A rapid quantitative method for protein content in the purification process of C1 esterase inhibitor

Abstract

The present invention relates to a rapid quantitative method for protein content in the purification process of C1 esterase inhibitor, and the method comprises the following steps: (1) diluting a CI-INH sample to be measured with water for injection; (2) detecting the absorbance Y of the diluted sample at a wavelength of 280 nm; (3) substituting into the corrected empirical formula to calculate the protein concentration, and the empirical formula is: protein concentration X = {10^[(LgY + 0.382) / 0.764] - 0.05} × dilution factor.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology. Specifically, it relates to a rapid quantification method for protein content in the purification process of C1 esterase inhibitor. Background Art

[0002] There is a protein called C1 esterase inhibitor (C1-INH) in human plasma, which is mainly synthesized by the liver. It is a single-chain glycoprotein composed of 478 amino acids, with a relative molecular mass of 104,000. It has a highly glycosylated structure, and the sugar content accounts for about 30% of the relative molecular mass. Its isoelectric point is 2.7 - 2.8, and its content in plasma is about 0.2 mg / ml. It has poor thermal stability. Its function is to inhibit the ester hydrolysis activity of complement factor 1 (C1), belonging to the serine protease inhibitor family. It is one of the important regulators of the human complement system and plays an important regulatory role in both the classical pathway and the alternative pathway of the complement system. In the kallikrein-kinin system, C1-INH can inhibit the activities of activated factor ⅩⅡ (FⅩⅡa) and kallikrein, thus affecting the formation of bradykinin. Bradykinin can cause capillary dilation and enhanced permeability, resulting in the exudation of vascular contents and the formation of local edema in human tissues, that is, hereditary angioedema (HAE). HAE is also caused by C1-INH deficiency or dysfunction. C1-INH deficiency leads to type I HAE, and C1-INH dysfunction leads to type II HAE. The clinical features are recurrent, self-limiting, localized, non-pitting edema of the skin and mucous membranes, mostly occurring in the limbs, face, and upper respiratory tract. When acute edema occurs in the upper respiratory tract, it often causes asphyxiation and death of patients due to laryngeal edema. The replacement therapy of C1-INH is used to restore the missing or dysfunctional protein in HAE patients, and the attacks of HAE are controlled by treating with blood-derived or recombinant C1-INH concentrates as needed. Among various therapeutic drugs for the disease, C1-INH concentrated preparations have few side effects, high safety and effectiveness, and are used for the long-term treatment of HAE. Its safety, effectiveness, and body tolerance have been verified, and plasma-derived C1-INH concentrated preparations have also become the first choice drugs for children, pregnant women, and lactating women patients.

[0003] The applicant is conducting research on the process of extracting C1-INH from plasma. Among them, protein content, titer, and purity are the main indicators for quality control throughout the production process. The protein content is detected using a specific protein detector according to the immunoturbidimetry method. The titer and purity are both detected using methods included in the European Pharmacopoeia, and the purity is detected by the HPLC method. The General Principles and Guidelines 0731 for the determination of protein content in the Third Edition of the Chinese Pharmacopoeia 2020 include six methods, namely: Kjeldahl method, Folin-Ciocalteu method (Lorry method), biuret method, BCA method, Coomassie brilliant blue method (Brandford method), and ultraviolet-visible spectrophotometry. According to the requirements of the European Pharmacopoeia, in the detection requirements for the specific activity of C1-INH concentrated preparations, the detection method for the total protein content is the Kjeldahl method. This method has a relatively long operation time, generally requiring 2 days, and is mostly used for finished product verification. During the process development, ultrafiltration, formulation, and other process operations need to be carried out according to the specific activity results. The Kjeldahl method cannot meet the need for rapid detection of process samples. Ultraviolet-visible spectrophotometry is based on the fact that proteins contain aromatic amino acids such as tyrosine and tryptophan with conjugated double bonds, which have the maximum absorbance at a wavelength of 280 nm. Within a certain range, the absorbance is proportional to the protein concentration. This method is simple and rapid to operate and is used for the detection of purified proteins. Generally, the concentration of the test sample is between 0.2 and 2 mg / ml, but it has poor accuracy and many interfering substances. The Pharmacopoeia recommends calculating the protein content in the test sample according to the absorption coefficient method or the reference substance comparison method of the ultraviolet-visible spectrophotometry in General Rule 0401. Based on our many years of experience in establishing quantitative detection methods and in the presence of high-purity C1-INH that can be used as a reference substance, we determined to use the reference substance comparison method as the basis and optimized it. We established a linear equation for the reference protein concentration and A280 over a wider protein concentration range, and then calculated by substituting the A280 of different dilutions of the test sample into the equation, and used the mean value as the protein concentration of the sample. At the same time, this method was verified and used for the determination of the protein content of high-purity samples in the latter half of the purification process.

