Method for detecting extinction coefficient of protein

Abstract

The invention discloses a method for detecting a protein extinction coefficient. The invention provides a protein concentration determination method, which comprises: (1) carrying out liquid exchange on a substance to be determined through a desalination column to obtain a desalination liquid; (2) hydrolyzing the desalted liquid obtained in the step (1) to obtain hydrolysate; (3) carrying out pre-column derivatization on the hydrolysate obtained in the step (2) to obtain a derivative solution; and (4) determining the protein concentration of the derivative solution in the step (3) through high performance liquid chromatography. According to the method disclosed by the invention, the influence of a preparation background solution on proteolysis and detection is reduced, the time for proteolysis is short, and the measured extinction coefficient is high in accuracy and high in efficiency.

Description

Technical Field

[0001] The invention relates to a method for detecting the extinction coefficient of protein. Background Art

[0002] In the process of protein drug research and development, production and quality control, the determination of protein concentration is of great significance. Accurate determination of the concentration of protein drugs helps to ensure the safety, effectiveness and quality controllability of drugs, and provide better protection for patient treatment. There are many methods for determining protein concentration. The widely used methods include Lowry, BCA, Bradford method, Kjeldahl nitrogen determination method and amino acid analysis method.

[0003] According to the Beer-Lambert law, protein concentration is proportional to the OD value of the protein sample at 280nm. Therefore, the protein extinction coefficient can be determined by measuring the exact protein concentration of the sample at a wavelength of 280nm and its corresponding OD value. Therefore, determining the accurate protein extinction coefficient is the key to protein concentration determination.

[0004] Currently, the commonly used methods for determining protein extinction coefficients include the Kjeldahl method and the amino acid analysis method. The Kjeldahl method is often limited by the sample requirement and the background buffer, and may not be applicable to specific proteins, which limits the application of this method to a certain extent. The amino acid analysis method is currently a widely used method. The amino acid analysis method measures protein concentration based on the absolute quantification of various amino acids that make up the protein. It has fewer interference factors, a high degree of automation, and strong applicability.

[0005] For the determination of absorbance of protein drugs, the traditional UV spectrophotometry method requires sample dilution and background correction, which is time-consuming. For amino acid analysis of protein drugs, the conventional PITC (phenyl isothiocyanate) derivatization method has high accuracy and low cost, but the entire sample processing time is long, of which the longest time is the hydrolysis time of the protein drug, which affects the analysis efficiency.

[0006] Therefore, for absorbance determination and amino acid analysis, there is room and need to shorten time and improve analysis efficiency while ensuring accuracy and minimizing cost increases. Summary of the invention

[0007] The problem to be solved by the present invention is to provide a method for detecting the extinction coefficient of protein. The method measures the extinction coefficient of protein by combining slope spectroscopy with high performance liquid chromatography, has the advantages of rapid acid hydrolysis of protein, accurate detection of the content of each amino acid, improved efficiency and accuracy of protein extinction coefficient determination, simple operation, etc., and has positive significance for the accurate determination of protein concentration in the research and development, production and quality control of protein drugs.

[0008] The present invention solves the above technical problems through the following solutions.

[0009] The present invention provides a method for determining protein concentration, which comprises the following steps:

[0010] (1) Exchanging the solution of the analyte through a desalting column to obtain a desalted solution;

[0011] (2) hydrolyzing the desalted liquid of step (1) to obtain a hydrolyzate;

[0012] (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution;

[0013] (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography;

[0014] The object to be detected is a solution containing protein.

[0015] In the assay method, the protein in the "protein-containing solution" may be a protein drug, such as bovine serum albumin (BSA) or trastuzumab for injection (Herceptin).

[0016] In the assay method, in step (1), the solvent of the analyte may be a phosphate solution, preferably a 20 mM phosphate solution.

[0017] In the determination method, the desalting column may be Thermo 89883.

[0018] In the determination method, the buffer used for the liquid exchange may be an aqueous solution of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate.

[0019] In the assay method, the concentration of the buffer used for the liquid exchange may be 20 mM.

[0020] In the determination method, in the step (1), the liquid replacement can be carried out by centrifugation.

[0021] In the determination method, in step (1), the liquid replacement comprises the following steps: placing the desalting column in an EP tube and centrifuging; adding a buffer and centrifuging again; adding the test substance and centrifuging again.

