sample loading tank, a deep filtration system, an automatic chromatography device, a first ultrafiltration membrane package equipment circulation tank, a second ultrafiltration membrane package equipment circulation tank, a centrifugal device, a filtering device, and pipelines and valves connected to each tank body; wherein the liquid preparation tank is connected to the liquid storage tank, the liquid storage tank is connected to the treatment tank and the automatic chromatography device, the deep filtration system is connected to the treatment tank, the treatment tank is connected to the centrifugal device, the centrifugal device is connected to the first ultrafiltration membrane package equipment circulation tank, the first ultrafiltration membrane package equipment circulation tank is connected to the filtering device, the filtering device is connected to the sample loading tank, the sample loading tank is connected to the automatic chromatography device, and the automatic chromatography equipment is connected to the second ultrafiltration membrane package equipment circulation tank.

2. According to claim 1, a kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, characterized in that: The liquid preparation tank is a 500L stainless steel tank, which is automatically controlled by a PLC control system for water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank.

3. According to claim 1, a kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, characterized in that: The liquid storage tank is a 500L stainless steel tank.

4. According to claim 1, a kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, characterized in that: The treatment tank is a 500L stainless steel tank, and the treatment tank performs octanoic acid precipitation.

5. According to claim 1, a kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agent, characterized in that: The centrifugal device is a continuous flow disc centrifuge.

6. According to claim 1, a kilogram-level automatic production device for high-concentration active heterophilic antibody blocking agents, characterized in that: The filtering device is a capsule filter.

7. A method for producing kilogram-level high-concentration active heterophilic antibody blocking agents using the device described in any one of claims 1 to 6, characterized in that: The steps include: 1) Liquid preparation: Various solutions are prepared in the liquid preparation tank. The PLC control system can automatically control water addition, stirring, conductivity, and pH adjustment. When the system detects that the pH reaches the set value and lasts for a period of time, and the conductivity meets the requirements, the solution is automatically discharged into the liquid storage tank; 2) 100L of mouse ascites obtained from immunized mice was initially filtered through filter cloth, and then the sample was deep filtered through 4-18μm to clarify; 3) Transfer the treated clarified raw material to a 500L treatment tank, add 40mM NaAc (pH4.0) diluent with a volume of 4 times the raw material, and stir evenly; add 10% n-octanoic acid according to the volume of the raw material, add slowly, and stir for 4 hours; 4) The stirred sample is centrifuged in a continuous flow disc centrifuge, and the supernatant is collected and placed in the first ultrafiltration membrane package equipment circulation tank for 2 hours, and the pH of the sample is adjusted to 7.2; 5) The sample after pH adjustment is dialyzed in an ultrafiltration membrane pack device, and glycerol and sodium chloride are added as protective agents during the liquid exchange and dialysis; 6) Filter the liquid replacement sample through a 0.45 μm capsule filter and transfer the filtered sample to the sample loading tank; 7) Prepare 200L of HP weak anion exchange medium and fill it into the chromatography column; use equilibration buffer 10mmPB+0.3M NaCl (pH7.4)+5% glycerol to equilibrate the HP DEAE column for 5 times column volume. After the column is equilibrated, adjust the protein UV detector display value to 0 as the baseline; 8) Load the sample at a loading rate of 10 mg / ml using an automated chromatography device at 2 L / min, collect the flow-through, and start collecting protein when the protein UV detector shows a value greater than 0.2; balance with 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol, and stop collecting protein when the protein UV detector shows a value less than 0.2; 9) The collected samples were exchanged with the second ultrafiltration membrane package (10 mm PB + 0.3 M NaCl (pH 7.4) + 2% Tween) and concentrated to 50 mg / ml. The samples were sterilized and filtered at 0.22 μm and then packaged; the product batch size was 1000 g.

8. The kilogram-level production method of high-concentration active heterophilic antibody blocking agent according to claim 7, characterized in that: Step 5) The ultrafiltration membrane was dialyzed with 10 mm PB + 0.3 M NaCl (pH 7.4) + 5% glycerol solution at a salt exchange ratio of 1:

10.

9. The kilogram-level production method of high-concentration active heterophilic antibody blocking agent according to claim 7, characterized in that: In step 7), the HP weak anion exchange medium is any one of agarose and polymethacrylate.

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