[0004] Based on this, the present invention is proposed Summary of the Invention

[0005] The present invention first relates to a method for determining the concentration of C1 esterase inhibitor (C1-INH), and the method comprises the following steps:

[0006] (1) Dilute the test CI-INH sample with water for injection;

[0007] (2) Detect the absorbance Y of the diluted sample at a wavelength of 280 nm;

[0008] (3) Substitute into the corrected empirical formula to calculate the protein concentration, and the empirical formula is:

[0009] Protein concentration X = {10^[(LgY + 0.382) / 0.764] - 0.05} × dilution factor.

[0010] Furthermore, use a microplate reader Spectromax Plus384 and quartz cuvettes to detect the absorbance Y of the sample at a wavelength of 280 nm.

[0011] Furthermore, in the test sample, the purity of C1-INH protein is higher than 85%, and the concentration range of C1-INH protein in the test sample is between 0.2 and 5.5 mg / ml.

[0012] The present invention also relates to the application of the described method in the protein purity detection during the production and purification of C1 esterase inhibitor.

[0013] The present invention also relates to the application of the described method in the protein content detection of C1 esterase inhibitor products.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. Most protein quantification methods use bovine serum albumin or immunoglobulin as standards to determine the protein content of test samples. When particularly high accuracy in protein quantification is required, a purified target protein needs to be used to plot a standard curve. Since there is currently no C1-INH protein concentration standard for C1-INH concentrated preparations on the market, we prepared an intermediate product of C1-INH with a purity > 95% as a control to plot a standard curve for detecting samples during the purification process.

[0016] 2. The A280 method uses quartz cuvettes as containers for detection on a microplate reader, and the data is processed with software to quickly obtain the recovery rates under different fitting equations to determine the best fitting equation and the linear range of protein concentration. In order to Meet the research requirements of a large number of samples and a wide concentration range in the development stage , we did not choose the coefficient method recommended by the pharmacopoeia, but established a double logarithmic equation using the control comparison method, with a protein concentration range of 0.2 - 3.5 mg / ml. Samples with different matrix solutions and target protein concentrations during the detection process were also tested, and an empirical formula with high accuracy was established to obtain the accurate protein concentration through the calculation of the A280 original value.

[0017] 3. Through experiments, it was found that freeze-drying The influence of the lyoprotectant added to the formulation on the protein concentration is within an acceptable accuracy range With the advantages of rapidity, accuracy and high throughput, it can be used for the detection of samples in the purification stage of CI-INH, as well as for the detection of bulk solution, semi-finished products and finished products (final verification). At the same time, it is not necessary to use different matrix solutions to make standard curves for each detection , so before the establishment of the Kjeldahl method, the A280 method can be used to estimate the total protein content to obtain the specific activity of the finished product.

[0018] 4. Generally, the A280 method is mostly used for detecting the protein content of samples in the purification stage during the R & D phase. For the finished products, stock solutions / semi-finished products of biological products, the Kjeldahl method and the biuret method are mostly used. During the R & D process of our new product, considering the need for consistency between the final verification method and the in-process verification, the results of the A280 method were compared with those of the Kjeldahl method. The conclusion is that the empirical formula established by the improved A280 method has which greatly simplifies the experimental process and saves detection time Figure 1 Figure 2 . BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The standard curve fitted with the original values of three experiments.