[0022] In the determination method, in the step (2), the hydrolysis comprises the following steps: mixing the desalted liquid with hydrochloric acid under nitrogen conditions.

[0023] In the determination method, in step (2), the concentration of the hydrochloric acid can be 6M to 8M; for example, 6M, 6.5M, 7M, 7.5M, 8M; preferably 8M.

[0024] In the determination method, in the step (2), the hydrolysis temperature can be 130°C to 150°C; for example, 130°C, 135°C, 140°C, 145°C, 150°C; preferably 140°C.

[0025] In the determination method, in the step (2), the hydrolysis time can be 2 h to 4 h; for example, 2 h, 2.5 h, 3 h, 4 h; preferably 2 h.

[0026] In the determination method, in the step (2), the hydrolysis further comprises the following steps: after the hydrolysis, diluting with water, mixing the diluted solution with a norleucine solution, concentrating, and mixing with mobile phase A to obtain a hydrolyzate; the norleucine solution is preferably a mixed solution of norleucine and 0.1M HCl; and the mobile phase A is preferably a sodium acetate solution.

[0027] In the determination method, in step (2), the concentration of the norleucine solution may be 1000 pmol / μL.

[0028] In the determination method, in step (2), the volume ratio of the norleucine solution to the diluted solution may be 0.045:1.

[0029] In the determination method, in step (2), the pH of the mobile phase A may be 6 to 7, preferably 6.5.

[0030] In the determination method, in the step (2), the concentration temperature may be 90°C.

[0031] In the determination method, in the step (2), the concentration can be carried out using a constant temperature mixer (Eppendorf / Thermo Mixer C), or any other instrument that can achieve the same conditions, such as a heating stirring module (Thermo).

[0032] In the assay method, in step (2), the dilution may include diluting to 10 times the volume, taking 1 / 10 of the solution and mixing it with the norleucine solution.

[0033] In the determination method, in the step (2), the hydrolysis comprises the following steps: under nitrogen conditions, the desalted liquid of step (1) is mixed with 8M hydrochloric acid, heated at 140°C for 2 hours for hydrolysis, the hydrolyzate is diluted with water, mixed with a norleucine solution, concentrated to dryness, and dissolved with mobile phase A to obtain a hydrolyzate.

[0034] In the determination method, in the step (3), the pre-column derivatization comprises the following steps: mixing the hydrolyzate, triethylamine solution and phenyl isothiocyanate (PITC) solution, adding n-hexane, shaking and letting stand, taking the lower layer solution for sample loading; the triethylamine solution is preferably a mixed solution of triethylamine and acetonitrile; the phenyl isothiocyanate (PITC) solution is preferably a mixed solution of phenyl isothiocyanate and acetonitrile.

[0035] In the determination method, in the step (3), the volume ratio of the triethylamine to the acetonitrile can be 1:(5-8); preferably 1:6.

[0036] In the determination method, in the step (3), the volume ratio of the phenyl isothiocyanate to the acetonitrile can be 1:(80-85); preferably 1:82.

[0037] In the determination method, in the step (3), the volume ratio of the triethylamine solution to the hydrolyzate is preferably 1:2.

[0038] In the determination method, in the step (3), the volume ratio of the phenyl isothiocyanate solution to the hydrolyzate is preferably 1:2.

[0039] In the determination method, in the step (3), the volume ratio of the n-hexane to the hydrolyzate is preferably 4:1.

[0040] In the determination method, in the step (3), the reaction time of the pre-column derivatization can be 60 minutes.

[0041] In the determination method, in step (4), the detection conditions of the high performance liquid chromatography are as follows:

[0042] The chromatographic column may be a Diamonsil AAA chromatographic column;

[0043] Mobile phase A can be 50 mM sodium acetate solution, pH 6.5 ± 0.1;

[0044] Mobile phase B can be methanol, acetonitrile and water in a volume ratio of 1:3:1;

[0045] The detection wavelength of the detector may be 254 nm.

[0046] In the determination method, in step (4), the high performance liquid chromatography uses an internal standard method to calculate the amino acid concentration; the internal standard substance of the internal standard method can be norleucine.

[0047] In the assay method, the molar concentration of the protein is calculated using the following formula:

[0048] ; Wherein, M is the molar concentration of each amino acid; N is the number of each amino acid.

