An evaluation method for the quality of the production process of recombinant hepatitis B vaccine

Abstract

The present disclosure relates to a method for evaluating the quality of the production process of a recombinant hepatitis B vaccine. The evaluation of the production process quality includes the quality evaluation of the recombinant hepatitis B vaccine bulk solution and the evaluation of the between-batch consistency of the recombinant hepatitis B vaccine bulk solution. The evaluation method comprises the following steps: (1) enriching the vaccine bulk solution to be tested, preparing an infrared detection sample for infrared spectroscopy detection; (2) analyzing and calculating the infrared spectrum of the vaccine bulk solution to be tested obtained by using the established quality evaluation model of the recombinant hepatitis B vaccine bulk solution to obtain multiple matching values, and then comparing the matching values with the quality evaluation criteria, and realizing the quality evaluation of the vaccine bulk solution to be tested through the comparison result; (3) evaluating the between-batch consistency of the vaccine bulk solution to be tested by using the consistency evaluation model. The evaluation method provided by the present disclosure can realize the quality evaluation of the intermediate product for preparing the recombinant hepatitis B vaccine, and can also evaluate the between-batch consistency of the vaccine bulk solution.

Description

Technical Field

[0001] The present disclosure relates to the technical field of vaccine quality evaluation, and particularly to a method for evaluating the quality of the production process of a recombinant hepatitis B vaccine. Background Art

[0002] Vaccines are biological products prepared by using biotechnology with pathogenic microorganisms or other components and metabolites as starting materials for preventing and treating corresponding human diseases. The types of vaccines include genetically engineered recombinant protein vaccines, subunit vaccines, nucleic acid vaccines, live attenuated vaccines, inactivated vaccines, etc. As preventive biological products for human use, vaccine safety is a crucial issue, and it is very necessary to establish a scientific and reasonable vaccine safety guarantee system.

[0003] Hepatitis B virus type B (referred to as hepatitis B) is caused by hepatitis B virus (HBV) infection. HBV is a liver tropic virus, and the infected person spreads the disease through blood or other body fluids, causing liver cell inflammation, necrosis and fibrosis. Hepatitis B is divided into acute hepatitis B and chronic hepatitis B. 90% of acute hepatitis B can heal itself in adults, and chronic hepatitis B may develop into liver cirrhosis or even liver cancer. Hepatitis B vaccine plays a crucial role in controlling the prevalence of hepatitis B.

[0004] Currently, all the marketed hepatitis B vaccines are recombinant hepatitis B vaccines, including hepatitis B surface antigen (HBsAg) expressed by recombinant Hansenula polymorpha, recombinant Saccharomyces cerevisiae, and recombinant CHO cells, which are all vaccines prepared by using genetic recombination technology to recombine the gene encoding the protective antigen of pathogenic microorganisms into bacteria, yeast or cells, and then extracting and purifying the expressed protective antigen after cultivation and proliferation.

[0005] The intermediate product obtained by extracting and purifying the product using protein separation technology is called vaccine bulk. The bulk is produced into a finished preparation that meets the human use conditions through a formulation process, such as an injection, an oral preparation, etc. The material basis of a genetically engineered recombinant protein vaccine is protein, and the target protein of a recombinant hepatitis B vaccine is the HBsAg protein (composed of the product of the 226-amino-S gene). During the production process, the consistency of the bulk product should be ensured to further ensure the quality of the final product.

[0006] Currently, the requirements for the production process control of human vaccines are divided into three levels: whole-process quality control, batch-to-batch consistency control, and control of target components and non-target components, that is, effectively controlling the entire production process of the vaccine from three aspects of raw materials, intermediate products, and products to ensure the quality of the final product.

[0007] The identification of the target protein is a key quality attribute of genetically engineered recombinant protein vaccines. Currently, for the identification of the bulk solution of genetically engineered recombinant protein vaccines, methods with high specificity such as N-terminal amino acid sequence determination and anti-idiotypic immunity are mainly used to identify the target protein, and it is stipulated that "it should be determined at least once a year". Although the amino acid sequencing method has strong specificity and accurate results, it has high costs and is time-consuming. More importantly, this method cannot characterize and evaluate the batch-to-batch consistency.

