Nucleoside Detection Method and Its Application

Abstract

The present invention provides a method for detecting nucleosides and its application. The method described in the present invention uses ultra-high performance liquid chromatography (UPLC) to detect the active ingredients in CHO host cell culture medium and cell culture supernatant. The method can quickly detect the components and contents of 6 nucleosides, namely cytidine, hypoxanthine, uridine, guanosine, thymidine and adenosine, simultaneously within 15 minutes.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for detecting nucleosides and its applications. Background Art

[0002] Nucleotides are compounds composed of purine or pyrimidine bases, ribose or deoxyribose, and phosphoric acid. Nucleotides are small molecules. In living organisms, nucleotides are very important low-molecular compounds with various important physiological functions. They can serve as precursors of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can also participate in the material metabolism activities of organisms as regulatory substances for physiological and biochemical processes, such as participating in the energy metabolism of organisms, and related adenosine triphosphate (ATP), dehydrogenase coenzymes, etc.

[0003] According to the different sugars, nucleotides can be divided into two categories: ribonucleic acid and deoxyribonucleic acid. According to the different bases, they can be divided into adenine nucleotides (adenosine monophosphate, AMP), guanine nucleotides (guanosine monophosphate, GMP), cytosine nucleotides (cytidine monophosphate, CMP), uracil nucleotides (uridine monophosphate, UMP), thymine nucleotides (thymidine monophosphate, TMP), and inosine nucleotides (inosine monophosphate, IMP), etc.

[0004] Nucleosides are glycosides formed by the condensation of nitrogenous bases and sugar components, including ribonucleosides and deoxyribonucleosides. Common nucleosides include adenosine, guanosine, cytidine, uridine, thymidine, and inosine, etc. As a type of energy source substance, nucleosides are also added to the culture medium of host cells during the production process of protein drugs.

[0005] At present, the preparation and production of biological drugs, especially the preparation and production of protein drugs, no longer use the early method of isolation from animal organs or tissues, but use the method of genetic engineering technology for preparation and production. Protein drugs obtained using genetic engineering technology greatly reduce the risk of drug use caused by animal sources, and can significantly reduce production costs, improve the production quality and yield of protein drugs.

[0006] In the process of preparing and producing protein drugs, a common method is to transfect an expression vector containing the gene sequence of the protein drug to be expressed into a suitable host cell, and then culture and express the host cell. Commonly used host cells include prokaryotic cells and eukaryotic cells. In the prior art, Escherichia coli, yeast, or mammalian cells are mostly used for large-scale production and expression of protein drugs. Among them, CHO cells (Chinese hamster ovary cell) are the most commonly used host cells.

[0007] The Chinese hamster ovary cell (CHO cell) mammalian expression system is widely used in the production of protein drugs. There are many factors affecting the expression level of foreign genes, which involve various levels such as gene replication, transcription, and post-transcriptional translation. Overall, the growth environment of host cells is also a very important influencing factor.

[0008] In the process of culturing host cells containing the gene of the target protein drug, what kind of host cell culture medium to use is a key control factor. In the production process of protein drugs, it is not only required that the host cells themselves can grow rapidly, but also that the host cells can better express the target protein drug. For host cells containing different foreign genes, different types of culture media are needed to meet their growth and the expression of foreign genes. According to the properties and structures of the target protein drugs to be expressed, different types of host cell culture media are selected.

[0009] Therefore, in the Chinese hamster ovary cell (CHO cell) mammalian expression system, the type of culture medium used by host cells is an important factor affecting the large-scale production and expression of protein drugs. The detection of various components in the culture medium is a key technical means to ensure the efficient expression of the expression system. Summary of the Invention

[0010] The present invention provides a method for detecting nucleosides and its application. The method of the present invention uses ultra-high performance liquid chromatography (UPLC) to detect the active components in CHO host cell culture medium and cell culture supernatant. The method can quickly detect the components and contents of 6 nucleosides, namely cytidine, hypoxanthine, uridine, guanosine, thymidine, and adenosine, simultaneously within 15 minutes.