[0049] In the assay method, the protein concentration is calculated using the following formula:

[0050] Protein concentration = protein molar concentration × protein relative molecular mass.

[0051] In the determination method, in step (4), the column temperature of the chromatographic column can be 42-48°C, preferably 45°C.

[0052] In the determination method, in the step (4), the flow rate of the mobile phase may be 1.0 mL / min.

[0053] In the determination method, in step (4), the elution mode of the high performance liquid chromatography is linear gradient elution; preferably:

[0054] Elution time (min) Mobile phase A percentage (%) Mobile phase B ratio (%) 0→39 95→52 5→48 39→40 52→0 48→100 40→45 0 100 45→46 0→95 100→5 46→60 95 5 .

[0055] The present invention provides a method for determining protein concentration, which comprises the following steps:

[0056] (1) exchanging the liquid of the object to be tested to obtain a liquid to be tested;

[0057] (2) hydrolyzing the test solution of step (1) to obtain a hydrolyzate;

[0058] (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution;

[0059] (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography;

[0060] The analyte is a solution containing protein;

[0061] The hydrolysis comprises the following steps: mixing the test solution of step (1) with 8M hydrochloric acid, and heating at 140° C. for 2 h.

[0062] In the determination method, steps (1), (2), (3) and (4) may be as described above.

[0063] The present invention provides a method for determining protein concentration, which comprises the following steps:

[0064] (1) exchanging the liquid of the object to be tested to obtain a liquid to be tested;

[0065] (2) hydrolyzing the test solution of step (1) to obtain a hydrolyzate;

[0066] (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution;

[0067] (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography;

[0068] The analyte is a solution containing protein;

[0069] The reaction time of the pre-column derivatization was 60 min.

[0070] In the determination method, steps (1), (2), (3) and (4) may be as described above.

[0071] The present invention also provides a method for measuring protein absorbance, which comprises the following steps:

[0072] I. passing the sample through a desalting column to obtain a desalted solution;

[0073] II. Determine the absorbance of the desalted solution by slope spectroscopy;

[0074] The analyte is a solution containing protein; the step I is as described in step (1) of the above-mentioned method for determining protein concentration.

[0075] In the determination method, the absorbance can be measured by combining a variable pathlength UV spectrophotometer (SYS-VPE-SOLO5) with a Cary 60 UV spectrophotometer.

[0076] In the measurement method, the measurement wavelength of the ultraviolet spectrophotometer is 280 nm.

[0077] The present invention also provides a method for detecting the extinction coefficient of protein, which is:

[0078]

[0079] Where A is the absorbance value, L is the optical path length, Measured by the above-mentioned protein absorbance determination method; C is the protein mass concentration, measured by the above-mentioned protein concentration determination method;

[0080] Preferably, the determination method comprises the following steps:

[0081] a. The analyte is passed through a desalting column to obtain a desalted solution;

[0082] b. The desalted solution was mixed with 8M hydrochloric acid and heated at 140 ° C for 2h to obtain a hydrolyzate;

[0083] c. subjecting the hydrolyzate to a pre-column derivatization reaction for 60 min to obtain a derivative solution;

[0084] d. Determining the protein concentration of the derivative solution by high performance liquid chromatography;

[0085] e. Determine the absorbance of the desalted solution by slope spectroscopy;

[0086] f. Calculate the protein extinction coefficient.

[0087] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0088] The reagents and raw materials used in the present invention are commercially available.

[0089] The positive and progressive effects of the present invention are:

[0090] The present invention uses a desalting column to replace the protein solution, which greatly reduces the influence of the preparation background solution on protein hydrolysis and detection. The slope spectroscopy method reduces the error of manual operation compared with the traditional UV method and improves the efficiency. The temperature and time of protein hydrolysis for each amino acid improve the work efficiency. The combination of the three improves the accuracy and efficiency of the protein detection extinction coefficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 is the standard curve determined by slope spectrometry;

[0092] Figure 2 This is the HPLC chromatogram of amino acid standards;

[0093] Figure 3 This is the high performance liquid chromatogram of Herceptin (trastuzumab);

[0094] Figure 4 This is the amino acid HPLC chromatogram of bovine serum protein;

[0095] Figure 5 The 10 experimental extinction coefficients of Herceptin (trastuzumab) measured by the method of the present invention and the theoretical extinction coefficient (1.481 L / g.cm -1 ) comparison. DETAILED DESCRIPTION

[0096] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0097] The material information in the following examples is as follows:

[0098] materials Manufacturer Model / Article No. Desalting columns Thermo 89883 Hydrolysis tube CNW VJEQ-38013CT-13100-100 Liquid Chromatograph Alliance e2695

[0099] Embodiment 1:

[0100] The protein sample to be tested is diluted with 20 mM phosphate solution or the sample is concentrated to about 20 mg / mL.