[0008] Therefore, the current identification methods cannot be used for daily inspection and process quality control during the production process. Currently, there is still a lack of a rapid process quality evaluation method with strong specificity, low cost, and simple operation. Summary of the Invention

[0009] To solve the above technical problems, the present disclosure provides a method for evaluating the quality of the production process of recombinant hepatitis B vaccines.

[0010] In a first aspect, the present disclosure provides a method for evaluating the quality of the production process of recombinant hepatitis B vaccines. The evaluation of the production process quality includes the quality evaluation of the bulk solution of recombinant hepatitis B vaccines and the evaluation of the batch-to-batch consistency of the bulk solution of recombinant hepatitis B vaccines. The evaluation method includes the following steps:

[0011] (1) Enrich the vaccine bulk solution to be tested and prepare an infrared detection sample for infrared spectroscopy detection;

[0012] (2) Analyze and calculate the infrared spectrum of the vaccine bulk solution to be tested obtained by using the established quality evaluation model of the bulk solution of recombinant hepatitis B vaccines to obtain multiple matching values, and then compare the matching values with the quality evaluation criteria to realize the quality evaluation of the vaccine bulk solution to be tested through the comparison results;

[0013] (3) Use the consistency evaluation model to evaluate the batch-to-batch consistency of the vaccine bulk solution to be tested.

[0014] The detection method provided by the present disclosure has strong specificity, low cost, and simple operation. At the same time, it can not only detect the quality of the vaccine bulk solution, but also characterize and evaluate the batch-to-batch consistency.

[0015] In the current detection methods, although infrared spectroscopy can achieve qualitative analysis of proteins and has strong specificity, its sensitivity is low; the content of the target protein in the bulk solution of hepatitis B vaccines is low, and water has a great influence on infrared spectroscopy, and the target proteins of the bulk solutions of hepatitis B vaccines with different matrices (production processes) have small differences. The conventional infrared spectroscopy method cannot meet the quality evaluation of the production process of genetically engineered recombinant protein vaccines.

[0016] The present disclosure combines chemometric methods, statistical process control techniques with the infrared detection method of the present disclosure to establish a quality evaluation model for the rapid recombinant hepatitis B vaccine bulk. Model parameters such as the quality evaluation spectral region and the reproducibility level corresponding to the quality evaluation spectral region are stored in the model. At the same time, in combination with the infrared spectrum set of the control recombinant hepatitis B vaccine bulk provided by the present disclosure, the method provided by the present disclosure can achieve rapid and accurate evaluation of the vaccine bulk to be tested and can also evaluate the batch-to-batch consistency of the vaccine bulk.

[0017] As a preferred technical solution of the present disclosure, in the evaluation method, if the quality evaluation of the vaccine bulk to be tested is of high quality risk, step (3) need not be carried out.

[0018] As a preferred technical solution of the present disclosure, the consistency evaluation model includes a quality target and an upper limit of quality variability.

[0019] As a preferred technical solution of the present disclosure, the quality target is the overall mean in the matching value statistical process parameters The upper limit of quality variability is the upper control limit UCL in the matching value statistical process parameters.

[0020] In the present invention, the quality target and the specific values of the upper limit of quality variability UCL are shown in Table 1:

[0021] Table 1

[0022]

[0023] Note: The recombinant hepatitis B vaccine bulk provided by Manufacturer 1 and Manufacturer 2 is of Saccharomyces cerevisiae matrix, Manufacturer 3 is of Hansenula polymorpha matrix, and Manufacturer 4 is of CHO cell matrix.

[0024] As a preferred technical solution of the present disclosure, the evaluation method for the batch-to-batch consistency of the vaccine bulk to be tested in step (3) includes:

[0025] According to the quality target and the upper limit of quality variability included in the consistency evaluation model, compare the distribution characteristics of the matching values of the vaccine bulk to be tested samples with the quality target and the upper limit of quality variability, and evaluate the batch-to-batch consistency and its corresponding quality risk.

