Method for obtaining crystallized fetal bovine serum containing agmatine

Abstract

The present invention relates to a method for obtaining crystallized fetal bovine serum by adding agmatine, which has the effect of increasing cell production and increasing protein expression per cell at a specific concentration. Furthermore, the method, with respect to a specific reconstitution form into a liquid, is shown for use without losing its original properties, allowing for storage without a cryogenic chain, reducing the risk of contamination, and enabling precise dosage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for crystallizing and reconstituting supplements for cell culture media.

Summary of the Invention

[0002] The object of the present invention is to construct a method for obtaining crystallized fetal bovine serum. In this method, agmatine is selected and added to obtain a crystallization efficiency of 98% for liquid fetal bovine serum. For this agmatine, various polyamines are tested and the one most suitable for increasing cell production and protein expression per cell is selected.

[0003] Also, a method for specifically regenerating in liquid for use without losing the original characteristics is presented. With this method, it becomes possible to accurately set the dosage relative to the usage amount, enabling storage without an ultra-low temperature cold chain and reducing the risk of contamination.

Background Art

[0004] In the culture of animal and plant cells, protein serum that makes nutrients available for growth must always be used. These nutrients are necessary for efficient cell growth and the production of the target protein or virus.

[0005] In order to efficiently develop biological agents such as viruses or recombinant proteins, it is important to obtain an optimal cell density and increase protein expression per cell to obtain the maximum agent yield.

[0006] ​Generally, the preparation of known fetal bovine serum requires a cold chain during transport and storage. The crystallized fetal bovine serum presented in this invention is a groundbreaking product with a fine crystalline structure, and the undesirable ratios of albumin, transferrin, mycoplasma, and bovine spongiform encephalopathy are appropriately adjusted, resulting in a safe formulation, especially when used in human drugs or vaccines.

[0007] Currently, alternatives are being sought to provide efficient culture conditions that significantly reduce contamination by thawing and collecting only what is needed for laboratory use. This invention arose from the need to provide a method to address some of these and other technical problems with currently available serums.

[0008] Batch-to-batch variability related to the concentration of serum components and their biological activity ultimately leads to experimental variability, limiting inter-institutional reproducibility, particularly with regard to the culture, proliferation, and differentiation of primary cells. This is where the main costs associated with cell culture arise.

[0009] Because the reproducibility of methods based on fetal bovine serum is questionable, their use for regulatory purposes is debated. For this reason, serum-supplemented media should be avoided whenever possible, and chemically defined media are preferred (van der Valk, et al., 2018).

[0010] Ideally, sufficient serum should be available for a complete test. Furthermore, when replacing vials, it is necessary to ensure that no other reagents have been changed, and that a sufficient amount of old serum is stored to investigate whether any unexpected results are due to the replacement.

[0011] For example, if a new serum batch affects the cell mitosis rate, it is easy to conclude that there is something wrong with the protocol for introducing DNA into the cells. Researchers should also record information provided by the supplier about the serum, such as the batch number (Baker, M, 2016).

[0012] Some researchers choose gamma irradiation to avoid serum contamination, as some common contaminants, such as mycoplasma, are sensitive even to low levels of radiation. However, this requires striking a balance.

[0013] This is because radiation also damages growth proteins and bioactive molecules that help cells grow. Raymond Nims, a cell culture consultant at RMC Pharmaceutical Solutions in Longmont, Colorado, advises anyone planning to use gamma-irradiated serum to first test whether the cells function as expected and to keep in mind that even serum free of contaminants cannot prevent infection from other sources (Baker, M, 2016).

[0014] The use of fetal bovine serum-free media can reduce animal suffering and potentially lead to more reproducible in vitro methods. Some of these media have been developed to serve as alternatives to or reduce animal testing, contributing to the replacement, reduction, and improvement of animal testing. Therefore, safety, as well as scientific and ethical considerations, provide incentives for developing and using fetal bovine serum-free media. (van der Valk, et al., 2018)

[0015] In this invention, the cold chain is not required in the use of the final formulation, contamination-free dose manipulation is possible, and storage at room temperature is possible, thereby significantly reducing costs for consumers. The crystallized fetal bovine serum developed in this invention is obtained by first classifying, mixing, and processing the blood of different fetal bovines to obtain fetal serum, then crystallizing this fetal serum, dissolving it into a liquid, and then crystallizing it again to obtain a solid form that, when incorporated into a culture medium, provides desirable efficiency and cell productivity.

[0016] This invention is based on a method for transforming the formulation form. This is because, while only known liquid fetal bovine serum is dominant in the market, this new invention, which will revolutionize the market, was obtained through a crystallization method.

