Preparation method and application of tumor antigen CA199
By using hollow fiber system culture and purification technology, the problems of complex preparation and low production capacity of tumor antigen CA199 have been solved, achieving efficient preparation and high purity of tumor antigen CA199, which is suitable for quality control and calibrators of clinical diagnostic reagents.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-04-03
AI Technical Summary
Existing methods for preparing the tumor antigen CA199 are complex, have low production capacity, and are costly, making it difficult to meet the growing market demand.
Cells secreting CA199 were cultured using a hollow fiber system. The supernatant from the external circulation cells was harvested, and the versatility of various detection reagents was evaluated. A suitable supernatant was then purified using a 5-100 kDa hollow fiber column to obtain high-purity tumor antigen CA199.
It has achieved efficient preparation of tumor antigen CA199, significantly increased production capacity, has good manufacturer compatibility, and is suitable for quality control and calibrators of clinical diagnostic reagents, meeting the needs of the in vitro diagnostic field.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to the preparation method and application of tumor antigen CA199. Background Technology
[0002] CA199 (carbohydrate antigen 199) is an important tumor marker for the clinical diagnosis of pancreatic cancer, with a sensitivity of approximately 70%–90% and a specificity of approximately 85%–95%, playing an irreplaceable role in the early diagnosis of pancreatic cancer. When pancreatic cancer is in stage I, about 60% of patients will show elevated CA199 levels, and the degree of elevation is positively correlated with tumor size and depth of invasion. In addition, CA199 also has some diagnostic value for cholangiocarcinoma and colorectal cancer. Combined detection of CA199 antigen with CA50 and CA242 is mainly used for the comprehensive diagnosis and staging assessment of colorectal cancer, with a sensitivity of 75%–85% and a specificity of 92%–96%. This not only improves the detection rate of early colorectal cancer but also allows for tumor staging by analyzing changes in the levels of these three markers. Significant elevations in all three often indicate that the tumor is in stage III-IV, with a poor prognosis.
[0003] Currently, the preparation of the tumor antigen CA199 mainly relies on traditional culture and purification techniques, including cell culture, supernatant collection, and multi-step purification processes. Although these methods can produce a certain amount of antigen, many challenges remain in terms of purity, yield, and cost control. Existing preparation methods have long preparation cycles, low production capacity, and high costs. The demand for efficient preparation of tumor antigens is increasing, making improved preparation methods a critical issue that urgently needs to be addressed. Summary of the Invention
[0004] In view of this, the present invention provides a method for preparing and applying the tumor antigen CA199 to solve the problems of complex preparation process and low universality of existing tumor antigen CA199 preparation methods.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for preparing tumor antigen CA199, comprising: culturing cells that secrete CA199 using a hollow fiber system, harvesting a supernatant solution containing CA199, detecting the supernatant solution using a variety of CA199 detection reagents to evaluate the universality of CA199, and purifying the supernatant solution based on the universality to obtain tumor antigen CA199.
[0007] In some specific embodiments of the present invention, the above preparation method includes:
[0008] S1: Seed CA199-secreting cells into a hollow fiber system for culture, change the internal circulation medium daily, and harvest the external circulation cell supernatant containing CA199.
[0009] S2: The supernatant of the external circulation cells is centrifuged to obtain a supernatant solution. The supernatant solution is then tested using various CA199 detection reagents to obtain the detection results.
[0010] S3: Based on the detection results, determine the universality of the harvested CA199, select the supernatant solution according to the universality, and purify it using a 5~100 kDa hollow fiber column to obtain tumor antigen CA199.
[0011] In some specific embodiments of the present invention, the cells that secrete CA199 in the above preparation method are COLO205 cells.
[0012] In some specific embodiments of the present invention, the number of cells inoculated in the above preparation method is 10. 6 ~10 8 It can be 2×10 6 4×10 6 6×10 6 8×10 6 10 7 2×10 7 4×10 7 6×10 7 Or 8×10 7 .
[0013] In some specific embodiments of the present invention, the culture conditions of S1 in the above preparation method are 5%~8% (can be 6% or 7%) CO2, 35~37.5℃ (can be 35.5℃, 36℃ or 36.5℃), the culture medium is DMEM containing 10% newborn calf serum, the culture period is not less than 70 days, and the supernatant of the external circulating cells is harvested every 5~10 days (can be 6 days, 8 days or 9 days).
[0014] In some specific embodiments of the present invention, the method for determining the universality of the above preparation method includes: the ratio of the detection values obtained by any two of the detection reagents is between 0.8 and 1.6, and can be 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5.