[0026] As a preferred technical solution of the present disclosure, in step (1), the enrichment method includes centrifuging using a cut-off centrifuge tube to obtain a retentate. Preferably, the cut-off centrifuge tube is a 50 kD cut-off centrifuge tube, and preferably the retentate is taken to prepare an infrared detection sample.

[0027] As a preferred technical solution of the present disclosure, the preparation method of the infrared detection sample is the dry film-making method. The dry film-making method includes: spotting the retentate or filtrate on a single-sided polished silicon plate, and then drying it in a constant temperature drying oven to obtain a dry film as the infrared detection sample.

[0028] For the recombinant hepatitis B vaccine bulk solution to be evaluated in the present disclosure, the target protein it contains is a macromolecular protein, and the remaining excipients are small molecule components. Therefore, a 50 kD cut-off centrifugal tube can be used to enrich the target protein, solving the problem of low sample content and inability to perform infrared detection; at the same time, the dry film-making method is adopted to avoid the influence of water on the infrared spectrum.

[0029] As a preferred technical solution of the present disclosure, the parameters in the quality evaluation model of the recombinant hepatitis B vaccine bulk solution include the quality evaluation spectral region and the reproducibility level corresponding to the quality evaluation spectral region.

[0030] As a preferred technical solution of the present disclosure, the infrared spectrum of a control recombinant hepatitis B vaccine bulk solution sample is also included in the quality evaluation model of the recombinant hepatitis B vaccine bulk solution.

[0031] As a preferred technical solution of the present disclosure, the quality evaluation model of the recombinant hepatitis B vaccine bulk solution includes the quality evaluation models of recombinant hepatitis B vaccine bulk solutions from different manufacturers.

[0032] When evaluating the quality of the vaccine bulk solution, it is necessary to select the corresponding quality evaluation model of the recombinant hepatitis B vaccine bulk solution for the vaccine bulk solution from different manufacturers (matrices). The specific parameters included in the quality evaluation models of the recombinant hepatitis B vaccine bulk solutions corresponding to different manufacturers are shown in Table 2:

[0033] Table 2

[0034]

[0035] Currently, the recombinant hepatitis B vaccines on the market are expressed by recombinant Hansenula polymorpha, recombinant Saccharomyces cerevisiae, and recombinant CHO cells respectively. For different manufacturers, due to the different matrices they use, the infrared spectra of their intermediate product - the recombinant hepatitis B vaccine bulk solution are different. The present disclosure includes all the vaccine bulk solutions of the four currently marketed vaccines in the scope of investigation, which can avoid incorrect evaluation of the vaccine bulk solution.

[0036] Therefore, as a preferred technical solution of the present disclosure, the control recombinant hepatitis B vaccine bulk solution sample includes the recombinant hepatitis B vaccine bulk solution expressed by Saccharomyces cerevisiae, the recombinant hepatitis B vaccine bulk solution expressed by Hansenula polymorpha, and the recombinant hepatitis B vaccine bulk solution expressed by CHO cells.

[0037] In the present disclosure, the control recombinant hepatitis B vaccine bulk solution sample is a recombinant hepatitis B vaccine bulk solution sample that has been detected as true by legal methods.

[0038] For the same reference substance, such as the recombinant hepatitis B vaccine bulk solution expressed by Saccharomyces cerevisiae from the same manufacturer, when sampling for infrared detection, in order to exclude accidental errors as much as possible, usually, at least three batches of vaccine bulk solution samples are taken, and at least six infrared detection samples are prepared from one sample. That is, for the same reference sample, at least 18 infrared detection spectra can be obtained.

