A quantitative detection method for CRM197 protein

Abstract

The present invention relates to the field of protein detection technology, and in particular to a quantitative detection method for CRM197 protein. The quantitative detection method comprises: using liquid chromatography to detect Corynebacterium diphtheriae fermentation broth, and the conditions include: mobile phase A is an aqueous solution containing 0.05~0.1v / v% trifluoroacetic acid; mobile phase B is an acetonitrile solution containing 0.1~0.15 v / v% trifluoroacetic acid; the chromatographic column adopts a C4 reverse phase chromatographic column; the elution process includes: at 0~5 minutes, the mobile phase B accounts for 65~70%; at 5.01~6 minutes, the mobile phase B accounts for 94~96%; at 6~18 minutes, the mobile phase B ratio is 100%; at 18.01~29 minutes, the mobile phase B ratio is 65~70%. The present invention adopts specific liquid chromatography conditions to carry out rapid quantification of CRM197 protein in Corynebacterium diphtheriae fermentation broth, and the detection result is accurate and the detection efficiency is high.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection, and in particular to a quantitative detection method for CRM197 protein. Background Art

[0002] Polysaccharide-protein conjugate vaccine is a vaccine made by coupling bacterial capsular polysaccharide with carrier protein. It has the components of carrier protein, which avoids the defect of polysaccharide antigen vaccine that cannot activate T lymphocytes and stimulates insufficient immune efficacy. It can activate T cells through the presentation pathway of carrier protein and then synergistically stimulate B lymphocytes to produce antibodies efficiently. The non-toxic mutant of diphtheria toxin CRM197 protein is currently one of the most commonly used carrier proteins for the polysaccharide-protein conjugate vaccine developed. Compared with other similar tetanus toxoids, diphtheria toxoids, Haemophilus influenzae D protein and group B meningococcal outer membrane protein, it has better immune effects and lower toxicity.

[0003] CRM197 protein is a non-toxic mutant of diphtheria protein. Due to a single base mutation, the 52nd amino acid Gly mutates to Glu, causing a change in the enzyme activity site, losing the ability to bind to ribosomal proteins, and thus losing its cytotoxicity. At the same time, CRM197 protein retains the immunogenicity of diphtheria toxin and is an ideal polysaccharide-conjugated vaccine carrier protein.

[0004] CRM197 contains 535 amino acid residues, which are divided into three domains, namely the catalytic region (C region) of about 21KD with 1-187 amino acid residues, the transmembrane region (T region) of 201-384 amino acid residues, and the receptor region (R region) of 387-535 amino acid residues. The T / R region is about 38KD in total. There is a loosely structured peptide segment between the C region and the T / R region, which contains 12 basic amino acid residues that are easily cleaved by serine proteases. This determines that the CRM197 protein is easily degraded into two fragments of 21KD and 38KD in solution. In addition, the T region is rich in hydrophobic amino acid residues, and the protein can be polymerized into polymers with the help of hydrophobic properties during solution storage.

[0005] The prior art usually uses a mutant strain of Corynebacterium diphtheriae C7 cell line or a genetically recombinant engineered bacterial strain to express the CRM197 protein, and then obtains the target protein through protein purification. Regardless of whether the mutant strain or the genetically recombinant engineered bacterial strain expresses the target protein, it is necessary to select the fermentation process for many conditions such as the culture medium composition, process parameters and appropriate culture time for process optimization research. After the process is basically formed, it is also necessary to implement real-time monitoring of the target protein expression in each batch of culture. The prior art generally uses SDS-PAGE electrophoresis to detect the expression of the target protein. This method is relatively intuitive, simple and low-cost. However, electrophoresis detection is time-consuming, generally requiring more than 4-5 hours. The main problem of this method is that it cannot accurately quantify the protein content. Another method for detecting the target protein content is the immunoflocculation method using diphtheria toxoid antiserum. The flocculent precipitation method can be used for semi-quantitative analysis, but it is cumbersome to operate, has high requirements for reagents, especially diphtheria toxin antiserum, and the reaction process is relatively complicated. The results of each reaction experiment fluctuate greatly, and the experimenter needs to pay attention to the reaction process at any time and record the precipitation time. If there is a slight mistake, the experiment needs to be repeated, and the experimental time required is generally 2-3 hours. Both of the above methods have the disadvantages of inaccurate quantification and long time, which is not conducive to monitoring the expression of target proteins during research and development and production. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for quantitative detection of CRM197 protein.