[0101] Desalting buffer (20 mM phosphate buffer): weigh 0.22 g sodium dihydrogen phosphate monohydrate and 0.35 g anhydrous disodium hydrogen phosphate in 200 mL ultrapure water, dissolve and mix. Storage conditions and validity period: store at 2-8°C for one month.

[0102] Triethylamine solution: Add 105 μL triethylamine to 645 μL acetonitrile and mix well. Prepare and use immediately.

[0103] PITC (phenyl isothiocyanate) solution: add 9 μL PITC to 741 μL acetonitrile and mix well. Discard after use.

[0104] 8M hydrochloric acid: Measure 80mL HCl, add 40mL UPW (ultrapure water), mix well. Storage conditions and shelf life: Store at room temperature for 6 months.

[0105] 0.1M hydrochloric acid: Take 0.5mL of hydrolysis solution (8M hydrochloric acid), add 39.5mL UPW, and mix well. Storage conditions and shelf life: Store at room temperature for 6 months.

[0106] 1000pmol / μL Nle (norleucine): weigh 13.1mg norleucine into a 100mL volumetric flask, add dissolving solution (0.1M HCl), mix and dilute to volume. Store at -20℃ for two years.

[0107] Mobile phase A (50 mM sodium acetate, pH 6.5): Weigh 8.2 g of sodium acetate and add it to 1.9 L of ultrapure water, dissolve and mix well, adjust the pH to 6.5 with acetic acid, and then make up to 2 L with ultrapure water.

[0108] Mobile phase B (methanol: acetonitrile: ultrapure water = 20:60:20): measure 200 mL of methanol, add 600 mL of acetonitrile, and then add 200 mL of ultrapure water, mix well, and filter with a 0.22 μm filter cup (or equivalent filter).

[0109] 1) Protein desalting

[0110] Break off the tail of the desalting column and loosen the lid, place the column in an EP tube, centrifuge, make a mark on the inclined tube wall of the compacted resin, and put the column into the centrifuge with the marked part facing outward in all subsequent centrifugation steps; add desalting buffer to the column, centrifuge at 1000rcf for 2 minutes, discard the buffer in the EP tube, and repeat balancing the column several times; place the column in a new EP tube, add 30-150μL of the sample to be tested, centrifuge at 1000rcf for 2 minutes, and discard the used column.

[0111] 2) Determination of UV absorption value

[0112] The absorbance of the desalted sample at 280 nm was determined using the slope spectrophotometry. The variable pathlength UV spectrophotometer (SYS-VPE-SOLO5) was combined with the Cary 60 UV spectrophotometer. The measurement parameters were: wavelength 280 nm; 10 data collection points; 3 repetitions, so 3 standard curves were obtained (the instrument automatically generates standard curves, such as Figure 1 ),like:

[0113]

[0114]

[0115] 3) Protein hydrolysis

[0116] Take 40μL (concentration is about 20mg / mL, if the concentration is insufficient, concentrate it) of desalted sample into a hydrolysis tube, add 1mL 8M hydrochloric acid, fill with nitrogen for 10min, mix well, and hydrolyze at 140℃ for 2 hours. Transfer the hydrolyzate to a 10mL volumetric flask, add water to the volume, take 1mL of the liquid after the volume is taken into a 1.5mL EP tube, add 45μL 1000pmol / μL Nle (norleucine), vacuum concentrate for 8 hours to dryness, and add 40μL mobile phase A to mix and dissolve.

