A method and system for simultaneous detection of protein expression level and charge heterogeneity of a cell culture

Abstract

The application provides a cell culture protein expression amount and charge heterogeneity synchronous detection method and system, and relates to the technical field of protein detection. Specifically, the method comprises the following steps: sequentially performing gradient elution and chromatography detection on the cell culture, wherein the chromatography column adopts a series connection of an affinity chromatography column and an ion exchange chromatography column. The synchronous detection method solves the blank of synchronous detection of antibody titer and charge heterogeneity in the prior art, improves the efficiency of the traditional analysis mode, and has the advantages of less sample consumption, shorter time consumption, less manpower and instrument resources required, simple structure of the synchronous detection system and convenient operation.

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, and more specifically, to a method and system for the simultaneous detection of protein expression levels and charge heterogeneity in cell cultures. Background Technology

[0002] Antibody drugs are a class of biotechnology drugs expressed through cells. During cell culture, protein expression levels change with culture time; therefore, it is necessary to monitor antibody titer changes simultaneously to find the optimal balance between input and output. Meanwhile, numerous post-translational modifications or alterations may occur during cell culture, causing heterogeneity in antibody charge distribution. These post-translational modifications affect antibody activity, immunogenicity, and pharmacokinetics; therefore, charge heterogeneity is also a key indicator for process control in antibody drug manufacturing.

[0003] Traditional methods for determining protein expression levels include nitrogen determination, biuret method, Lowry method, UV absorption method, and Coomassie brilliant blue method. However, these methods can only detect the total protein content in a sample and cannot determine the concentration of the active target protein, and the values ​​of the two methods differ significantly. In contrast, enzyme-linked immunosorbent assay (ELISA), Protein A-HPLC / Protein G-HPLC, and biomembrane interferometry (BLI) can specifically bind to the target protein and accurately determine its concentration. Among these methods, ELISA has a low detection limit, making it suitable for detecting trace samples; HPLC is characterized by accuracy, a wide linear range, and high recognition; and BLI offers advantages such as high throughput, small error, and simple operation.

[0004] Common methods for detecting charge heterogeneity include ion exchange chromatography (IEX), isostatic focusing electrophoresis (IEF), capillary zone electrophoresis (CZE), capillary isostatic focusing electrophoresis (CIEF), and imaging capillary isostatic focusing electrophoresis (iCIEF). Among these, IEX, CIEF, and CIEF offer better resolution and quantification, while CZE is versatile, has a short analysis time, and exhibits considerable separation and accuracy.

[0005] Currently, in the development of antibody drug cell lines, the traditional analytical approach involves first detecting the antibody titer (equivalent to the protein expression level, or protein concentration, antibody concentration, as discussed in this invention) in the cell supernatant. The cell supernatant is then purified using affinity chromatography before being used for antibody charge heterogeneity detection. This approach has drawbacks such as high sample consumption, labor costs, and instrumental resources.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for the simultaneous detection of protein expression levels and charge heterogeneity in cell cultures. Such a process for simultaneously detecting protein expression levels and charge heterogeneity is not currently reported in the market. This process will significantly reduce sample consumption, improve detection efficiency, and lower experimental costs.

[0008] The second objective of this invention is to provide a system for the simultaneous detection of protein expression levels and charge heterogeneity in cell cultures, which is adapted to the aforementioned method for the simultaneous detection of protein expression levels and charge heterogeneity in cell cultures.

[0009] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted: a method for simultaneous detection of protein expression levels and charge heterogeneity in cell cultures, comprising the following steps: sequentially subjecting cell cultures to gradient elution and chromatographic detection, wherein the chromatographic column uses a series connection of an affinity chromatography column and an ion exchange chromatography column, and the chromatographic detection is performed using high-performance liquid chromatography (HPLC). Specifically, the protein quantification result, i.e., protein expression level, is obtained through detection in the affinity chromatography column; and the charge heterogeneity detection result, i.e., charge qualitative and quantitative results, is obtained through detection in the ion exchange chromatography column.