[0007] The prior art uses HPLC to detect the concentration of CRM197 protein, but it generally detects the concentration of CRM197 protein after purification. When these methods are applied to the detection of CRM197 protein concentration in fermentation broth, they encounter various problems. This is because the prior art usually needs to monitor the CRM197 protein during the fermentation process to optimize and determine the components of the fermentation medium, optimize and confirm various fermentation parameters, determine the fermentation endpoint, and determine the expression level of the supernatant, etc. However, the fermentation broth produced during the fermentation process is not purified, and contains not only various impurities, but also fragments produced by the degradation of CRM197 protein, and these fragments are continuously produced, which makes it difficult for the existing methods to distinguish and accurately quantify CRM197 protein.

[0008] The present invention uses specific liquid chromatography conditions to perform quantitative detection of CRM197 protein in diphtheria Corynebacterium fermentation broth, and can directly achieve rapid and accurate quantification of CRM197 protein in the fermentation broth (real-time monitoring is possible without purification). It can be applied to the monitoring of CRM197 protein expression in fermentation process research in the research and development stage, facilitating the optimization and determination of fermentation medium components, the optimization and confirmation of various fermentation parameters, the judgment of the fermentation endpoint and the determination of the supernatant expression amount, taking into account convenience, accuracy and speed. At the same time, this method can also be used for the calculation of the target protein harvest and recovery rate in each step of the purification process, providing an accurate protein quantification method for optimizing purification process parameters.

[0009] In a first aspect, the present invention provides a method for quantitatively detecting CRM197 protein, comprising: using liquid chromatography to detect Corynebacterium diphtheriae fermentation broth, the conditions comprising: Mobile phase A is an aqueous solution containing 0.05-0.1 v / v% trifluoroacetic acid; Mobile phase B was acetonitrile solution containing 0.1-0.15 v / v% trifluoroacetic acid; The chromatographic column used was a C4 reverse phase column; The elution process includes: At 0-5 minutes, mobile phase B accounts for 65-70%; At 5.01-6 minutes, mobile phase B accounted for 94-96%; At 6-18 minutes, the mobile phase B ratio was 100%; At 18.01-29 minutes, the mobile phase B ratio was 65-70%.

[0010] Furthermore, the mobile phase A is an aqueous solution containing 0.08-0.1 v / v% trifluoroacetic acid; and the mobile phase B is an acetonitrile solution containing 0.1-0.12 v / v% trifluoroacetic acid.

[0011] Furthermore, the elution process comprises: At 0 minutes, mobile phase B accounted for 65%; At 5 minutes, mobile phase B accounted for 65%; At 5.01 minutes, the mobile phase B ratio was 95%; At 6 minutes, mobile phase B accounted for 100%; At 18 minutes, mobile phase B accounted for 100%; At 18.01 minutes, mobile phase B accounted for 65%; At 29 minutes, mobile phase B accounted for 65%.

[0012] Furthermore, the conditions of the liquid chromatography method also include: The column flow rate was 0.6~1mL / min, the detection wavelength was 214nm, the column temperature was 24~26℃, and the injection volume was 20~25μL.

[0013] Furthermore, the diphtheria Corynebacterium is diphtheria Corynebacterium C7.

[0014] Furthermore, the Corynebacterium diphtheriae fermentation broth is prepared by the following method: The fermentation culture medium of Corynebacterium diphtheriae was fermented at 36-37°C and 200-250 rpm. Aspartic acid, L-asparagine, L-glutamine, L-glycine, L-methionine, L-histidine, L-lysine hydrochloride, L-phenylalanine, L-proline, L-serine, L-threonine, L-valine, beta-alanine, L-isoleucine, L-tryptophan, L-arginine, beta-leucine, L-cystine, L-glutamic acid, L-tyrosine, magnesium sulfate, zinc sulfate, manganese chloride, copper sulfate, vitamin B3, vitamin B7, vitamin B1, vitamin B5, vitamin B6, maltose and CaCl2.

[0015] Further, Aspartic acid, L-asparagine, L-glutamine, L-glycine, L-methionine, L-histidine, L-lysine hydrochloride, L-phenylalanine, L-proline, L-serine, L-threonine, L-valine, β-alanine, L-isoleucine, L-tryptophan, L-arginine, β-leucine, L-cystine, L-glutamic acid and L-tyrosine are all 0.5~2.0g / L.