[0011] The existing methods for detecting nucleosides are mostly capillary electrophoresis and high performance liquid chromatography (HPLC), which can only detect a few nucleosides simultaneously, take a long time, and the types of nucleosides detected are insufficient. Moreover, there is currently no method specifically for detecting nucleosides in CHO host cell culture medium and cell culture supernatant.

[0012] The methods for detecting nucleosides provided in the prior art are time-consuming, costly, and there is no method that can simultaneously detect the components and contents of 6 nucleosides, namely cytidine, hypoxanthine, uridine, guanosine, thymidine, and adenosine, at one time.

[0013] The nucleoside detection method provided by the present invention can simultaneously detect the components and contents of 6 nucleosides, namely cytidine, hypoxanthine, uridine, guanosine, thymidine and adenosine, at one time. The nucleoside detection method provided by the present invention can quickly and accurately detect the types and contents of nucleotides in a sample to be detected in a short time by using ultra-high performance liquid chromatography (UPLC). The nucleoside detection method provided by the present invention is applicable to samples with many interfering components such as serum and the culture medium of protein expression host cells.

[0014] The present invention discloses a nucleoside detection method, which uses ultra-high performance liquid chromatography analysis to detect nucleosides. The mobile phase of the detection method includes mobile phase A, mobile phase B and mobile phase C. Mobile phase A is ammonium acetate or triethylamine, mobile phase B is acetonitrile, and mobile phase C is ultrapure water.

[0015] For the nucleoside detection method as described above, mobile phase A is 0.01% - 0.1% triethylamine, and mobile phase B is 80% - 100% acetonitrile. For the nucleoside detection method as described above, a preferred embodiment is that mobile phase A is 0.1% triethylamine and mobile phase B is 100% acetonitrile.

[0016] For the nucleoside detection method as described above, mobile phase A is 1 mM - 10 mM ammonium acetate, and mobile phase B is 80% - 100% acetonitrile. For the nucleoside detection method as described above, a preferred embodiment is that mobile phase A is 10 mM ammonium acetate and mobile phase B is 100% acetonitrile.

[0017] For the nucleoside detection method as described above, the detection column temperature of the ultra-high performance liquid chromatography detection method is 25°C - 45°C. A preferred embodiment is that the detection column temperature of the ultra-high performance liquid chromatography detection method is 25°C.

[0018] For the nucleoside detection method as described above, the detection flow rate of the ultra-high performance liquid chromatography detection method is 0.1 ml / min - 0.7 ml / min. A preferred embodiment is that the detection flow rate of the ultra-high performance liquid chromatography detection method is 0.3 ml / min.

[0019] For the nucleoside detection method as described above, the elution gradient of the ultra-high performance liquid chromatography detection method is:

[0020] Time (min) % A % B %C 0.00 99 .9 0.1 0.0 3.00 95 .0 5.0 0.0 8.50 95 .0 5.0 0.0 9.00 0.0 50 .0 50 .0 10 .30 0.0 50 .0 50 .0 10 .31 99 .9 0.1 0.0 15 .00 99 .9 0.1 0.0

[0021] For the application of the nucleoside detection method as described above, the detection method is used for the detection of nucleosides in serum, or for the detection of nucleosides in the culture medium of host cells for the production of protein drugs and in the culture supernatant. A preferred embodiment is that the detection method is used for the detection of nucleosides in the culture medium of CHO host cells for the production of antibody drugs and in the culture supernatant. Description of the Drawings

[0022] Figure 1 、Detection chromatogram of the reference standard when the column temperature of AccQ-Tag Ultra C18 1.7μm 2.1*100 mm chromatographic column is 25°C;

[0023] Figure 2 、Detection chromatogram of the reference standard when the column temperature of AccQ-Tag Ultra C18 1.7μm 2.1*100 mm chromatographic column is 45°C;