[0015] In some specific embodiments of the present invention, the ratio of the above preparation method is between 0.9 and 1.3.
[0016] In some specific embodiments of the present invention, the molecular weight cutoff of the hollow fiber column prepared by the above method can be 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa or 100 kDa.
[0017] The present invention also provides a method for preparing a calibration substance for tumor antigen CA199, which includes the step of preparing CA199 according to the above preparation method.
[0018] The present invention also provides a method for preparing a detection reagent, which includes the step of preparing CA199 according to the above preparation method.
[0019] The tumor antigen CA199 prepared using this invention, after identification as a positive control, meets the requirements for use in terms of potency and stability. The tumor antigen CA199 obtained using the method of this invention can be used as an immunogen, a quality control for clinical diagnostic reagents, a calibrator, a positive substance, and for antibody purification, providing a reliable basis for clinical diagnosis and treatment. This invention introduces hollow fiber column technology, making it possible to optimize the preparation process. The resulting antigen has a large production capacity and good manufacturer compatibility, playing an important role in the field of in vitro diagnostics and meeting the growing market demand. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.
[0021] Figure 1 The curve of tumor antigen CA199 secreted by COLO205 cells is shown. Detailed Implementation
[0022] This invention discloses a method for preparing and applying the tumor antigen CA199. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0023] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.
[0024] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.
[0025] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.
[0026] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.
[0027] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.
[0028] In some implementations, the method for preparing the tumor antigen CA199 of the present invention includes: using a method of in vitro culture of hollow fibers in tumor cells, growing and expressing the tumor antigen, and then purifying to obtain a highly active CA199 antigen.
[0029] In practice, the steps include:
[0030] (1) Cell culture: Colon cancer cells were cultured in vitro using hollow fiber three-column parallel technology;
[0031] (2) Antigen expression: The internal circulating culture medium is changed every day, and the external circulating antigen is harvested to achieve continuous culture and long-term harvest of CA199 antigen;
[0032] (3) Antigen purification: Collect the supernatant of cell culture, centrifuge to remove residual cells and obtain a clear supernatant solution, test the manufacturer's universality of the supernatant solution, obtain a supernatant solution with good manufacturer universality, and use a 5~100 kD hollow fiber column for washing, filtration and concentration purification.
[0033] In one example, the culture conditions in step (2) can be: 5% CO2, 37°C, culture medium is DMEM + 10% newborn calf serum, culture period is 90 days, and cell supernatant is harvested every 7 days.
[0034] In one example, cell culture involves seeding 10 cells. 6 ~10 8 Orders of magnitude of cells are contained within hollow fiber columns.
[0035] In one example, the cells used for culturing were COLO205 cells.
[0036] In one example, the tumor antigen CA199 obtained by the present invention is applied in the field of diagnostic reagents, and in particular, can be used as a calibrator or quality control product.
[0037] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.
[0038] The present invention will be further illustrated below with reference to the embodiments.
[0039] Example 1: Preparation of cell supernatant for tumor antigen CA199
[0040] Connect a hollow fiber system (Fibercell, model P3202), and sterilize it after verification. After sterilization, transfer the hollow fiber system to the incubator. Use a tube connector / sealer to aseptically connect the internal refill bottle, waste bottle, external refill bottle, and harvest bottle to the hollow fiber system. Start the peristaltic pump or elevate the refill bottle to fill the hollow fiber system tubing with culture medium, and then start the peristaltic pump to begin rinsing. Resuscitate one vial of COLO205 cells (ATCC, CCL-222), 75 cm. 2 The cells were cultured in T225 flasks at 37°C and 5% CO2 for 2 days. Then, they were subcultured at a 1:3 ratio into T225 flasks and cultured at 37°C and 5% CO2 for 4 days. After further expansion culture, eight T225 flasks were subcultured at a 1:5 ratio and cultured at 37°C and 5% CO2 for 4 days. Seed cells were then harvested by trypsin digestion and centrifuged to obtain at least 2 × 10⁻⁶ cells. 8 Cells were seeded onto hollow fiber columns using DMEM medium containing 15% newborn calf serum. Internal and external circulation were activated, and the medium was replaced promptly based on the consumption of the medium during internal and external circulation. Cell supernatant was routinely harvested weekly after seeding, and the CA199 antigen activity in the cell supernatant was measured.