[0016] Further, magnesium sulfate 75~105g / L, zinc sulfate·7H2O 0.4~0.8g / L, manganese chloride·4H2O 0.3~0.5g / L, copper sulfate 0.3~0.5g / L, vitamin B3 4.0~8.0g / L, vitamin B7 0.1~0.2g / L, vitamin B1 0.2~0.6g / L, vitamin B5 1.5~2.5g / L, vitamin B6 0.05~0.15g / L, maltose 25~35g / L and calcium chloride 0.15~0.4g / L.

[0017] Another key point of the present invention is the optimization of the fermentation medium. The present invention has obtained a peptone-free and serum-free medium suitable for Corynebacterium diphtheriae C7 through exploration. In this case, other impurities except CRM197 protein and degradation fragments can be reduced to a great extent. In this way, the difficulty of real-time monitoring of CRM197 protein during the fermentation process is mainly reflected in the interference of its degradation fragments. This problem has been solved by the aforementioned detection method of the present invention.

[0018] Further, after the liquid chromatography, the method further comprises: The peak area of ​​the CRM197 protein detected was compared with the standard curve to obtain the content of the CRM197 protein in the diphtheriae fermentation broth; The standard curve is constructed based on the peak area and protein content of the reference protein.

[0019] The present invention further provides application of the quantitative detection method in monitoring the expression level of CRM197 protein in fermentation engineering.

[0020] The present invention has the following beneficial effects: The present invention studies and obtains a specific method for detecting CRM197 protein in Corynebacterium diphtheriae fermentation broth. Through specific liquid chromatography conditions, CRM197 protein can be separated and detected with high efficiency, and the detection result is accurate and the detection efficiency is high.

[0021] The detection method of the CRM197 protein provided by the present invention can be used for efficient and accurate quantitative analysis of protein expression during the fermentation process, which is of great significance in the field of fermentation production of the CRM197 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is a schematic diagram of the CRM197 protein structure provided in Example 1 of the present invention.

[0024] Figure 2 It is a schematic diagram of the electrophoresis results of CRM197 protein at different fermentation time periods provided in Example 1 of the present invention.

[0025] Figure 3 This is the electrophoresis diagram of samples at each stage of purification provided in Example 1 of the present invention.

[0026] Figure 4 It is a graph of the electrophoresis detection results of CRM197 protease cleavage provided in Example 1 of the present invention; lanes 1 and 2 are purified target proteins, 3 is the reduction electrophoresis of 9, 4 is a marker, from large to small, they are 116, 66, 45, 35, 25, 18.4 and 14.4 KDa, 5-10 are target protein and enzyme ratios of 1:5000, 4000, 3000, 2000, 1000 and 500, respectively, mixed and digested at 37°C for 1 hour.

[0027] Figure 5 This is a chromatogram after CRM197 protease cleavage provided in Example 1 of the present invention.

[0028] Figure 6 This is the C4 reverse column experimental result provided in Example 1 of the present invention.

[0029] Figure 7 This is the C8 reverse column experimental result provided in Example 1 of the present invention.

[0030] Figure 8 This is a comparison diagram of the separation effects of C4 and C8 chromatographic columns RP-HPLC chromatography on protein aggregates provided in Example 1 of the present invention.

[0031] Fig. 9 It is an overlay graph of the standard curves of the five quantitative standards provided in Example 1 of the present invention.

[0032] Fig.10 1 is the standard curve of the five quantitative standards provided in Example 1 of the present invention.

[0033] Fig.11 This is a detection chart of CRM197 in the fermentation broth supernatant provided in Example 1 of the present invention.

[0034] Fig.12 This is a detection diagram of the purified CRM197 protein stock solution provided in Example 1 of the present invention.

[0035] Fig.13 This is a detection diagram of the purified CRM197 protein stock solution provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0038] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example: Example 1 The present invention provides a method for treating CRM197 protein (structure such as Figure 1 The rapid quantitative method of the present invention comprises the following steps: 1. Preparation of target protein The present invention uses Corynebacterium diphtheriae C7 (also known as Corynebacterium diphtheriae C7) strain to obtain the target protein through strain activation, fermentation seed liquid preparation, fermentation culture, centrifugation to collect fermentation supernatant, filtration clarification, two-step ammonium sulfate precipitation and ion exchange chromatography. The process includes the following: (1) Strain activation: Take out the C7 strain seeds from the -80°C refrigerator, thaw at room temperature, inoculate them into a triangular shake flask containing low-iron culture medium on a clean bench, and culture them in a constant temperature shaking incubator at 36°C and 180 rpm overnight.