[0117] 4) Take 25 μL of the hydrolyzed sample, add 12.5 μL of triethylamine solution, mix well, add 12.5 μL of PITC (phenyl isothiocyanate) solution, mix well and let stand. Add 100 μL of n-hexane, shake vigorously and let stand for 10 minutes. Take 20 μL of the lower layer solution and mix with 180 μL of mobile phase A, centrifuge at 12000 rpm for 5 minutes and prepare for sample loading.

[0118] 5) Preparation of amino acid standards

[0119] Prepare amino acid standard solution according to the table below, vacuum concentrate for 8 hours to dryness, add 40μL of mobile phase A and mix well, store at -20℃, and the shelf life is 2 years. (Amino acid mixed standard is a mixed solution of 17 amino acid standards, and its solvent is 0.1M hydrochloric acid).

[0120]

[0121] The standard solution was prepared according to 4).

[0122] 6) Preparation of quality control solution (10 mg / mL)

[0123] Weigh 100 mg BSA (bovine serum albumin), add UPW (ultrapure water) to dissolve and dilute to 10 mL, mix well. Store at -20℃ for one year. Prepare the control solution according to 3) and 4).

[0124] 7) Blank solution: Prepare according to step 4) by using mobile phase A instead of the hydrolyzed sample.

[0125] 8) Chromatographic column Diamonsil AAA chromatographic column

[0126] 9) Detection wavelength 254nm

[0127] 10) Column temperature 45±3℃

[0128] 11) Sample chamber temperature 8±3℃

[0129] 12) Injection volume 10 μL

[0130] 13) Liquid chromatograph: Alliance e2695 with 2998 Photodiode Array (PDA) Detector

[0131] 14) The operation mode adopts the following gradient mode

[0132] Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0→39 1.0 95→52 5→48 39→40 1.0 52→0 48→100 40→45 1.0 0 100 45→46 1.0 0→95 100→5 46→60 1.0 95 5

[0133] 15) Flow rate 1.0 mL / min

[0134] 16) Running time 60 minutes

[0135] Standard curve establishment:

[0136] The amino acid standards used include aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), tyrosine (Tyr), valine (Val), methionine (Met), cysteine ​​(Cys), isoleucine (Ile), leucine (Leu), phenylalanine (Phe) and lysine (Lys). The content of each amino acid was determined by high performance liquid chromatography (HPLC). Figure 2 ), according to the concentration ratio of each amino acid: internal standard (norleucine) in the standard solution as the Y axis, the peak area ratio of amino acid: internal standard (norleucine) as the X axis, a standard curve is established as follows:

[0137]

[0138]

[0139] The 17 standard curves are consistent with the determination coefficient R 2 ≥0.99 requirement.

[0140] The molar concentration of protein was calculated using the following formula:

[0141]

[0142] Wherein, M is the molar concentration of each amino acid; N is the molecular weight of each amino acid.

[0143]

[0144] By combining the absorbance and protein concentration, the extinction coefficient of the protein can be obtained according to A=C×EC×L.

[0145] Difference percentage D% = │(actual value - theoretical extinction coefficient) │ / theoretical extinction coefficient × 100%

[0146] In this example, the slope spectroscopy method combined with high performance liquid chromatography was used to analyze the extinction coefficient of trastuzumab for injection (Herceptin) and the extinction coefficient of bovine serum albumin (BSA) (see the high performance liquid chromatography diagram). Figure 3-Figure 4 ), and 10 sets of data were analyzed respectively (Table 1); at the same time, the extinction coefficient of bovine serum albumin was analyzed by traditional method, and 10 sets of data were also analyzed (Table 2).

[0147] Table 1 Ten groups of extinction coefficients of trastuzumab and bovine serum albumin detected by the new method

[0148]

[0149]

[0150]

[0151]

[0152] Traditional experimental methods:

[0153] (1) Hydrolyze bovine serum albumin at 6 M HCl and 100 °C for 24 h;

[0154] (2) Determine absorbance using a spectrophotometer;

[0155] (3) The remaining test conditions were performed according to the conventional procedures in the art; the protein extinction coefficient was calculated as shown in the following table:

[0156] Table 2 Ten groups of extinction coefficients of BSA (bovine serum albumin) detected by traditional methods

[0157]

[0158]

[0159]

[0160] Experimental results:

[0161] 1. The RSD% of the sample concentrations of Herceptin (trastuzumab) and BSA (bovine serum albumin) analyzed using this method were all within 3%.