[0010] In current detection processes, protein expression level detection and charge heterogeneity detection are usually performed independently, and there are a series of technical obstacles to combining them into a simultaneous process. The target protein (antibody) first binds to an affinity chromatography column, removes impurities, and then elutes from the affinity column, immediately binding to an ion exchange chromatography column. However, the affinity column requires acidic conditions (pH = 2.5) for elution, and the eluted protein cannot bind to the ion exchange column, instead flowing directly through it. Through extensive innovative practice and improvement by the inventors, by studying the buffer volume and buffer time required for the target protein to move from the affinity column to the ion exchange column, and by adding a mobile phase to the affinity column, using a combination of mobile phases A, B, and C to balance the column, the aim is to achieve the goal of not affecting the subsequent binding of the protein to the ion exchange column.

[0011] Meanwhile, the detection of charge heterogeneity is molecularly specific and challenging to develop; for example, the selection of chromatographic columns, mobile phases, and elution conditions all present development difficulties, making it impossible to achieve a universal solution through a platform-based approach. Furthermore, when charge heterogeneity and expression levels are detected simultaneously, the column tolerance conditions required for the two detections are inconsistent (e.g., differences in flow rate and column temperature), further increasing the development difficulty. The technical solution adopted in this invention provides a universal simultaneous detection method with good practical performance and application prospects.

[0012] Preferably, the proteins in the cell culture include at least one of IgA, IgG, IgM, Kappa light chain, Lambda light chain, and cytokines;

[0013] More preferably, the protein in the cell culture is a monoclonal IgG1 antibody.

[0014] Preferably, the method further includes pre-treating the cell culture before synchronous detection; the pre-treatment includes centrifuging the supernatant of the cell culture and using the supernatant after centrifugation as the sample to be tested.

[0015] More preferably, the centrifugation speed is 10,000 rpm to 15,000 rpm, and the centrifugation time is 8 min to 12 min.

[0016] Preferably, the affinity chromatography column has one or more of the following technical features:

[0017] (1) The gradient elution mobile phase includes a combination of mobile phase A, mobile phase B and mobile phase C; wherein, mobile phase A includes glycine and sodium chloride; mobile phase B includes CX-1 pH gradient A; and mobile phase C includes CX-1 pH gradient B.

[0018] More preferably, the mobile phase B comprises (0.8–1.2) × CX⁻¹ pH gradient Buffer A; and the mobile phase C comprises (0.8–1.2) × CX⁻¹ pH gradient Buffer B.

[0019] More preferably, the mobile phase B comprises (0.5–1.5) × CX⁻¹ pH gradient Buffer A; and the mobile phase C comprises (0.5–1.5) × CX⁻¹ pH gradient Buffer B.

[0020] More preferably, in the mobile phase A, the concentration of glycine is 80 mmol / L to 120 mmol / L, and the concentration of sodium chloride is 130 mmol / L to 170 mmol / L;

[0021] More preferably, the concentration of glycine includes, but is not limited to, 80, 85, 90, 95, 100, 105, 110, 115, and 120 (mmol / L), and the concentration of sodium chloride includes, but is not limited to, 130, 135, 140, 145, 150, 155, 160, 165, and 170 (mmol / L).

[0022] (2) The affinity chromatography column includes POROS A / 20 or protein A affinity chromatography column.

[0023] (3) The flow rate of the mobile phase in gradient elution is 0.5 mL / min, the column temperature is 25℃, the injector temperature is 5℃, and the detection wavelength is 280 nm.

[0024] Preferably, the ion exchange column has one or more of the following technical features:

[0025] (1) The gradient elution mobile phase includes: a combination of mobile phase X1 and mobile phase X2, or a combination of mobile phase Y1 and mobile phase Y2; wherein, mobile phase X1 includes phosphate buffer, mobile phase X2 includes phosphate buffer and sodium chloride; mobile phase Y1 includes CX-1 pH gradient Buffer A, and mobile phase Y2 includes CX-1 pH gradient Buffer B.

[0026] More preferably, the mobile phase Y1 comprises (0.8–1.2) × CX⁻¹ pH gradient Buffer A, and the mobile phase Y2 comprises (0.8–1.2) × CX⁻¹ pH gradient Buffer B;

[0027] More preferably, the mobile phase Y1 comprises (0.5–1.5) × CX⁻¹ pH gradient Buffer A; and the mobile phase Y2 comprises (0.5–1.5) × CX⁻¹ pH gradient Buffer B.