[0039] As a preferred technical solution of the present disclosure, the method for establishing the quality evaluation model of the recombinant hepatitis B vaccine bulk solution described in step (2) includes:

[0040] (A) Enrich the control recombinant hepatitis B vaccine bulk solution samples, prepare infrared detection samples for infrared detection, prepare multiple infrared detection samples for each control recombinant hepatitis B vaccine bulk solution sample, screen all infrared spectra, and obtain available infrared spectra;

[0041] (B) Divide the available infrared spectra into a training set and a validation set, and perform first derivative preprocessing on each infrared spectrum respectively;

[0042] (C) According to all the infrared spectra included in the training set, select at least one continuous spectral segment with small differences in the same matrix and large differences in different matrices as the quality evaluation spectral region;

[0043] (D) Perform vector normalization processing on each quality evaluation spectral region of all the infrared spectra included in the training set, and calculate the reproducibility level of each quality evaluation spectral region to obtain the primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution;

[0044] (E) First, use the cross-validation mode, calculate the matching values with other spectra in the training set using the primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution, then optimize the primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution based on the calculated matching values, and then further verify and optimize the quality evaluation model of the recombinant hepatitis B vaccine bulk solution using the validation set to obtain the final quality evaluation model of the recombinant hepatitis B vaccine bulk solution;

[0045] (F) Use the reverse validation light set to verify the quality evaluation model of the recombinant hepatitis B vaccine bulk solution, determine the critical matching value according to the output matching value and determine the quality evaluation standard;

[0046] (G) Separate the infrared spectra of the recombinant hepatitis B vaccine bulk solution samples with different manufacturers and different matrices in the model, and establish quality evaluation models for the recombinant hepatitis B vaccine bulk solution of different manufacturers respectively.

[0047] The method for establishing a quality evaluation model of the recombinant hepatitis B vaccine bulk solution provided by the present disclosure can not only establish a quality evaluation model of the recombinant hepatitis B vaccine bulk solution, but also be maintained through verification (positive verification and negative verification). When detecting the vaccine bulk solution to be tested, on the one hand, the evaluation of the vaccine bulk solution to be tested can be realized, and at the same time, it is also the maintenance of the quality evaluation model of the recombinant hepatitis B vaccine bulk solution. That is, the method provided by the present disclosure can realize the rapid and accurate evaluation of the recombinant hepatitis B vaccine bulk solution by continuously optimizing parameters.

[0048] As a preferred technical solution of the present disclosure, the available infrared spectroscopy quality inspection method satisfies the following conditions:

[0049] In the range of 1560 - 1800 cm -1 , the minimum spectral intensity ≥ 0.03, and the maximum spectral intensity ≤ 0.3;

[0050] In the range of 1560 - 1800 cm -1 , the signal-to-noise ratio is 25, and the signal / water is 50;

[0051] In the range of 1442 - 1505 cm -1 , the signal-to-noise ratio is 20, and the signal / water is 20;

[0052] In the range of 2000 - 2100 cm -1 , the maximum noise is below 0.0002;

[0053] In the range of 1837 - 1847 cm -1 , the maximum value of the water signal is below 0.0003;

[0054] In the range of 2000 - 2300 cm -1 , the interference fringes are 0.0002.

[0055] Specifically, as shown in Table 3:

[0056] Table 3

[0057]

[0058] As a preferred technical solution of the present disclosure, for the calculation of the reproducibility level described in the present disclosure, the present disclosure provides the following calculation method:

[0059] (i) Calculate the Pearson correlation factor r of two infrared spectra in each modeling spectral region using the formula shown in Equation I:

[0060]

[0061] where k is the frequency of the quality evaluation spectral region, cm -1 ; a mand b m is the average spectral intensity, a n (k) and b n (k) is the spectral intensity corresponding to the mass evaluation spectral region after vector normalization.