[0162] 2. The extinction coefficients of Herceptin (trastuzumab) and BSA (bovine serum albumin) analyzed by this method have a difference percentage D% between the experimental extinction coefficient and the theoretical extinction coefficient within + / -10% ( Figure 5 ).

[0163] 3. Based on the traditional PITC derivatization method, this optimized method can detect the extinction coefficient of protein efficiently, with high quality and stably.

[0164] Embodiment 2:

[0165] 1. According to the experimental process of Example 1, the protein hydrolysis conditions, including hydrolysis temperature, hydrolysis time, and hydrochloric acid concentration during hydrolysis, were explored. The exploration plan is as follows:

[0166] The extinction coefficient of bovine serum albumin (prepared by ourselves) with a concentration of about 20 mg / mL was determined and compared with the theoretical extinction coefficient (0.667 L / g.cm -1 ) and calculate the difference percentage.

[0167] Table 3 Hydrolysis conditions schedule

[0168]

[0169]

[0170] The experiment was carried out according to the plan in Table 3 (derivatization time was 60 min), and the final experimental results were as follows:

[0171] Table 4 Experimental results of hydrolysis conditions: hydrolysis time at 130°C and different hydrochloric acid concentrations during hydrolysis

[0172]

[0173] Table 5 Experimental results of hydrolysis conditions: hydrolysis time at 140°C and different hydrochloric acid concentrations during hydrolysis

[0174]

[0175] Table 6 Experimental results of hydrolysis conditions: hydrolysis time at 150°C and different hydrochloric acid concentrations during hydrolysis

[0176]

[0177] Based on the above experimental results, the following conclusions can be drawn:

[0178] When the hydrolysis time was 2 h, the extinction coefficient D% was within 10%, meeting the basic experimental requirements.

[0179] Under the condition of the same hydrolysis time (2h), when the hydrolysis temperature is increased to 140-150°C, the D% of the extinction coefficient is within 5%, which meets the experimental requirements.

[0180] Under the conditions of the same hydrolysis time (2h) and the same hydrolysis temperature, the D% of the extinction coefficient of 8M hydrochloric acid is less than that of 6M hydrochloric acid.

[0181] Therefore, the conditions for protein hydrolysis into single amino acids can be tentatively determined as follows: hydrolysis temperature 140°C, hydrolysis time 2h, and hydrochloric acid concentration 8M during hydrolysis.

[0182] 2. According to the experimental process of Example 1, after the protein is hydrolyzed into single amino acids, the single amino acids are derivatized using PITC (phenyl isothiocyanate), and the derivatization time is explored. The exploration plan is as follows:

[0183] The amino acids under various hydrolysis temperature conditions were derivatized, and the design is as follows:

[0184] Table 7 Derivatization experimental design table

[0185]

[0186] The experiment was carried out according to the plan in the table, and the final experimental results are as follows:

[0187] Table 8

[0188]

[0189] As can be seen from the table, amino acids cannot be completely derivatized within 15 minutes and 30 minutes, and some experimental results meet the requirements within 45 minutes. However, the time difference between the derivatization of 45 minutes and 60 minutes is not much, so it is still believed that the derivatization experiment of 60 minutes is more stable.

[0190] Example 3

[0191] According to the experimental process of Example 1, after the protein is hydrolyzed into single amino acids, the protein solution is concentrated. The sample contains concentrated hydrochloric acid, and a constant temperature mixer (Eppendorf / Thermo Mixer C) is used for heating and concentration at 90°C instead of vacuum concentration, thereby avoiding corrosion of the instrument by concentrated hydrochloric acid and also avoiding the influence of rust entering the sample on the results.

[0192] Table 9 Derivatization Experimental Design

[0193]

[0194]

[0195] The experiment was carried out according to the plan in the table, and the final experimental results are as follows:

[0196] Table 10

[0197]

[0198] As can be seen from the table, the hydrolyzed sample was concentrated to dryness using a constant temperature mixer at 90°C, and the experimental results were in line with expectations.