[0041] Example 2: Detection of tumor antigen CA199 activity in cell supernatant at different culture stages
[0042] The CA199 antigen cell supernatant was diluted with 10 mM PBS pH 7.2 to different gradients (1 / 10, 1 / 50, 1 / 100). If the activity content at any of these dilutions was outside the standard curve range, the dilution factor could be adjusted as needed, and at least five dilution gradients should be tested. The CA199 activity content was determined according to the instructions of Antu's "Carbohydrate Antigen CA199 Detection Kit (Magnetic Microparticle Chemiluminescence)" to calculate the antigen titer. The results are shown in Table 1. Figure 1 (See the corresponding curve). After 8 days of COLO205 cell culture, the activity of CA199 antigen began to rise, plateaued between 40 and 120 days, and then showed a downward trend.
[0043] Table 1: Detection of CA199 tumor antigen activity in cell supernatant at different culture stages
[0044]
[0045] Example 3: Consistency test of tumor antigen CA199 in cell supernatant at different culture stages
[0046] Using the titer calculated from the Antu kit, the CA199 antigen cell supernatant was diluted with 10 mM PBS pH 7.2 to prepare an antigen activity target value of 80-120 U / mL. Then, the CA199 activity was determined according to the instructions of Roche's "Carbohydrate Antigen 19-9 Assay Kit (Electrochemiluminescence Assay)" and Abbott's "Carbohydrate Antigen 19-9 Assay Kit (Chemiluminescent Magnetic Particle Immunoassay)". The activity ratios of each manufacturer were calculated: Ratio 1 was Antu / Abbott, calculated as Abbott activity detection value / Antu activity detection value; Ratio 2 was Antu / Roche, calculated as Roche activity detection value / Antu activity detection value. The total CA19-9 antigen activity was ≥6000 U / mL. Cell supernatants with an Antu / Abbott ratio between 0.8 and 1.6, and those with an Antu / Roche ratio between 0.9 and 1.3, were used for purification. The results are shown in Table 2. The CA199 antigen secreted by COLO205 cells throughout the entire culture cycle showed good consistency between Antu and Roche. However, the CA199 antigen test results using the Abbott kit showed a trend of first increasing and then decreasing. From 43 to 134 days of culture, the consistency between Antu and Abbott ranged from 0.8 to 1.6. These results indicate that the CA199 antigen secreted by COLO205 cells varies between different manufacturers at different culture stages. Based on the combined test results, the cell supernatant from 43 to 134 days of culture was selected for purification.
[0047] Table 2: Activity assay results of tumor antigen CA199 in cell supernatant at different culture stages from various manufacturers (Antu, Roche, and Abbott).
[0048]
[0049] Example 3: Purification of tumor antigen CA199
[0050] Measure the volume of cell supernatant and assemble the 50 kD hollow fiber system; adjust the filtration rate of the hollow fiber system to 90-100 mL / min, wash continuously with 10 mM PBS pH 7.2 buffer (10 times the volume of the concentrated sample), concentrate 5-10 times; centrifuge at 10000 rpm for 30 min at 4℃, and collect the concentrated supernatant.
[0051] Example 4: Application of tumor antigen CA199 in CA199 quality control products
[0052] Potency assay: The purified antigen was diluted to different gradients (1 / 200, 1 / 400, 1 / 800) using the quality control diluent. If the activity content at any of these dilutions was outside the standard curve range, the dilution factor could be adjusted as needed. The CA199 activity content of the samples was determined according to the instructions of Antu's "Carbohydrate Antigen CA199 Detection Kit (Magnetic Microparticle Chemiluminescence)" to calculate the antigen titer. The results are shown in Table 3. The purified CA199 antigen titer was 186591 U / mL.
[0053] Table 3: Results of CA199 Tumor Antigen Titer Detection
[0054]
[0055] Manufacturer consistency: Based on the calculated potency, the self-made antigen was added to a certain amount of diluent and prepared at three levels: low, medium, and high, Q1, Q2, and Q3 (antigen activity target values of 20~40 U / mL, 60~80 U / mL, and 100~140 U / mL, respectively). The prepared samples were tested using reagent kits from Antu, Roche, and Abbott. The controls were Lyphochek Tumor Marker Enhanced Quality Control Level 1 (Bio-Rad, 367), abbreviated as Bio-Rad Q1; Lyphochek Tumor Marker Enhanced Quality Control Level 2 (Bio-Rad, 368), abbreviated as Bio-Rad Q2; and Lyphochek Tumor Marker Enhanced Quality Control Level 3 (Bio-Rad, 369), abbreviated as Bio-Rad Q3. The consistency of antigens among different manufacturers was assessed, and the results are shown in Table 4. The Q1 and Q3 levels of Bio-Rad showed inconsistencies in the tests conducted by Antu, Roche, and Abbott. However, the results for the tumor antigen CA199 were consistent across the three platforms and were superior to the control Bio-Rad, thus meeting the performance requirements for quality control products.