[0024] Figure 3 、Detection chromatogram of the reference standard when mobile phase A of ACQUITY UPLC HSS T3 1.7μm 2.1*100 mm chromatographic column is 10 mM ammonium acetate;

[0025] Figure 4 、Detection chromatogram of the reference standard when mobile phase A of ACQUITY UPLC HSS T3 1.7μm 2.1*100 mm chromatographic column is 0.1% triethylamine;

[0026] Figure 5 、Comparative chromatogram of 10 mg / L reference standard and nucleosides in CHOM-B01 medium detected by UPLC;

[0027] Figure 6 、Detection chromatogram of nucleosides in CHO host cell medium CHOM-B02;

[0028] Figure 7 、Detection chromatogram of nucleosides in CHO host cell culture supernatant sample 1;

[0029] Figure 8 、Detection chromatogram of nucleosides in CHO host cell culture supernatant sample 2. Detailed implementation method

[0030] Example 1: Preparation of 6 nucleoside reference standards

[0031] The nucleoside reference standard contains 6 kinds of nucleosides, namely cytidine, hypoxanthine, uridine, guanosine, thymidine and adenosine. The concentration of each nucleoside is 0.5 g / L. Hydrochloric acid is added to assist dissolution, and theobromine is selected as the internal standard with a concentration of 0.5 g / L.

[0032] The 0.5 g / L nucleoside reference standard is diluted to prepare reference standards with different concentrations. The linear range of the standard curve is 0.1 - 50 mg / L. Accordingly, the concentrations of the reference standards are set to 50, 10, 5, 0.5 and 0.1 mg / L respectively. Theobromine with a final concentration of 10 mg / L is added to different concentration reference standards.

[0033] Method for dissolving theobromine: Weigh 50 mg of theobromine, add about 12 ml of concentrated hydrochloric acid. After dissolution, add water to make the volume up to 100 ml.

[0034] The dilution method of the standard product is shown in Table 1.

[0035] Table 1. Dilution method of the standard product

[0036]

[0037] Example 2: Detection of UPLC chromatographic column

[0038] 1). AccQ-Tag Ultra C18 1.7 μm 2.1*100 mm chromatographic column

[0039] Mobile phase A: 0.1% triethylamine; Mobile phase B: 100% acetonitrile; Mobile phase C: Milli Q water (ultrapure water). (Note: Ultrapure water can be replaced by injection water pre-cooled to room temperature, but it needs to be filtered with a 0.22 μm filter membrane before use).

[0040] Column temperature of UPLC chromatographic column: 25°C or 45°C; Injection volume: 1 μl; Flow rate: 0.3 ml / min; Detection wavelength: 260 nm.

[0041] Detect the elution conditions, gradient elution, and the parameters are shown in Table 2.

[0042] Table 2. Gradient elution parameters of UPLC chromatographic column

[0043] Time (min) Flow rate (ml / min) % A % B %C 0 0.3 99 .9 0.1 0.0 3.0 0.3 95 .0 5.0 0.0 8.50 0.3 95 .0 5.0 0.0 9.00 0.3 0.0 50 .0 50 .0 10 .30 0.3 0.0 50 .0 50 .0 10 .31 0.3 99 .9 0.1 0.0 15 .00 0.3 99 .9 0.1 0.0

[0044] After the above UPLC chromatography conditions, the detection chromatograms are shown in Figure 1 and Figure 2 , Figure 1 is the standard product detection chromatogram when the column temperature of the chromatographic column is 25°C, Figure 2 is the standard product detection chromatogram when the column temperature of the chromatographic column is 45°C.

[0045] 2). ACQUITY UPLC HSS T3 1.7 μm 2.1*100 mm chromatographic column

[0046] Mobile phase A: 10 mM ammonium acetate or 0.1% triethylamine; Mobile phase B: 100% acetonitrile; Mobile phase C: Milli Q water (ultrapure water). (Note: Ultrapure water can be replaced by injection water pre-cooled to room temperature, but it needs to be filtered with a 0.22 μm filter membrane before use).