[0039] (2) Preparation of fermentation seed solution: In a clean bench, take the activated seed solution after the culture is completed, inoculate it into a triangular flask containing complete amino acid culture medium, and culture it in a constant temperature shaking incubator at 36°C and 250 rpm for 8 hours. Take the seeds after the culture is completed for microscopic examination.

[0040] (3) Fermentation culture, The fermentation medium adopts a protein-free and serum-free chemically defined medium. The fermentation medium formula is as follows: Aspartic acid 2.0g / L, L-asparagine 0.5g / L, L-glutamine 0.5g / L, L-glycine 2.0g / L, L-methionine 0.8g / L, L-histidine 1.5g / L, L-lysine hydrochloride 2.0g / L, L-phenylalanine 1.0g / L, L-proline 2.0g / L, L-serine 2.0g / L, L-threonine 2.0g / L, L-valine 1.5g / L, β-alanine 1.5g / L, L-isoleucine 2.0g / L, L-tryptophan 0.5g / L, L-arginine 1.0g / L, L-leucine 1.0g / L, L-cystine 1.5g / L, L-glutamic acid 2.0g / L, L-tyrosine 0.5g / L, magnesium sulfate 90.0g / L, zinc sulfate 7H2O 0.6g / L, manganese chloride 4H2O 0.4g / L, copper sulfate 0.4g / L, vitamin B3 6.0g / L, vitamin B7 0.15g / L, vitamin B1 0.4g / L, vitamin B5 2.0g / L, vitamin B6 0.1g / L, maltose 30.0g / L and calcium chloride 0.25g / L.

[0041] First, various amino groups were dissolved in sequence and then the pH was adjusted to 6.90 with 1 mol / L NaOH solution. Various vitamins dissolved separately and mixed well, inorganic salts and trace elements dissolved separately, and maltose dissolved separately were added, and finally K2HPO4 and KH2PO4 were mixed to adjust the pH to 7.30±0.1.

[0042] All the above culture medium components are dissolved in sequence, filtered and sterilized, and poured into the fermentation tank that has been sterilized by high pressure to culture C7 diphtheria bacteria. The filtered and sterilized fermentation medium is poured into the fermentation tank through the inoculation port under the cover of flame, and the tank temperature is set to 36°C. Take the seed liquid after the culture is completed, check under a microscope for contamination by foreign bacteria, and calculate the OD value after the seeding. 600 The value is about 0.3 to calculate the inoculation volume, and the seed liquid is inoculated into the fermentation tank to start fermentation. The initial ventilation volume is set to 0.8m 3 / h, speed 200rpm, temperature 36℃, dissolved oxygen 20% and serial speed. Connect the feeding pipelines of acid, alkali, feed and defoamer to the corresponding peristaltic pumps. During the fermentation process, feed liquid with the same amino acid concentration as the fermentation formula is added in a timely manner, and the amount is about 10% of the total fermentation volume. The fermentation is terminated at 22:00.

[0043] (4) After the culture is completed, the fermentation liquid is collected and the volume of the fermentation liquid is recorded. The supernatant is collected by centrifugation at 8000 rpm and 4°C for 20 minutes. The centrifuged supernatant is clarified and filtered using a 0.45 μm filter. The clarified liquid is concentrated to 1 / 10 of the volume of the fermentation liquid using an ultrafiltration membrane package and then filtered with 10 mmol / L PB until the conductivity of the permeate is reduced.

[0044] (5) Slowly add solid ammonium sulfate to the ultrafiltration concentrate until it reaches a lower saturation, stir to fully dissolve it, let it stand at 2-8°C until precipitation is complete, and then centrifuge at 8000 rpm and 4°C for 20 minutes to collect the supernatant.

[0045] (6) After clarifying and filtering the supernatant of the first step ammonium sulfate precipitation, solid ammonium sulfate is added to a high saturation, stirred to fully precipitate, and then centrifuged at 8000 rpm and 4°C for 20 minutes to collect the centrifugal precipitate.