[0056] The prepared samples were tested on three batches of Antu CA199 antigen detection kits to assess the inter-batch matrix effect of the antigen reagents. The results are shown in Table 5. The inter-batch matrix effect of both Bio-Rad triple-level and tumor antigen CA199 triple-level reagents was within ±10%, meeting the quality control requirements.
[0057] Table 4: Consistency test results of tumor antigen CA199 from various manufacturers
[0058]
[0059] Table 5: Results of inter-batch matrix effect detection of CA199 tumor antigen reagent
[0060]
[0061] Stability: Based on the calculated potency, CA199 antigen was added to a certain amount of diluent to prepare three levels (Q1, Q2, and Q3) adjusted to low, medium, and high (antigen activity target values of 20–40 U / mL, 60–80 U / mL, and 100–140 U / mL, respectively). After lyophilization, the potency change after reconstitution and accelerated heating at 2–8℃ and 37℃ for 14 days was assessed. Stability data are shown in Table 6. The potency change of tumor antigen CA199 after accelerated heating at 2–8℃ and 37℃ for 14 days was within 10%, and the stability changes after accelerated heating at 37℃ for Q1 and Q2 were slightly better than the control, Bio-Rad, meeting the performance requirements of the quality control product.
[0062] Table 6: Results of stability testing for tumor antigen CA199
[0063]
[0064] Cross-reactivity: The CA199 antigen activity was adjusted to 100-160 ng / mL, and reactivity was tested on 20 tumor markers (CA125, CA15-3, CA242, CA50, AFP, CA72-4, CEA, Cyfra21-1, Ferritin, fPSA, HE4, NSE, PGI, PGII, ProGRP, SCCA, Tg, tPSA, β2-Microglobulin, β-HCG). All kits were purchased from Zhengzhou Antu Biotechnology Co., Ltd. The test results required that the kits contain no or minimal tumor markers other than the CA199 antigen. After preparation, other markers contained in the antigen should not significantly affect the quality control product. Cross-reactivity results are shown in Table 7. The results of the CA199 tumor antigen and the Bio-Rad quality control on the 20 tumor markers were comparable to those of the matrix solution, and neither affected the CA199 detection, indicating no cross-reactivity and meeting the requirements for quality control use.
[0065] Table 7: Results of cross-detection of tumor antigen CA199
[0066]
[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing tumor antigen CA199, characterized in that, include: Cells secreting CA199 were cultured using a hollow fiber system, and a supernatant containing CA199 was harvested. The supernatant was then tested using various CA199 detection reagents to assess the universality of CA199. Based on the universality, the supernatant was purified to obtain the tumor antigen CA199.
2. The preparation method according to claim 1, characterized in that, include: S1: Seed CA199-secreting cells into a hollow fiber system for culture, change the internal circulation medium daily, and harvest the external circulation cell supernatant containing CA199. S2: Centrifuge the supernatant of the external circulation cells to obtain a supernatant solution, and use a variety of detection reagents for CA199 to detect the supernatant solution and obtain the detection results; S3: Based on the detection results, determine the universality of the harvested CA199, select the supernatant solution according to the universality, and purify it using a 5~100 kDa hollow fiber column to obtain tumor antigen CA199.
3. The preparation method according to claim 1, characterized in that, The cells that secrete CA199 are COLO205 cells.
4. The preparation method according to claim 2, characterized in that, The number of cells inoculated is 10. 6 ~10 8 .
5. The preparation method according to claim 2, characterized in that, The culture conditions for S1 are 5%~8% CO2, 35~37.5℃, and the culture medium is DMEM containing 5%~20% newborn calf serum. The culture period is not less than 70 days, and the supernatant of the external circulation cells is harvested every 5~10 days.
6. The preparation method according to claim 2, characterized in that, The culture conditions for S1 are 5% CO2, 37°C, and the culture medium is DMEM containing 10% newborn calf serum. The culture period is no less than 70 days, and the supernatant of the external circulation cells is harvested every 7 days.
7. The preparation method according to claim 2, characterized in that, The method for determining universality includes: the ratio of the detection values obtained by any two of the detection reagents is between 0.8 and 1.
6.
8. The preparation method according to claim 7, characterized in that, The ratio is 0.9 to 1.
3.
9. A method for preparing a calibrator for tumor antigen CA199, characterized in that, The method includes the step of preparing CA199 according to any one of claims 1 to 8.
10. A method for preparing a detection reagent, characterized in that, The method includes the step of preparing CA199 according to any one of claims 1 to 8.