[0028] More preferably, for any of the phosphate buffers, the concentration of hydrogen phosphate is 15 mmol / L to 25 mmol / L, and the concentration of sodium chloride is 130 mmol / L to 170 mmol / L.

[0029] More preferably, the concentration of hydrogen phosphate includes, but is not limited to, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 (mmol / L), and the concentration of sodium chloride includes, but is not limited to, 130, 135, 140, 145, 150, 155, 160, 165, and 170 (mmol / L).

[0030] (2) The ion exchange column is a cation exchange column, preferably Thermo Mabpac SCX; those skilled in the art can also select a column with higher specificity for the type of protein.

[0031] (3) The flow rate of the mobile phase in gradient elution is 0.5 mL / min, the column temperature is 25℃, the injector temperature is 5℃, and the detection wavelength is 280 nm.

[0032] A system for simultaneous detection of protein expression levels and charge heterogeneity in cell cultures includes a series-connected affinity chromatography column and ion exchange chromatography column, as well as a column oven.

[0033] Preferably, the affinity chromatography column and the ion exchange chromatography column are connected by a polyetheretherketone (PEEK) tube;

[0034] Preferably, the column oven is provided with an inlet and an outlet on both sides, respectively;

[0035] Those skilled in the art may also install conventional basic or functional components or assemblies, such as solvent pumps, injectors, detectors, etc., as needed.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method and system for detecting the expression level and charge heterogeneity of proteins in cell culture supernatant, which can also be used as an identification method; it fills the gap in the prior art regarding the simultaneous detection of antibody titer and charge heterogeneity, and improves the efficiency of traditional analysis modes; the simultaneous detection method of the present invention consumes less sample, takes less time, and requires less manpower and instrument resources, and the simultaneous detection system has a simple structure and is easy to operate. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the synchronous detection system used in an embodiment of the present invention;

[0039] Figure 2 This is the chromatogram of sample A to be tested in an embodiment of the present invention;

[0040] Figure 3 This is a magnified chromatogram of sample A in an embodiment of the present invention;

[0041] Figure 4 This is the chromatogram of sample B to be tested in this embodiment of the invention;

[0042] Figure 5 This is a magnified chromatogram of sample B to be tested in an embodiment of the present invention;

[0043] Figure 6 This is the chromatogram of sample C to be tested in an embodiment of the present invention;

[0044] Figure 7 This is a magnified chromatogram of sample C to be tested in an embodiment of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0046] Information on some of the instruments and reagents used in the following embodiments of the present invention is as follows:

[0047] High-performance liquid chromatograph (DAD, VWD, MWD or FLR detector): Manufacturer: Agilent; Model: Agilent 1260;

[0048] Electronic balance: Manufacturer: Mettler Toledo; Model: MS204 / ML204T;

[0049] Refrigerated centrifuge: Manufacturer: Eppendorf; Model: 5424R;

[0050] Affinity chromatography column: Life Technologies POROS A / 20 (Stainless steel) 2.1mm × 30mm (1-5024-12);

[0051] Ion exchange chromatography column: Thermo Mabpac SCX, 10um, 4*250;

[0052] Mobile phase: CX-1 pH gradient A, Thermo / 302779; CX-1 pH gradient B, Thermo / 302780;

[0053] Sodium chloride: analytical grade, Sinopharm / 10019318;

[0054] Glycine: analytical grade, Sigma / G8790;

[0055] Hydrochloric acid: analytical grade, Sinopharm / 10011018.

[0056] Example

[0057] (1) Host cells CHO were cultured in three different culture media, and the cell cultures were all monoclonal antibody IgG1. Among them, sample A was cultured in its own ExpiCHO medium, sample B was cultured in ExpiCHO medium, and sample C was cultured in Actipro medium.

[0058] (2) Take three samples and centrifuge the cell culture supernatant at 13000 rpm for 10 min. Take the supernatant after centrifugation as the test sample, and obtain test sample A, test sample B and test sample C respectively.

[0059] (3) The protein A affinity column and the IEX column were connected in series via a peak tube to obtain the high-performance liquid chromatography detection system used in this embodiment, such as... Figure 1 As shown; the tandem connection of the chromatographic columns is achieved in the column oven of the high-performance liquid chromatograph.