[0046] The centrifugal precipitate was fully dissolved with 10mmol / L PB buffer, and the permeate was washed with an ultrafiltration membrane coated with 10mmol / L PB to a lower conductivity value, and then the sample was collected, clarified and filtered, and then purified by chromatography.

[0047] (7) Chromatographic purification using ion exchange columns The column was equilibrated with 10 mmol / L PB buffer until the conductivity and pH value were stable before loading the sample.

[0048] After loading, continue to rinse with loading buffer until the UV 280nm absorption peak is stable, then elution can be performed.

[0049] The target protein was eluted with a loading buffer containing NaCl and collected under ultraviolet monitoring at 280 nm.

[0050] (8) The eluted target protein is sterilized and filtered using a 0.22 μm filter. The filtrate is the CRM197 protein stock solution and is temporarily stored at 2-8°C.

[0051] The electrophoresis results of CRM197 protein expression at different fermentation time periods are shown in Figure 2. Figure 2 The electrophoresis diagrams of samples at each stage of purification are shown in Figure 3 shown.

[0052] 2. Determination of target protein structure and preparation of physicochemical reference substances The CRM197 protein stock solution obtained in step 1 was tested by various methods. The mass spectrometry test results showed that the molecular weight of the target protein was 58413Da, and the multi-angle laser (SEC-MALS) detection molecular weight was 58.6kDa. The results of amino acid composition, C-terminal amino acid sequence, N-terminal amino acid sequence, isoelectric point analysis were consistent with the theoretical value of CRM197 protein, so it can be determined that the target protein obtained is CRM197.

[0053] The CRM197 protein content was determined by Lowry protein quantification, diluted to 1.0 mg / mL with 10 mmol / L PB, and then packaged and freeze-dried for use as a physical and chemical reference for drawing the HPLC protein quantification standard curve.

[0054] 3. Obtaining related impurity proteins The structure and physicochemical characteristics of the CRM197 protein determine the impurities associated with it, which are mainly protein polymers and degradation fragments. The latter includes a 38KD large fragment and a 21KD small fragment. Other impurities are impurities formed by other process-related protein residues and may vary from batch to batch.

[0055] The purified CRM197 protein was properly digested with trypsin. After the degradation detected by electrophoresis was in accordance with the expectation, the intact CRM197 protein, the 38KD large fragment and the 21KD small fragment were separated by molecular sieve chromatography. After being collected and concentrated in steps and confirmed by electrophoresis, the impurity proteins related to the 38KD large fragment and the 21KD small fragment were prepared.

[0056] (1) Study on enzyme digestion conditions CRM197 protein and Trypsin protease were diluted with 1% ammonium bicarbonate solution. The designed substrate and protease ratios were 1:500, 1000, 2000, 3000, 4000, 5000, and the enzyme digestion was compared under non-reducing and reducing conditions. The reaction was carried out in a 37°C water bath for 1 hour, and the digestion was detected by SDS-PAGE electrophoresis after the reaction.

[0057] (2) If Figure 4As shown, the non-reducing reaction conditions of lane 5 with trypsin addition of 1:5000 are the most suitable. After CRM197 was digested with trypsin according to the above reaction conditions, the protein digest solution was loaded with 20mM PBS containing 20mM DTT, and the XK26 / 100 chromatography column with a height of about 85 cm and Superdex-75 chromatography medium was separated to obtain the elution peaks in each tube, and the protein peaks were collected in parts under UV detection.

[0058] (3) If Figure 5 As shown, electrophoresis detection showed that the complete CRM197 protein was collected at 210 mL, and the 38kD and 21kD CRM197 fragments were collected at 230 mL and 250 mL, respectively. The degradation fragments were collected step by step, lyophilized and stored for reference of related impurities.

[0059] 4. HPLC chromatography method selection 1: Comparison of the separation effects of C4 and C8 columns on protein degradation fragments The present invention studies the physicochemical characteristics of CRM197 protein, and compares the separation effects of degradation fragments by using C4 and C8 chromatographic columns RP-HPLC chromatography, specifically including the following process: Purified CRM197 samples and prepared molecular weight 21kD and 38kD fragments were injected into C4 or C8 liquid chromatography for separation and determination. The liquid chromatography determination conditions are shown in the following table: Table 1 Liquid chromatography determination conditions

[0060] The results are as follows Figure 6 and Figure 7 As shown in the figure, the C4 column elutes faster in terms of retention time, and the C8 column has a slightly better separation of degradation fragments in terms of sample separation, but both column and sample have achieved ideal separation effects.