[0017] The crystallized fetal bovine serum according to the present invention maintains the same properties as a liquid formulation, does not require freezing, offers higher efficiency and performance during handling, and is provided in the form of fine crystals.

[0018] The advantages of this formulation include eliminating the need for a cold chain, improving the shelf life of the formulation as inventory, allowing the formulation to be stored at room temperature below 30°C, eliminating the need for refrigeration, enabling contamination-free dosage setting without compromising the integrity of the formulation, and consequently making distribution and sales methods more efficient.

[0019] The crystallized fetal bovine serum on which this invention is based is characterized by the fact that, although it is necessary to prepare the required amount based on a dilution quick reference table to prepare the necessary culture medium, it is possible to obtain fetal bovine serum with the same properties as known liquid serum simply by regenerating the crystals with a depyrogenated aqueous solution.

[0020] Naturally, fetal bovine serum is a liquid preparation, but it must be stored at a minimum temperature of -20°C, and its properties are lost upon thawing, meaning it can only be used once after thawing. Therefore, it is a delicate and sensitive preparation to handle. Furthermore, the preparation of commercially available fetal bovine serum requires a cold chain during transportation.

[0021] The fetal bovine serum obtained by the crystallization method of the present invention is an innovative formulation with a fine crystalline structure, and the proportions of undesirable substances such as albumin, transferrin, mycoplasma, and bovine spongiform encephalopathy virus are reduced, making it a safe formulation, especially when used in human drugs or vaccines.

[0022] The present invention enables uniform cell proliferation by using crystallized fetal bovine serum, thereby increasing the yield of desired products, particularly proteins. A key feature of the present invention is the use of agmatine, a short-chain polyamine that synergistically increases cell proliferation, cell-specific productivity, and final cell density in the method of crystallizing bovine serum.

[0023] Among the patents selected from the patent database, particularly from the International Patent Classification A 61 in the consumer goods sector, there is a reference to prior art in section A61K35 / 16, Patent No. US2009124011A1, Serum Production System, which describes a bovine serum composition having controlled bovine serum properties, and a method for producing such a composition from the whole blood of female offspring of bovine mammals. This patent describes the control of bovine fetal serum properties by production conditions, but does not mention a crystallization method.

[0024] In sections A61D1 / 00, A61D1 / 08, A61K35 / 16, B01D17 / 00, B01D35 / 00, and B01D57 / 00, Patent No. CN101112334A, a technology for producing fetal bovine serum, refers to fetal bovine serum. In particular, it refers to a method for producing fetal bovine serum, and describes the collection, separation, filtration, and freezing for subsequent storage in the invention.

[0025] This patent does not describe a crystallization method as described in the patent according to the present invention. None of the patents describe a method for crystallizing fetal bovine serum containing agmatine, particularly in the specifications detailed in the present invention.

[0026] Interestingly, the fetal bovine serum crystallized by the present invention was found to be more advantageous in terms of protein expression and cell proliferation rate compared to natural fetal bovine serum, which is considered a standardized formulation in terms of its production. [Modes for carrying out the invention]

[0027] Regarding the present invention, features, content, and functions will be described in detail while referring to each step and figure. Similarly, differences from known ones will also be mentioned.

Example

[0028] Various analyses were performed to determine various parameters required for control fetal bovine serum, such as the amounts of proteins and albumin. This enabled adaptation to or improvement of preparations in different forms such as crystallized serum.

Table 1

[0029] The following results were obtained.

Table 2

[0030] After identifying these fetal bovine blood batches, they were mixed to conform to the physicochemical properties of fetal bovine blood, fibrin was removed, and centrifugation was performed at 8,000 rpm. Using a Beckman Coulter centrifuge model Avanti JxN-26 at 4°C for 30 minutes, ultrafiltration was carried out by nitrogen push using a Pall 0.10 micron two-layer Acropack filter and placed in a sterile PETG container.

[0031] Next, the most important batches that yielded almost satisfactory results during the implemented tests are listed.

Table 3

[0032] A mixture with characteristics similar to control fetal bovine serum was obtained. At this time, verification was performed between the analysis in this example and the analysis performed by an accredited testing institution such as LABORATORIOS LEI where parameters such as proteins, albumin, immunoglobulins, endotoxins, osmolarity, hemoglobin, and hydrogen ion index were confirmed (the results from the testing institution are attached as Appendix "A").