[0062] In the present disclosure, r ranges from [-1, 1]. When r is 1, it indicates that the two infrared spectra are completely positively correlated. When r is -1, it indicates that the two spectra are completely negatively correlated;

[0063] (ii) Calculate the spectral distance D using the formula shown in Equation II:

[0064] D = (1 - r) × 1000;

[0065] Equation II;

[0066] In the present disclosure, D ranges from [0, 2000]. When D is 0, it indicates that the two infrared spectra are completely correlated. When D is 2000, it indicates that the two spectra are completely negatively correlated;

[0067] (iii) Calculate the reproducibility level reprolevel using the formula shown in Equation III:

[0068]

[0069] where and σ are the statistics of the spectral distances pairwise correlated for the same kind (or the same batch) of recombinant hepatitis B vaccine bulk solution included in the training set - the mean value and the standard deviation;

[0070] For the recombinant hepatitis B vaccine bulk solution quality evaluation model (the model includes vaccine bulk solutions of different matrices from different manufacturers), its reproducibility level corresponds to the quality evaluation spectral region, and the reproducibility level of each quality evaluation spectral region is the maximum value of the reproducibility levels calculated for several control recombinant hepatitis B vaccine bulk solutions.

[0071] If the infrared detection spectral maps in its training set include samples of multiple batches, the reproducibility level corresponding to the quality evaluation spectral region is the maximum value of the reproducibility levels calculated for each batch of samples.

[0072] For the calculation of the matching value D NR refer to the formula shown in Equation IV:

[0073]

[0074] where reprolevel i is the reproducibility level corresponding to the i-th quality evaluation spectral region, D i is the spectral distance corresponding to the i-th quality evaluation spectral region, and n is the number of quality evaluation spectral regions.

[0075] As a preferred technical solution of the present disclosure, the samples used for the reverse verification light set include 18AA compound amino acid injection and human albumin injection.

[0076] As a preferred technical solution of the present disclosure, the critical matching value is 0.2.

[0077] The reverse verification samples include currently common and potential fake vaccine stock solution samples, as well as other samples that are easily confused with the target protein. The established model can be verified and analyzed by using the reverse verification of the fake vaccine stock solution samples.

[0078] In the present disclosure, in fact, the verification of the recombinant hepatitis B vaccine stock solution identification model includes forward verification and reverse verification. For forward verification, when establishing the quality evaluation model of the recombinant hepatitis B vaccine stock solution, by dividing the infrared spectrum into a training set and a verification set, the verification performed on the verification set is forward verification. At the same time, the infrared spectra of samples such as 18AA compound amino acid injection and human albumin injection are taken for reverse verification.

[0079] The present disclosure determines the critical matching value in a manner combining forward verification and reverse verification, which can prevent the defects of false positives or false negatives in the results output by the recombinant hepatitis B vaccine stock solution quality evaluation model. Among them, forward verification is to verify the established model with the infrared spectrum of the true recombinant hepatitis B vaccine stock solution, and the variation range of the recombinant hepatitis B vaccine stock solution product is examined through the matching value to determine the critical matching value; reverse verification is to verify the model with non-recombinant hepatitis B vaccine stock solutions not involved in the model, and the rationality of the critical matching value is examined through the matching value.

[0080] As a preferred technical solution of the present disclosure, the method for establishing the consistency evaluation model in step (3) includes: using the matching value output by the cross-validation of the recombinant hepatitis B vaccine stock solution quality evaluation model and applying the statistical process control method to establish the consistency evaluation model.

[0081] As a preferred technical solution of the present disclosure, the method for determining the parameters of the consistency evaluation model includes:

[0082] (A) Group and statistically analyze the matching values calculated when establishing the evaluation model, and calculate the median of the matching values in each group to represent the quality levels of different manufacturers;

[0083] (B) Take the average of the quality levels of different manufacturers (matrices) as the quality target;

[0084] (C) Estimate the quality discreteness with the median of the average absolute deviation;

[0085] (D) Calculate the upper limit of quality variability with the quality target and three times the quality discreteness.