[0020] Figure 2 The standard curve fitted with the extinction values of three experiments. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] MATERIALS AND METHODS

[0022] Samples

[0023] Purification process flow: raw material sample → rough purification capture → fine purification → stock solution → semi-finished product → finished product

[0024] Intermediate samples of the purification process (C1-INH purity > 85%): samples in the fine purification step, stock solutions and semi-finished products.

[0025] Reference substance: sample after ultrafiltration (Kjeldahl determination result is 10.7 mg / ml);

[0026] All of the above samples were prepared by the applicant himself / herself.

[0027] MAIN REAGENTS AND INSTRUMENTS

[0028] Microplate reader Spectromax Plus384 and supporting Softmax 5.2 software; colorimetric cuvette.

[0029] Example 1. Establishment and verification of the quantitative detection method

[0030] 1.1. Establishment of the quantitative detection method

[0031] The reference substance was stepwise diluted with water for injection to a total of 12 concentrations, covering the concentration range of 0.214 - 5.35 mg / ml. 1500 μl of each concentration sample was prepared to cover 2 / 3 of the volume of the quartz cuvette, and the A280 value was read, repeated 6 times. Similarly, the operation of the blank control was carried out. Subtract the A280 value of the blank control from the A280 value of the reference substance to obtain the absorbance difference, that is, the extinction value. Fit the extinction value and the theoretical protein concentration with different equations. The X-axis of the standard curve is the theoretical protein concentration, and the Y-axis is the corresponding extinction value. The recovery rate of each dilution degree % = (back-calculated value / theoretical value) * 100, the CV of the extinction value of each dilution product, and the correlation coefficient R of the curve 2 。

[0032] The acceptable criteria for the best-fitting curve equation are: the recovery rates of each dilution degree are between 100 ± 15%, and the R 2 value should be ≥ 0.990.

[0033] Dilute the test sample to the linear range determined by this method, substitute the extinction value into the equation for calculation, the CV of the test result < 10%, and take the average value of different dilution degrees as the final protein concentration of the test sample.

[0034] The A280 result shows that when the protein concentration of the C1-INH reference substance is between 0.214 - 5.35 mg / ml, its A280 value is between 0.1 - 1.2.

[0035] The results in Table 1 show that

[0036] If linear fitting is performed according to the four-parameter equation, its linear range is 0.214 - 5.35 mg / ml, and the R 2 is 1;

[0037] If fitting is performed according to the quadratic equation and the double logarithmic equation, its linear range is 0.214 - 3.567 mg / ml, and the R 2 are 1 and 0.996 respectively;

[0038] If linear fitting is performed according to the linear function, its linear range is 0.357 mg / ml - 2.14 mg / ml, and the R 2 is 0.997.

[0039] Although the four-parameter equation has the widest linear range that meets the requirements, the actual calculation process is complex, so it is discarded;

[0040] Although the actual calculation process of the linear function is the simplest, the linear range is relatively narrow, so it is discarded;

[0041]

[0042] Table 1. Results of fitting the standard curve with different functions

[0043]

[0044] *The recovery rate in this interval exceeds the upper limit at the highest concentration, and the actual usable range is 0.214~3.567mg / ml.

[0045] After determining the double logarithmic fitting method, we performed double logarithmic fitting of the original value (without subtracting the blank control) and the extinction value (absorbance difference) for the 12 concentrations of the standard curve. From the recovery rate of each concentration, the recovery rate of the original value fitting ranged from 92.264% to 103.907%, R 2 =0.999, the recovery rate fitted by extinction value is between 88.408% and 106.288%, R 2 =0.996. The accuracy and recovery rate of the original value fitting are slightly better than those of the extinction value fitting. The results are shown in Table 2.