[0047] Column temperature of UPLC chromatographic column: 25°C; Injection volume: 1 μl; Flow rate: 0.3 ml / min; Detection wavelength: 260 nm.

[0048] Detect the elution conditions, gradient elution, and the parameters are shown in Table 2.

[0049] After the above UPLC chromatography conditions, the detection chromatograms are shown in Figure 3 and Figure 4 , Figure 3 which is the detection chromatogram of the standard product when mobile phase A is 10 mM ammonium acetate, Figure 4 and which is the detection chromatogram of the standard product when mobile phase A is 0.1% triethylamine.

[0050] The elution peak order of each standard product and internal standard under the above different UPLC columns and different detection conditions is as follows: cytidine, hypoxanthine, uridine, guanosine, thymidine, adenosine, theobromine. See Figures 1 to 4 .

[0051] Example 3. Detection of nucleosides in the CHO host cell medium and culture supernatant during antibody production

[0052] 1). Sample preparation: After centrifuging the suspension at 1000 rpm for 10 min, take the supernatant and store it at -20°C. Thaw it at room temperature before use.

[0053] Sample dilution: The medium and culture supernatant of CHO host cells during antibody production do not need to be diluted, but theobromine with a final concentration of 10 mg / L needs to be added (add 10 μl of 0.5 g / L theobromine to every 490 μl, which is equivalent to a dilution of 1.02 times). Then use a 3K ultrafiltration centrifugal tube (Millipore, product number UFC500396) for ultrafiltration treatment. After ultrafiltration, label it as "ultrafiltered" or "UF" (Ultrafiltration).

[0054] Treatment of 3k ultrafiltration centrifugal tube before use: A new ultrafiltration centrifugal tube can be directly used after leak detection. If it is a used ultrafiltration centrifugal tube, discard the liquid in it and rinse it twice with purified water (do not rinse directly against the filter membrane). Then add 500 μl of purified water and centrifuge at 14000 g for 15 min, discard the filtrate, and spin off the remaining water droplets in the centrifugal tube. The ultrafiltration centrifugal tube needs to be leak-tested before use (both new and used centrifugal tubes need to be leak-tested, and the sample is directly used for leak detection). Add 200 μl of cell culture supernatant and centrifuge at 1000 rpm for 2 min. If no liquid filters through, the ultrafiltration centrifugal tube is intact.

[0055] Ultrafiltration centrifugation: Centrifuge at 14000 g for 15 min, and take the filtered solution for nucleoside detection.

[0056] Treatment of ultrafiltration centrifugal tube after use: Discard the liquid in the centrifugal tube and rinse it twice with purified water; soak it twice with 0.1 mol / L NaOH for 15 min each time; if the ultrafiltration centrifugal tube is not used on the same day, change to soak it with 20% ethanol for 15 min, discard the ethanol in the centrifugal tube and add 20% ethanol for storage at 4°C.

[0057] UPLC Detection: Before running the sample, run the equilibration process twice and equilibrate the chromatographic column twice.

[0058] The method group is "nucleoside", the instrument method is "nucleoside UPLC elution method", the treatment method is "nucleoside", the termination program is "nucleoside detection termination program", and the chromatographic column position is maintained at position 4. Note that when running the termination program, the function is set to "equilibrate the chromatographic column" and the time is 75 min.

[0059] Take 100 μl of the ultrafiltration-treated sample and add it to the full recovery bottle (pay special attention to flicking the bubbles at the bottom of the bottle as bubbles will cause injection failure), and then perform on-machine detection of the nucleoside content.

[0060] After the results of the first standard sample chromatogram come out, check whether the number of peaks and their corresponding positions are normal. If not, there may be problems with the mobile phase or instrument settings, and the problems need to be found and solved.