[0060] (4) The three test samples were independently detected sequentially. The test sample was drawn up through a syringe and flowed through a series of affinity and ion exchange columns, followed by gradient elution and detection. Specific characteristics and parameter information are as follows:

[0061] Affinity chromatography column: Mobile phase A: 100mM glycine, 150mM sodium chloride, pH 2.5±0.2; Mobile phase B: 1x CX-1 pH gradient A, pH 5.6; Mobile phase C: 1x CX-1 pH gradient B, pH 10.2; The same mobile phase composition and detection conditions were used for all three samples, with a gradient elution flow rate of 0.5mL / min, column temperature of 25℃, injection plate temperature of 5℃, and detection wavelength of 280nm.

[0062] Ion exchange chromatography column (for sample B): Mobile phase Y1: 20mM phosphate buffer, mobile phase Y2: 20mM phosphate buffer, 150mM NaCl, column oven temperature: 35℃, injector temperature: 5℃, flow rate: 0.6mL / min;

[0063] Ion exchange chromatography column (for sample A and sample C): Mobile phase Z1: 1×CX-1 pH gradient Buffer A, pH 5.6, Mobile phase Z2: 1×CX-1 pH gradient Buffer B, pH 10.2, Column oven temperature: 30℃, Injector temperature: 5℃, Flow rate: 0.6mL / min;

[0064] The elution gradients for each sample are shown in Tables 1-3 below.

[0065] Table 1 Elution gradient of sample A to be tested

[0066]

[0067]

[0068] Table 2 Elution gradient of sample B to be tested

[0069] Time (min) Mobile phase A / % Mobile phase B / % Mobile phase C / % 0.00 0.0 50.0 50.0 5.00 0.0 50.0 50.0 5.10 0.0 100.0 0.0 10.00 0.0 100.0 0.0 10.10 100.0 0.0 0.0 16.00 100.0 0.0 0.0 16.10 0.0 90.0 10.0 20.00 0.0 90.0 10.0 48.00 0.0 65.0 35.0 48.10 0.0 0.0 100.0 53.00 0.0 0.0 100.0 53.10 0.0 50.0 50.0 55.00 0.0 50.0 50.0

[0070] Table 3 Elution gradient of sample C to be tested

[0071] Time (min) Mobile phase A / % Mobile phase B / % Mobile phase C / % 0.00 0.0 0.0 100 5.00 0.0 0.0 100 10 0.0 100.0 0.0 14 0.0 100.0 0.0 16.1 100.0 0.0 0.0 21 100.0 0.0 0.0 25 0.0 65.0 35.0 25.1 0.0 65.0 35.0 65 0.0 0.0 100 65.1 0.0 0.0 100.0 70 0.0 0.0 100.0 75 0.0 0.0 100.0

[0072] Optimizing the elution gradient can enable the target protein to bind to and elute the affinity column, and can also improve the separation of acid and basic peaks of charge isomers.

[0073] (5) The obtained chromatogram is as follows: Figures 2-7 As shown, where, Figure 2 This is a full-view image of sample A to be tested. Figure 3 This is a magnified view of a portion of sample A to be tested. Figure 4 This is a full-view image of sample B to be tested. Figure 5 This is a magnified view of a portion of sample B to be tested. Figure 6 This is a full-view image of the sample C to be tested. Figure 7 This is a magnified view of a portion of the sample C to be tested.

[0074] The expression level of monoclonal antibody IgG1 in each test sample was calculated using the external standard method. The standard used was a solution of 2.0 mg / mL monoclonal antibody IgG1. The calculation formula was: antibody expression level = 2 * total antibody peak area / total standard peak area, and the final results are shown in Table 5 below. Meanwhile, the results of heterogeneity detection (percentage area of ​​acid-base peaks and the main peak) are shown in Table 6 below.

[0075] Comparative Example 1

[0076] Protein expression levels were independently detected using the same test samples A, B, and C as in the examples. The analysis was performed using high-performance liquid chromatography (HPLC), with mobile phase X1 consisting of 150 mM phosphate buffer and 150 mM sodium chloride at pH 7.0, and mobile phase X2 consisting of 100 mM glycine and 150 mM sodium chloride at pH 2.5. An affinity column was used. Gradient elution modes X1 and X2 were employed in HPLC, with elution conditions shown in Table 4. The expression level of monoclonal antibody IgG1 in each test sample was calculated using the external standard method (the specific method is consistent with that described in step (5) of the examples). Table 5 lists the simultaneous detection method of the examples and the protein expression levels (i.e., antibody concentrations) obtained when the comparative examples were independently detected.