[0046] Table 2. Comparison of double logarithmic fitting results of standard curve using original value and extinction value

[0047]

[0048] 1.2. Validation of quantitative detection methods

[0049] After the linear range and fitting method were initially determined, the method was verified. The reference substance was diluted 3 times with water for injection as the starting concentration, followed by 2-fold step dilution, with a total of 5 protein concentrations: 3.567 mg / ml (3-fold dilution), 1.783 mg / ml (6-fold dilution), 0.892 mg / ml (12-fold dilution), 0.446 mg / ml (24-fold dilution), 0.223 mg / ml (48-fold dilution), which is the standard series;

[0050] At the same time, the reference substance was diluted 4 times, 15 times, 40 times and 50 times with WFI, corresponding to three quality control substances of high (2.675 mg / ml), medium (0.713 mg / ml) and low (0.268 mg / ml) and the minimum detection limit (0.214 mg / ml).

[0051] Each of the above concentrations was measured 6 times in parallel, and the average was taken for double logarithmic fitting to obtain the standard curve and equation, and the CV and recovery rate of the protein content of the standard and quality control products were calculated.

[0052] The process was repeated 3 times. The slope and intercept of the standard curves of different experimental batches were compared to determine the linear range and repeatability.

[0053] Accuracy acceptance criteria: intra-batch and inter-batch protein content recovery rate = (measured value / theoretical value)*100 is 100±15%;

[0054] Precision acceptance criteria: The CV of protein content within and between batches is ≤ 10%.

[0055] The precision and accuracy results of the quality control products and the lowest detection limit are shown in Tables 3 and 4. The results show that the recovery rates are all within 100 ± 6%, and the CVs are all within 3%, indicating that the equations obtained by double logarithmic fitting using the original values and extinction values are both valid. The repeatability comparison of the standard curves fitted with the original values three times is shown in Protein concentration X = {10^[(LgY + 0.382) / 0.764] - 0.05} × dilution factor , and the repeatability comparison of the standard curves fitted with the extinction values is shown in ​ . It can be seen that the standard curves of the three experiments almost coincide, indicating good stability and repeatability.

[0056] Table 3. Summary of the results of precision and accuracy verification calculated by the original value equation

[0057] Table 4. Summary of the results of precision and accuracy verification calculated by the extinction value equation

[0058]

[0059]

[0060] 1.3 Determination of the empirical formula

[0061] Take the average value of A280 at each concentration of the standard curves for the three linear range verifications, and then use double logarithmic fitting again to obtain the following equations:

[0062] Original average value equation: LgY = -0.382 + 0.764LgX, R 2 = 0.998; (One of the alternative curves of the empirical formula)

[0063] Extinction average value equation: LgY = -0.441 + 0.872LgX, R 2 = 0.994. (The second alternative formula of the empirical formula)

[0064] Since using the extinction value equation requires diluting the standard product with different matrix solutions, eliminating the blank control, and then refitting the standard curve, which is not conducive to rapid on-line detection. Therefore, we use the original value fitting equation with higher accuracy to explore the empirical formula. We detected A280 of various matrix solutions containing the target protein and calculated the protein concentration using the original value formula. The results showed that the A280 values of matrix solutions with different salt ion concentrations and amino acid components were in the range of 0.038 - 0.051. According to the original mean equation, the corresponding protein concentrations were between 0.044 mg / ml and 0.064 mg / ml, with an average of 0.05 mg / ml. Therefore, the original A280 value (Y value) of the sample to be detected can be directly substituted into the original mean equation LgY = -0.382 + 0.764LgX to calculate the protein concentration X (unadjusted value). Subtract the average protein concentration of the matrix solution, 0.05 mg / ml, from the X result and then multiply by the dilution factor to obtain the protein concentration of the corresponding sample to be detected, that is, the empirical formula:

[0065] ​ 。

[0066] At the same time, we also carried out a sample addition experiment to verify the accuracy again, to verify that the empirical formula is applicable to the detection of samples with different matrix solutions, that is, only the original absorbance value of the sample needs to be detected, and the accurate protein concentration can be obtained through the empirical formula calculation, without having to remake the standard curve. Using the above protein concentration empirical formula avoids dissolving the reference substance with different matrix solutions to make a standard curve for calculation.