[0033] The following results were obtained from the second batch using an effective mixture. [Table 4]

[0034] This batch was sent to the Department of Immunology at the Instituto Mexicano del Seguro Social Siglo XXI for similar testing. The same institution had already conducted cell proliferation analyses using HUVEC, RAJI, and THP-1 cells (the results from the testing institution are attached as Appendix "B"). [Table 5] [Table 6]

[0035] The following mixture was prepared for the next batch. The samples were sent again to the Department of Immunology at the Instituto Mexicano del Seguro Social Siglo XXI, where physicochemical and cell proliferation tests were continued. Similarly, they were also sent to the Department of Biotechnology at the Instituto Politécnico Nacional, where cell culture was performed using heLa cells (the results from these laboratories are attached as Appendix "A").

[0036] The following mixture was prepared for the next batch. [Table 7] SFB: Fetal bovine serum. SE: 40% ethanol solution. [Examples]

[0037] Work was initiated to improve the physical properties of the formulation, such as stability and color. Freeze-drying was considered and carried out at the Universidad Autónoma del Estado de Hidalgo (UAEH), but the results were not as favorable as those of conventional fetal bovine serum, with neither appearance nor physicochemical properties being satisfactory. [Examples]

[0038] We also obtained important results in the next batch. [Table 8] We began by using several chemical formulations to remove water and the resulting solids, and explored other polyamines and polyamides until we obtained the expected results. We repeated small error tests until we concluded which organic reagent yielded the desired results. [Examples]

[0039] The following mixture was prepared for the next batch. [Table 9] The process was repeated under the same conditions as batch DQ-297 / 14-12-2020, but using a larger quantity resulted in the same outcome as before. However, this time, ethyl alcohol was added for purification, excess was removed in a water bath, dried at 30°C, and sterilized by irradiation with ultraviolet light for 30 to 45 minutes. [Examples]

[0040] To use this formulation, prepare the required amount based on the dilution chart for preparing the necessary culture medium. By simply regenerating the crystals with a depyrogenated aqueous solution, you can obtain fetal bovine serum with the same properties as the known liquid form. [Table 10]

[0041] The table above is based on 48.50 grams per liter of fetal bovine serum crystals, and dilution is performed based on this data. Due to the efficiency of the method of the present invention, each batch of crystallized fetal bovine serum obtained can contain a certain amount of protein.

[0042] After crystallization of fetal bovine serum, supply the specified amount of diluent as shown in the dilution quick reference table above, adjusting it to the ratio shown in the corresponding culture medium. This dilution ratio should be 10%. The equivalence of the finished product with the liquid form is shown in the same table in milliliters of liquid fetal bovine serum. The following describes a preferred embodiment of the present invention selected to obtain crystallized fetal bovine serum.

[0043] I began my search for polyamides and encountered several aliphatic polyamides. According to the literature, they polymerize very easily, and even more so with changes in pH. Therefore, I decided to continue my search for polyamines.

[0044] According to the literature, polyamines are preferable if they have a higher affinity for proteins, and even better if they have a low molecular weight and a low melting point. Therefore, we decided to mix them with aliphatic compounds and conducted various tests, varying stability, appearance, color, quantity, handling, polyamine concentration, alcohol concentration, temperature, dehydration, positive and negative chemical and biological effects, and crystallization variations.

[0045] When beginning work with polyamines, agmatine, spermine, and spermidine were found to be available. The same variables were used for each, including stability, appearance, color, quantity, handling, polyamine concentration, ethyl alcohol concentration, temperature, dehydration, positive and negative chemical and biological effects, and crystallization. The best results were those that demonstrated positive chemical and biological properties and were deemed worth considering.

[0046] To actually carry out the collection of crystallized fetal bovine serum according to the present invention, each step of a preferred embodiment is described in full and clearly below.

[0047] Identify the origin of the animal (female cow).

[0048] Verify that the animals meet health requirements. Ensure that records of illnesses, vaccinations, and feed are complete.

[0049] 3. Select cows that meet the following characteristics. a) The pregnancy must be within the first seven months. b) Not being sick. c) Having received all vaccinations. d) The animals must be nutritionally healthy through a grass-based diet.

[0050] 4. To ensure that animals are slaughtered without being subjected to cruel treatment, numbing is performed using CASH brand non-penetrating stunning tools.

[0051] 5. The animal is slaughtered by vertically slitting its throat.

[0052] 6. Perform drainage of the bovine fetus.

[0053] 7. Then, the fetus is removed vertically.

[0054] 8. Collect fetal blood vertically and collect it in a polypropylene bag under hygienic conditions to ensure it is free from contamination and ready for processing.

[0055] 9. Check the blood for external contaminants. Visual and manual inspection will be performed to check whether the fetal blood was collected under adequately clean conditions, whether coagulation has already begun, and to check the blood's characteristics such as color and consistency.