[0199] Comparative Example 1

[0200] The advantages of using desalting columns to desalt versus ultrafiltration tubes (Merck, 30K centrifugal Filters, catalog number: UFC903096) for fluid exchange in this method are as follows:

[0201] Table 11

[0202]

[0203] From the perspective of protein recovery rate, the method of changing the liquid in the desalting column will have a higher protein recovery rate than that of changing the liquid in the ultrafiltration tube. In addition, the method of changing the liquid in the desalting column only requires one centrifugation of the sample to complete the desalting, while the method of changing the liquid in the ultrafiltration requires the sample to be centrifuged three times + replenishing the buffer (specific operation steps: wash the ultrafiltration tube with ultrapure water and centrifuge at 15000rpm for 2min, repeat this step and wash it once, and then add 120uL of the sample. Centrifuge at 15000rpm for 2min, add 20mM phosphate buffer to make up to 120uL, repeat this centrifugation and adding phosphate buffer 3 times. Then turn the ultrafiltration tube upside down into a new centrifuge tube, centrifuge at 15000rpm for 2min, and add 20mM phosphate buffer to make up to 120uL). It can be seen that the operation method of changing the liquid in the desalting column is relatively simpler.

Claims

1. A method for determining protein concentration, characterized in that: It includes the following steps: (1) Exchanging the solution of the analyte through a desalting column to obtain a desalted solution; (2) hydrolyzing the desalted liquid of step (1) to obtain a hydrolyzate; (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution; (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography; The object to be detected is a solution containing protein.

2. The measuring method according to claim 1, characterized in that The step (1) satisfies one or more of the following conditions: (1) The protein in the "protein-containing solution" is a protein drug, such as bovine serum albumin or trastuzumab for injection; (2) The solvent of the analyte is a phosphate solution; preferably a 20 mM phosphate solution; (3) The desalting column is Thermo 89883; (4) The buffer used for the liquid replacement is an aqueous solution of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate; preferably, the concentration of the buffer used for the liquid replacement is 20 mM; (5) The liquid change is carried out by centrifugation; preferably, the liquid change comprises the following steps: placing the desalting column in an EP tube and centrifuging; adding a buffer and centrifuging again; adding the test substance and centrifuging again.

3. The measuring method according to claim 1, characterized in that In the step (2), the hydrolysis comprises the following steps: mixing the desalted liquid with hydrochloric acid under nitrogen; Preferably, the hydrolysis satisfies one or more of the following conditions: (1) The concentration of the hydrochloric acid is 6M to 8M; for example, 6M, 6.5M, 7M, 7.5M, 8M; preferably 8M; (2) The hydrolysis temperature is 130°C to 150°C; for example, 130°C, 135°C, 140°C, 145°C, 150°C; preferably 140°C; (3) The hydrolysis time is 2 h to 4 h; for example, 2 h, 2.5 h, 3 h, 4 h; preferably 2 h; (4) The hydrolysis further comprises the following steps: after the hydrolysis, diluting with water, mixing the diluted solution with a norleucine solution, concentrating, and mixing with mobile phase A to obtain a hydrolyzate; the norleucine solution is preferably a mixed solution of norleucine and 0.1 M HCl; and the mobile phase A is preferably a sodium acetate solution.

4. The assay method according to claim 3, wherein The hydrolysis satisfies one or more of the following conditions: (1) The concentration of the norleucine solution is 1000 pmol / μL; (2) the volume ratio of the norleucine solution to the diluted solution is 0.045:1; (3) The pH of the mobile phase A is 6 to 7, preferably 6.5; (4) The concentration temperature is 90° C. (5) The concentration is carried out using a thermostatic mixer (Eppendorf / Thermo Mixer C), such as a heating stirring module (Thermo); (6) the dilution includes diluting to 10 times the volume, taking 1 / 10 of the solution and mixing it with the norleucine solution; (7) The hydrolysis comprises the following steps: under nitrogen conditions, mixing the desalted solution of step (1) with 8M hydrochloric acid, heating and hydrolyzing at 140°C for 2h, diluting the hydrolyzate with water, mixing with a norleucine solution, concentrating to dryness, adding mobile phase A to dissolve, and obtaining a hydrolyzate.