[0067] The specific method is as follows:

[0068] We diluted the reference substance 10 times with different matrix solutions, detected its original absorbance value, and calculated the protein concentration and recovery rate using the original mean and extinction mean equations and the empirical formula. The results showed that the recovery rate of the protein concentration calculated according to the empirical formula was between 96.636% and 109.533%, which was better than the original mean and extinction mean equations (the recovery rate of sample formulation 3 with matrix solution containing amino acids exceeded 85% - 115%), meeting the requirements of accuracy verification. The results are shown in Table 5.

[0069] Table 5 Summary of results of different matrix solutions and corresponding standard addition experiments

[0070]

[0071]

[0072] Example 2, Detection and Verification of Actual Samples

[0073] 2.1. The empirical formula of the A280 method is used for the detection of samples at different purification stages of different batches and compared with the results of the Kjeldahl method

[0074] The results showed that the results calculated by the improved A280 method using the empirical formula were in good agreement with those of the Kjeldahl method. The coefficient values of A280 / Kjeldahl for samples 2 - 7 were between 0.93 and 1.04. Sample 1 was a crude pure sample. Since the purity of this sample was less than 85%, the coefficient value of A280 / Kjeldahl was relatively high. Therefore, the empirical formula for the rapid quantification of A280 is applicable to the calculation of the C1-INH protein content in the highly purified stage with a purity higher than 85%, and is used to guide the development of process research. The specific results are shown in Table 6.

[0075] Table 6 Comparison of the detection results between the A280 method and the Kjeldahl method

[0076]

[0077] 2.2 Detection of protein content in samples at the process highly purified stage using the A280 method empirical formula:

[0078] The protein content of key samples at the highly purified stage was calculated using different equations, and the accuracy of the results obtained at different dilution degrees was compared. The rapid detection results of the A280 method for key samples in the process are shown in Table 7. Among them, the Kjeldahl result of the nanofiltration sample was 10.6 mg / ml. The average value of the results calculated by the original value formula for this sample was 11.4445 mg / ml; the average value of the extinction formula was 11.960 mg / ml; the result calculated by the empirical formula was 10.695 mg / ml. The average value of the protein content results calculated by the empirical formula was closest to the detection result of the Kjeldahl method. The protein concentrations calculated by the original average formula and the extinction average formula were on the high side. At the same time, since the amino acids contained in the finished product formula had little influence on the detection results of the A280 method, in the case where the Kjeldahl method for detecting amino acids (trichloroacetic acid method, protein content = total nitrogen - non-protein nitrogen) was not established, the A280 method empirical formula could be used to initially obtain the protein content of semi-finished and finished products, which further highlighted the importance of the A280 method empirical formula.

[0079] Table 7 Detection of protein content in process samples

[0080]

[0081] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. A rapid determination method for the concentration of C1 esterase inhibitor (C1-INH), the method comprising the following steps: (1) Dilute the CI-INH sample to be tested with water for injection; (2) Detect the absorbance Y of the diluted sample at a wavelength of 280 nm; (3) Substitute into the corrected empirical formula to calculate the protein concentration, and the empirical formula is: Protein concentration X = {10^[(LgY + 0.382) / 0.764] - 0.05} × dilution factor; the protein concentration X ranges from 0.2 to 5.5 mg / ml; and in the sample to be tested, the purity of C1-INH protein is higher than 85%.

2. The method according to claim 1, wherein Use a microplate reader Spectromax Plus384 and quartz cuvettes to detect the absorbance Y of the sample at a wavelength of 280 nm.

3. Use of the method according to claim 1 or 2 in the detection of protein purity in the production and purification process of C1 esterase inhibitor.

4. Use of the method according to claim 1 or 2 in the detection of protein content in C1 esterase inhibitor products.

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