[0056] 10. Allow the blood to coagulate for 5 hours. Keep the coagulated blood in ice to prevent microbial growth.

[0057] 11. Mechanically removes fibrin from the blood and dissolves all types of clots.

[0058] 12. For serum separation, the blood is centrifuged at 8,000 rpm for 30 minutes at 4°C using a Beckman Coulter Avanti JxN-26 centrifuge.

[0059] 13. Perform decantation.

[0060] 14. Fetal bovine serum is obtained.

[0061] 15. The serum is placed in an elliptical homogenization tank with a capacity of 10 to 20 liters, and homogenized in it by mechanical means, namely circular motion, horizontal motion, vertical motion, and vibration motion.

[0062] 16. A homogeneous serum is obtained.

[0063] 17. Transfer the serum to a cylindrical stainless steel container with an inward-sloping design. The outside of the container is fitted with a manometer for measuring the pressure of the nitrogen push, a lid for sealing, and a flexible fitting that extends from the cylindrical container to a container with a capacity of 10 to 20 liters. It must be connected to a system with a nitrogen push for packaging based on the set dose.

[0064] 18. The serum is triple filtered using a Pall 0.1 micron two-layer Acropack filter to remove toxins and unwanted chemicals.

[0065] 19. Package the product for distribution under hygienic and completely sterile conditions.

[0066] 20. After packaging, irradiate the serum with ultraviolet light for 30 to 45 minutes to ensure complete sterilization of the preparation.

[0067] 21. Store the serum frozen at -21°C.

[0068] 22. Thaw the pure fetal bovine serum at room temperature between 8 and 12 degrees Celsius.

[0069] 23. Add 80 ml of 30% agmatine with water to bring the pH to 6. This mixture is equivalent to a solution of 600 ml of fetal bovine serum, 80 ml of 30% agmatine, and 100 ml of 40% ethanol solution, all adjusted to pH 6.

[0070] Incubate at 24.-5°C for 2 hours.

[0071] 25. To separate the solids, centrifuge at a speed of 8,000 rpm at -6°C.

[0072] 26. If solid matter is present after centrifugation, remove it from the sample.

[0073] 27. A 12.8% solution of 40% ethyl alcohol is added to the agmatine-containing fetal bovine serum compound to help maintain the properties of the fetal bovine serum without affecting its chemical composition.

[0074] 28. Separate the solids by centrifuging at a maximum temperature of -5°C and 8,000 rpm.

[0075] 29. To dehydrate the formulation without losing its original chemical properties, gradually increase the temperature until the maximum temperature reaches 18°C, taking the time necessary to remove the water.

[0076] 30. The sample forms fine pink crystals. Fine pink crystals of approximately 1 micron are obtained.

[0077] 31. The crystallized compound is packaged in a sterile container made of PETG polyester glycol or glass.

[0078] 32. Irradiate the preparation with ultraviolet light for 30 to 45 minutes to ensure complete sterilization.

[0079] 33. Test key parameters to confirm compliance with the quality and sterility of the formulation.

[0080] 34. For use of this product, prepare the required amount based on the dilution quick reference chart for preparing the necessary culture medium. By simply regenerating the crystals with a depyrogenated aqueous solution, you can obtain fetal bovine serum with the same properties as the known liquid form.

[0081] Based on the test batches performed, the optimal amount for crystal regeneration and use is 0.242 g of crystallized fetal bovine serum for 50 ml of depyrogenated aqueous solution, 0.485 g of crystallized fetal bovine serum for 100 ml of the same solution, 1.2125 g of crystallized fetal bovine serum for 250 ml of the same solution, 2.425 g of crystallized fetal bovine serum for 500 ml of the same solution, and 4.85 g of crystallized fetal bovine serum for 1000 ml of the same solution.