5. The measuring method according to claim 1, characterized in that In the step (3), the pre-column derivatization comprises the following steps: mixing the hydrolyzate, triethylamine solution and phenyl isothiocyanate solution, adding n-hexane, shaking and letting stand, taking the lower layer solution for sample loading; the triethylamine solution is preferably a mixed solution of triethylamine and acetonitrile; the phenyl isothiocyanate solution is preferably a mixed solution of phenyl isothiocyanate and acetonitrile; Preferably, the pre-column derivatization satisfies one or more of the following conditions: (1) The volume ratio of the triethylamine to the acetonitrile is 1:(5-8); preferably 1:6; (2) The volume ratio of the phenyl isothiocyanate to the acetonitrile is 1:(80-85); preferably 1:82; (3) the volume ratio of the triethylamine solution to the hydrolyzate is 1:2; (4) the volume ratio of the phenyl isothiocyanate solution to the hydrolyzate is 1:2; (5) The volume ratio of the n-hexane to the hydrolyzate is 4:1; (6) The reaction time of the pre-column derivatization is 60 min.

6. The measuring method according to claim 1, characterized in that In the step (4), the detection conditions of the high performance liquid chromatography are as follows: The chromatographic column is a Diamonsil AAA column; Mobile phase A was 50 mM sodium acetate solution, pH 6.5 ± 0.1; Mobile phase B was methanol, acetonitrile, and water in a volume ratio of 1:3:1; The detection wavelength of the detector is 254 nm; Preferably, step (4) satisfies one or more of the following conditions: (1) The high performance liquid chromatography uses an internal standard method to calculate the amino acid concentration; the internal standard substance of the internal standard method is norleucine; (2) The molar concentration of the protein is calculated using the following formula: ； Wherein, M is the molar concentration of each amino acid; N is the number of each amino acid; (3) The protein concentration is calculated using the following formula: Protein concentration = protein molar concentration × protein relative molecular mass; (4) The column temperature of the chromatographic column is 42-48°C, preferably 45°C; (5) The flow rate of the mobile phase is 1.0 mL / min; (6) The elution mode of the high performance liquid chromatography is linear gradient elution; preferably: 。 7. A method for determining protein concentration, characterized in that: It includes the following steps: (1) exchanging the liquid of the object to be tested to obtain a liquid to be tested; (2) hydrolyzing the test solution of step (1) to obtain a hydrolyzate; (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution; (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography; Wherein, the analyte is a solution containing protein; The hydrolysis comprises the following steps: mixing the test solution of step (1) with 8M hydrochloric acid, and heating and hydrolyzing at 140° C. for 2 h; Preferably, steps (1), (2), (3) and (4) are independently as described in any one of claims 1 to 6.

8. A method for determining protein concentration, characterized in that: It includes the following steps: (1) exchanging the liquid of the object to be tested to obtain a liquid to be tested; (2) hydrolyzing the test solution of step (1) to obtain a hydrolyzate; (3) subjecting the hydrolyzate of step (2) to pre-column derivatization to obtain a derivatized solution; (4) determining the protein concentration of the derivatized solution of step (3) by high performance liquid chromatography; Wherein, the analyte is a solution containing protein; The reaction time of the pre-column derivatization is 60 min; Preferably, steps (1), (2), (3) and (4) are independently as described in any one of claims 1 to 6.

9. A method for measuring protein absorbance, characterized in that: It includes the following steps: I. passing the sample through a desalting column to obtain a desalted solution; II. Determine the absorbance of the desalted solution by slope spectroscopy; The analyte is a solution containing protein, and step I is as described in step (1) of the method for determining the protein concentration according to claim 1 or 2; Preferably, the absorbance is measured by combining a variable pathlength UV spectrophotometer with a Cary 60 UV spectrophotometer; preferably, the measuring wavelength of the UV spectrophotometer is 280 nm.

10. A method for detecting the extinction coefficient of a protein, characterized in that: It is: Where A is the absorbance value, L is the optical path length, Measured by the method for determining protein absorbance according to claim 9; C is the protein mass concentration, measured by the method for determining protein concentration according to any one of claims 1 to 6; Preferably, the determination method comprises the following steps: a. The analyte is passed through a desalting column to obtain a desalted solution; b. The desalted solution was mixed with 8M hydrochloric acid and heated at 140 ° C for 2h to obtain a hydrolyzate; c. subjecting the hydrolyzate to a pre-column derivatization reaction for 60 min to obtain a derivative solution; d. Determining the protein concentration of the derivative solution by high performance liquid chromatography; e. Determine the absorbance of the desalted solution by slope spectroscopy; f. Calculate the protein extinction coefficient.

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