[0077] Table 4

[0078]

[0079]

[0080] Table 5

[0081] Sample Name Total peak area Tandem antibody concentration Detecting antibody concentration separately Antibody B - Example 1882801 2.0 NA Antibody B - Comparative Example 1 2421137 2.6 2.5 Antibody A - Example 713862 1.0 0.9 Antibody A - Comparative Example 1 1480925 2.0 NA Antibody C - Example 11685515 3.3 2.9 Antibody C - Comparative Example 1 7177555 2.0 NA

[0082] Comparative Example 2

[0083] Using the same test samples A, B, and C as in the examples, charge heterogeneity was independently detected; the detection was performed using high-performance liquid chromatography (HPLC) under the same conditions as the ion-exchange column detection conditions in step (4) of the examples. Table 5 below lists the simultaneous detection method of the examples and the percentage area of ​​the main peak, acidic peak, and basic peak obtained when the comparative examples were detected independently.

[0084] Table 6

[0085]

[0086]

[0087] Since the sample properties of the examples and the comparative examples are different, there are certain differences in the acid-base peaks and the percentage of the main peak area between the two, which is normal. At this time, by comparing the spectra, it can be seen that the separation of the acid-base peaks is good, and the peak shapes of the samples and the reference are comparable. This confirms that the method of tandem protein A and IEX chromatographic columns can realize the detection of the charge heterogeneity of proteins in cell supernatant.

[0088] Furthermore, those skilled in the art can see from the process flow of the above-described embodiments and comparative examples that the experimental time required for the comparative examples is much longer than that for the embodiments. Specifically, the experimental process described in the embodiments is actually equivalent to the sum of the experimental processes performed in comparative examples 1 and 2. When performing the two comparative examples, it is necessary to prepare two types of test samples, seven mobile phase solutions, start the chromatographic equipment twice, and perform two sets of result processing and summarization respectively. However, the method of the embodiments can significantly shorten the time required to produce the two results and significantly shorten the experimental time, which has good prospects for practical application.

[0089] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A method for simultaneous detection of protein expression levels and charge heterogeneity in cell cultures, characterized in that, Includes the following steps: The cell culture was subjected to gradient elution and chromatographic detection sequentially, wherein the chromatographic column used was a series connection of an affinity chromatography column and an ion exchange chromatography column; the protein in the cell culture was IgG1; and the detection wavelength for the chromatographic detection was 280 nm. Before performing synchronous detection, the method further includes: pre-treating the cell culture, and the pre-treatment includes: centrifuging the supernatant of the cell culture and taking the supernatant after centrifugation as the sample to be tested; The host cells CHO were cultured in two different culture media: sample B was cultured in ExpiCHO medium and sample C was cultured in Actipro medium to obtain the cell culture. The gradient elution mobile phase is a combination of mobile phase A, mobile phase B, and mobile phase C; wherein, mobile phase A is glycine and sodium chloride; mobile phase B is CX-1 pH gradient A; mobile phase C is CX-1 pH gradient B; in mobile phase A, the concentration of glycine is 80 mmol / L to 120 mmol / L, and the concentration of sodium chloride is 130 mmol / L to 170 mmol / L; the pH of mobile phase B is 5.6, and the pH of mobile phase C is 10.2; The affinity chromatography column is a LifeTechnologies POROSA / 20, and the ion exchange chromatography column is a ThermoMabpacSCX; The gradient elution for sample B is as follows: ； The gradient elution for sample C is as follows: 。 2. The method for simultaneous detection of protein expression levels and charge heterogeneity in cell cultures according to claim 1, characterized in that, The centrifugation speed is 10,000 rpm to 15,000 rpm, and the centrifugation time is 8 min to 12 min.

3. The method for simultaneous detection of protein expression levels and charge heterogeneity in cell cultures according to claim 1, characterized in that, The chromatographic detection conditions include: a mobile phase flow rate of 0.5 mL / min in gradient elution, a column temperature of 25 °C, and an injector temperature of 5 °C.

Images