Claims

1. A method for obtaining crystallized fetal bovine serum containing agmatine, comprising the following steps.

1. Identification of the origin of the female cattle, 2. Confirmation of the health requirements of the cows, 3. Selection of female cows, 4. Insensitization of cows using CASH brand non-penetrating stunning tools, 5. Slaughter by vertically slitting the throat, 6. Drainage of bovine fetuses, 7. Vertical extraction of bovine fetuses, 8. Vertical collection of fetal blood and hygienic recovery in a polypropylene bag, 9. Confirm that there are no external contaminants in the blood, 10. Leaving blood unattended, 11. Mechanical fibrin removal of blood to dissolve all types of coagulation, 12. Separation of serum by centrifugation, 13. Decantation of fetal bovine serum, 14. Collection of fetal bovine serum, 15. Addition of fetal bovine serum to the homogenization tank, 16. Collection of homogeneous fetal bovine serum, 17. Transferring bovine fetal serum into a stainless steel container, 18. Filtration of fetal bovine serum for the removal of toxins and unwanted chemicals, 19. Packaging of fetal bovine serum for distribution under hygienic and completely sterile conditions, 20. Sterilization of fetal bovine serum, 21. Freezing of fetal bovine serum, 22. Thawing of pure bovine fetal serum, 23. Collection of a mixture of fetal bovine serum, agmatine, and water, 24. Incubation of the mixture, 25. Centrifugation of the mixture, 26. Removal of as much solid matter as possible from the sample after centrifugation, 27. Addition of a 12.8% solution of 40% ethyl alcohol to agmatine-containing fetal bovine serum to help maintain the properties of fetal bovine serum without affecting its chemical composition and to remove residual polyamines, 28. Centrifugation for separating solids, 29. Drying to dehydrate the mixture, 30. Collection of fine crystals, 31. Packaging of crystallized compounds, 32. Disinfection of compounds by ultraviolet light, 33. Conducting tests for quality control, 34. Crystal regeneration.

2. A method for obtaining crystallized bovine fetal serum containing agmatine according to claim 1, characterized in that, in step 3, the selection of cows is based on their age up to 7 months of gestation.

3. A method for obtaining a crystallized compound of fetal bovine serum containing agmatine according to claim 1, characterized in that, in step 10, the blood is left in ice for 5 hours.

4. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 12, the serum is separated by centrifugation at 8,000 rpm for 30 minutes at 4°C in a Beckman Coulter Avanti JxN-26 centrifuge.

5. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that, in step 15, an elliptical stainless steel tank with a capacity of 10 to 20 liters is used, and homogenization is carried out therein by mechanical means, namely circular motion, horizontal motion, vertical motion and vibration motion.

6. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 17, the serum is transferred to a cylindrical stainless steel container which needs to be connected to a system with a nitrogen push for packaging based on a set dose.

7. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 18, triple filtration by nitrogen push is performed using a 0.10 micron two-layer Acropack filter from Pall.

8. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 20, the fetal bovine serum is sterilized by ultraviolet light for 30 to 45 minutes.

9. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that, in step 21, the fetal bovine serum is stored at -21 degrees Celsius.

10. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 22, the pure fetal bovine serum is thawed at room temperature between 8 and 12 degrees Celsius.

11. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 23, a mixture is obtained by adjusting the pH of a solution of 600 ml of fetal bovine serum, 80 ml of 30% agmatine, and 100 ml of 40% ethanol solution to 6.

12. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 24, the mixture is incubated at -5°C for 2 hours.

13. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 25, the mixture is centrifuged at -6°C at a speed of 8,000 rpm for the separation of solids.

14. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 27, ethyl alcohol is added to the compound of fetal bovine serum and agmatine, and the concentration of ethyl alcohol is 40%.

15. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 28, centrifugation is performed at a maximum temperature of -5°C and 8,000 rpm for the separation of solids.

16. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that, in step 29, the drying temperature for dehydrating the compound is gradually increased up to a maximum temperature of 18°C ​​to prevent loss of the original chemical properties.

17. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 30, fine pink crystals with a size of 1 micron are obtained, and the size of the crystals is not limited to 1 micron.

18. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 31, the packaging of the compound may be made of sterile PETG polyester glycol or a glass container.

19. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that the compound is sterilized by ultraviolet light in step 32.

20. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 32, the sterilization time of the compound is 30 to 45 minutes.

21. A method for obtaining crystallized fetal bovine serum containing agmatine according to claim 1, characterized in that in step 34, the amount of crystals for regeneration and use may be 0.242 grams of crystallized fetal bovine serum in the case of 50 ml of depyrogenated aqueous solution, 0.485 grams of crystallized fetal bovine serum in the case of 100 ml of the same aqueous solution, 1.2125 grams of crystallized fetal bovine serum in the case of 250 ml of the same aqueous solution, 2.425 grams of crystallized fetal bovine serum in the case of 500 ml of the same aqueous solution, and 4.85 grams of crystallized fetal bovine serum in the case of 1000 ml of the same aqueous solution.

22. Crystallized fetal bovine serum containing agmatine obtained by the method described in claims 1 to 20.

23. Use of crystallized fetal bovine serum containing agmatine in cell culture to promote cell proliferation and increase protein expression per cell.

24. Use in cell culture of crystallized fetal bovine serum containing agmatine according to claim 1 for storage without using an ultra-low temperature cold chain.

25. Use of crystallized fetal bovine serum containing agmatine according to claim 1 in cell culture, using the precise dose setting according to claim 2, which reduces the risk of contamination relative to the amount used.

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