[0061] As for the separation effect of the target protein and related impurities, the liquid chromatography method based on C4 and C8 columns has a good separation effect on CRM197 protein, CRM197 protein aggregates, 38KD large fragments and 21KD small fragments related impurity proteins. In order to eliminate the AUC difference caused by different extinction coefficients between different impurity proteins and target proteins, the ultraviolet wavelength of 214nm was selected so that the AUC area is only related to the protein content, but not to the protein type.

[0062] 5. HPLC chromatography method selection 2: Comparison of the separation effects of C4 and C8 columns on protein aggregates The present invention further uses protein aggregate samples to compare the separation effects of C4 and C8 chromatographic columns. The protein aggregate samples are derived from partial aggregates formed by themselves after long-term storage at 2-8°C, and are a mixture of CRM197 protein monomers and aggregates.

[0063] The liquid phase determination conditions are shown in the following table: Table 2 Liquid chromatography determination conditions

[0064] The results are as follows Figure 8 As shown in the figure, the C4 reverse phase column has a better separation effect on protein aggregates and monomers than C8. Taking all factors into consideration, C4 is the best choice. On the basis of achieving better separation effect on various components, the chromatography time can be shortened and the amount of acetonitrile can be reduced.

[0065] 6. The final HPLC-C4 chromatography operation process is as follows: (1) Dilution solution: 110mmol / L NaCl + 10mmol / L PB, pH = 6.8.

[0066] (2) Standard curve determination: Take the CRM197 protein standard and dilute it to 20, 40, 80, 120, and 160 μg / mL with diluent. Filter it with a 0.45 μm water filter membrane and store it in a sample vial at 4°C for later use.

[0067] (3) Test solution treatment: Use C7 fermentation broth as the test sample or protein samples obtained in other purification steps, filter through a 0.45 μm water filter membrane, and store in a sample vial at 4°C for later use.

[0068] (4) Sample determination: Accurately measure the test sample solution and each point of the set standard curve and inject them into the liquid chromatograph, and record the chromatogram.

[0069] The standard curve is made with the concentration of each point as the horizontal axis and the peak area of ​​each point as the vertical axis. 2 >0.995.

[0070] Substitute the peak area of ​​the test solution into the curve to obtain the sample concentration.

[0071] The single-point calibration method can also be used to calibrate the instrument and correct errors.

[0072] (5) Liquid chromatography conditions: The final HPLC conditions are shown in the following table: Table 3 Liquid chromatography detection conditions

[0073] 7. Quantitative determination and calculation of target protein 7.1 Determination of protein content in fermentation broth (1) Dilution solution: 110mmol / L NaCl + 10mmol / L PB, pH 6.8 (2) Standard curve sample processing: Take CRM197 standard and dilute it to 20, 40, 80, 120, and 160 μg / ml with diluent. Filter it with a 0.45 μm water filter membrane and store it in a sample vial at 4°C for later use.

[0074] (3) Treatment of fermentation broth supernatant solution: Take 500 μl of the fermentation broth supernatant solution, filter it through a 0.45 μm water filter membrane, and place the filtrate in a liquid phase vial for later use.

[0075] (4) Sample determination: Inject the prepared standard curve solution and fermentation broth supernatant into the liquid chromatograph in sequence, and determine according to the liquid phase determination conditions in Table 4. Record the chromatogram. Fig. 9 The standard curve is made with the concentration of each point as the horizontal axis and the peak area of ​​each point as the vertical axis, see Fig.10 , R 2 >0.995. Sample test chart see Fig.11 , substitute the peak area of ​​the test solution into the curve to obtain the sample concentration.

[0076] Table 4 Liquid chromatography determination conditions

[0077] (5) Statistical analysis of experimental results For data processing, the slope in the integral parameters was set to 500, and the remaining parameters were set to default values.

[0078] The standard curve results are shown in the following table.

[0079] Table 5 Standard curve determination results

[0080] Table 6 Fermentation broth sample test results

[0081] The results showed that the CRM197 content in the fermentation broth supernatant was 69.2 mg / L.

[0082] 7.2 Determination of protein content of CRM197 stock solution after purification (1) Dilution solution: 110mmol / L NaCl + 10mmol / L PB PH = 6.8.