[0061] Taking the CHO host cell culture medium CHOM-B01 in the antibody production process as an example, use an ACQUITY UPLC HSS T3 1.7 μm 2.1 * 100 mm chromatographic column. Mobile phase A is: 10 mM ammonium acetate; mobile phase B is: 100% acetonitrile; mobile phase C is: Milli Q water (ultrapure water) (note: ultrapure water can be replaced by injection water pre-cooled to room temperature, but it needs to be filtered through a 0.22 μm filter membrane before use).

[0062] UPLC chromatographic column temperature: 25 °C; injection volume: 1 μl; flow rate: 0.3 ml / min; detection wavelength: 260 nm.

[0063] Detect the elution conditions, gradient elution, and the parameters are shown in Table 2.

[0064] The results of detecting nucleosides in the CHO host cell culture medium CHOM-B01 are shown in Figure 5 , Figure 5 which is a comparative chromatogram of the 10 mg / L standard sample detected by UPLC and nucleosides in the CHOM-B01 culture medium.

[0065] The results of detecting nucleosides in the CHO host cell culture medium CHOM-B02 are shown in Figure 6 .

[0066] The results of detecting the CHO host cell culture supernatant sample 1 are shown in Figure 7 .

[0067] The results of detecting the CHO host cell culture supernatant sample 2 are shown in Figure 8 .

Claims

1. A nucleoside detection method, characterized in that, The CHO host cell culture medium and the culture supernatant were added with theobromine at a final concentration of 10 mg / L, and after ultrafiltration treatment, six nucleosides including cytidine, hypoxanthine, uridine, guanosine, thymidine and adenosine were simultaneously detected by ultra-high performance liquid chromatography analysis. The culture medium of the CHO host cell was CHOM-B01 or CHOM-B02; the mobile phase of the detection method included mobile phase A as ammonium acetate or triethylamine, mobile phase B as acetonitrile, and mobile phase C as ultrapure water; the chromatographic column used for the ultra-high performance liquid chromatography analysis was ACQUITY UPLC HSS T3 1.7 μm 2.1*100 mm, and the detection wavelength was 260 nm; the elution gradient of the ultra-high performance liquid chromatography detection method was: 。 2. The nucleoside detection method according to claim 1, characterized in that, Mobile phase A was 0.01% - 0.1% triethylamine, and mobile phase B was 80% - 100% acetonitrile.

3. The nucleoside detection method according to claim 2, characterized in that, Mobile phase A was 0.1% triethylamine, and mobile phase B was 100% acetonitrile.

4. The nucleoside detection method according to claim 1, characterized in that, Mobile phase A was 1 mM - 10 mM ammonium acetate, and mobile phase B was 80% - 100% acetonitrile.

5. The nucleoside detection method according to claim 4, characterized in that, Mobile phase A was 10 mM ammonium acetate, and mobile phase B was 100% acetonitrile.

6. The nucleoside detection method according to claim 1, characterized in that, The detection column temperature of the ultra-high performance liquid chromatography detection method was 25°C - 45°C.

7. The nucleoside detection method according to claim 6, characterized in that, The detection column temperature of the ultra-high performance liquid chromatography detection method was 25°C.

8. The nucleoside detection method according to claim 1, characterized in that, The detection flow rate of the ultra-high performance liquid chromatography detection method was 0.1 ml / min - 0.7 ml / min.

9. The nucleoside detection method according to claim 8, characterized in that, The detection flow rate of the ultra-high performance liquid chromatography detection method was 0.3 ml / min.

10. The application of the nucleoside detection method according to any one of claims 1-9, characterized in that, The detection method was used for the detection of nucleosides in the host cell culture medium CHOM-B01 or CHOM-B02 and the culture supernatant for the production of protein drugs.

11. The application of the nucleoside detection method according to claim 10, characterized in that, The detection method was used for the detection of nucleosides in the culture medium CHOM-B01 or CHOM-B02 and the culture supernatant of CHO host cells for the production of antibody drugs.

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