[0083] (2) Sample treatment and standard curve determination are performed in the same manner as above. After purification, the sample is diluted to approximately 0.05 mg / ml and 0.1 mg / ml (based on Lowry quantitative determination data), filtered through a 0.45 μm water filter membrane, and the filtrate is placed in a liquid phase vial for later use. Samples are loaded separately. The standard curve is the same as in step 7.1.

[0084] (3) Calculation of protein content in samples.

[0085] Table 7 Determination results of purified samples

[0086] The protein content of CRM197 stock solution determined by this method is basically consistent with the Lowry quantitative determination data. Fig.12 and Fig.13 .

[0087] The above results illustrate that the method provided by the present invention can achieve accurate and rapid quantification of CRM197 protein in the fermentation broth. The present invention further applies it to the optimization of various parameters and fermentation medium composition in the fermentation production process of CRM197 protein, so that the normal expression level of CRM197 protein is increased from 60~70mg / L to 100~110mg / L, which is of great value in the field of CRM197 protein production.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitative detection of CRM197 protein, characterized in that: include: Liquid chromatography was used to detect the diphtheria Corynebacterium fermentation broth, and the conditions included: Mobile phase A is an aqueous solution containing 0.05-0.1 v / v% trifluoroacetic acid; Mobile phase B was acetonitrile solution containing 0.1-0.15 v / v% trifluoroacetic acid; The chromatographic column used was a C4 reverse phase column; The elution process includes: At 0-5 minutes, mobile phase B accounts for 65-70%; At 5.01-6 minutes, mobile phase B accounted for 94-96%; At 6-18 minutes, the mobile phase B ratio was 100%; At 18.01-29 minutes, the mobile phase B ratio was 65-70%.

2. The quantitative detection method according to claim 1, characterized in that: The mobile phase A is an aqueous solution containing 0.08-0.1 v / v% trifluoroacetic acid; the mobile phase B is an acetonitrile solution containing 0.1-0.12 v / v% trifluoroacetic acid.

3. The quantitative detection method according to claim 1, characterized in that: The elution process comprises: At 0 minutes, mobile phase B accounted for 65%; At 5 minutes, mobile phase B accounted for 65%; At 5.01 minutes, the mobile phase B ratio was 95%; At 6 minutes, mobile phase B accounted for 100%; At 18 minutes, mobile phase B accounted for 100%; At 18.01 minutes, mobile phase B accounted for 65%; At 29 minutes, mobile phase B accounted for 65%.

4. The quantitative detection method according to any one of claims 1 to 3, characterized in that: The conditions of the liquid chromatography method also include: The column flow rate was 0.6~1mL / min, the detection wavelength was 214nm, the column temperature was 24~26℃, and the injection volume was 20~25μL.

5. The quantitative detection method according to any one of claims 1 to 4, characterized in that: The diphtheria Corynebacterium is diphtheria Corynebacterium C7.

6. The quantitative detection method according to claim 5, characterized in that: The diphtheria bacillus fermentation liquid is prepared by the following method: The fermentation culture medium of Corynebacterium diphtheriae was fermented at 36-37°C and 200-250 rpm. Aspartic acid, L-asparagine, L-glutamine, L-glycine, L-methionine, L-histidine, L-lysine hydrochloride, L-phenylalanine, L-proline, L-serine, L-threonine, L-valine, beta-alanine, L-isoleucine, L-tryptophan, L-arginine, beta-leucine, L-cystine, L-glutamic acid, L-tyrosine, magnesium sulfate, zinc sulfate, manganese chloride, copper sulfate, vitamin B3, vitamin B7, vitamin B1, vitamin B5, vitamin B6, maltose and CaCl2.

7. The quantitative detection method according to claim 6, characterized in that: The Corynebacterium diphtheriae fermentation liquid is filtered using a 0.4-0.5 μm filter membrane before detection.

8. The quantitative detection method according to any one of claims 1 to 3, characterized in that: After the liquid chromatography, the method further comprises: The peak area of ​​the CRM197 protein detected was compared with the standard curve to obtain the content of the CRM197 protein in the diphtheriae fermentation broth; The standard curve is constructed based on the peak area and protein content of the reference protein.

9. Use of the quantitative detection method according to any one of claims 1 to 8 in monitoring the expression level of CRM197 protein in fermentation engineering.

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