[0086] For the method for determining the quality target and the upper limit of quality variability described in the present disclosure, the present disclosure provides the following determination method:

[0087] (A) Group and count the matching values, and calculate the median of each group of matching values respectively using the formula shown in Equation V to represent the quality levels of different manufacturers;

[0088]

[0089] Among them, X i med represents the median of each group of matching values, and x ij represents the jth matching value;

[0090] (B) Take the average of the quality levels of different manufacturers as the quality target, and the specific calculation method is shown in Equation VI;

[0091]

[0092] Among them, represents the quality target; X i med represents the median of each group of matching values, and m represents that there are m matching values;

[0093] (C) Estimate the quality discreteness using the median of the average absolute deviation, and the specific calculation method is shown in Equation VII;

[0094]

[0095] Among them, represents the quality discreteness, x ij represents the jth matching value, represents the average value of the matching values of the ith manufacturer (matrix);

[0096] (D) Calculate the upper limit of quality variability (UCL) using the quality target and 3 times the quality discreteness, and the specific calculation method is shown in Equation VIII;

[0097]

[0098] As a preferred technical solution of the present disclosure, the method for evaluating batch-to-batch consistency includes:

[0099] According to the quality target and the upper limit of quality variability included in the consistency evaluation model of the vaccine bulk to be tested, compare the relationship between the distribution characteristics of the matching values of the vaccine bulk sample to be tested and the quality target and the upper limit of quality variability, and evaluate the batch-to-batch consistency and its corresponding quality risks.

[0100] In the present disclosure, interval consistency actually refers to quality risk. If the interval consistency is good, there is no quality risk. If the interval consistency is poor, it indicates the existence of quality risk in the production process.

[0101] As a specific implementation manner of the present disclosure, the quality evaluation method described in the present disclosure includes:

[0102] (1) Enrich the vaccine stock solution to be tested using a 50kD cut-off centrifuge tube. If no retentate is obtained, that is, the sample does not contain macromolecules above 50kD, directly determine that the vaccine stock solution to be tested is unqualified. If retentate is obtained, that is, the sample contains macromolecules above 50kD, then prepare an infrared detection sample for infrared spectroscopy detection;

[0103] (2) Establish a quality evaluation model for recombinant hepatitis B vaccine stock solution, which includes the infrared spectrum (training set spectrum) of the control recombinant hepatitis B vaccine stock solution sample, the quality evaluation spectral region, and the reproducibility level corresponding to the quality evaluation spectral region;

[0104] (3) Verify the quality evaluation model for recombinant hepatitis B vaccine stock solution to determine the quality evaluation criteria;

[0105] (4) Analyze and calculate the infrared spectrum of the vaccine stock solution to be tested obtained using the established quality evaluation model for recombinant hepatitis B vaccine stock solution to obtain multiple matching values, and then compare the matching values with the quality evaluation criteria to achieve the quality evaluation of the vaccine stock solution to be tested through the comparison results.

[0106] As a preferred technical solution of the present disclosure, the method for analyzing and calculating the infrared spectrum of the vaccine stock solution to be tested using the quality evaluation model for recombinant hepatitis B vaccine stock solution in step (2) includes:

[0107] Calculate the matching values between the infrared spectrum of the vaccine stock solution to be tested and each infrared spectrum in the training set according to the reproducibility level included in the quality evaluation model for recombinant hepatitis B vaccine stock solution to obtain multiple matching values.

[0108] As a preferred technical solution of the present disclosure, the method for comparing the matching values with the quality evaluation criteria in step (2) includes:

[0109] Arrange the multiple matching values described in step (2) from small to large. If the first three matching values are all greater than 0.2, the quality risk of the vaccine stock solution sample to be tested is high, and an alarm should be triggered during production;

[0110] On the contrary, further use the consistency evaluation model to evaluate the batch-to-batch consistency of the vaccine stock solution to be tested and its corresponding quality risk.

[0111] When evaluating the quality of the production process of the recombinant hepatitis B vaccine bulk using the method provided by the present disclosure, since the process control is completed within the enterprise, during the evaluation process of the method described in the present disclosure, different models need to be selected for different manufacturers. For example, for the recombinant hepatitis B vaccine bulk with a CHO matrix (manufacturer 4), the quality model corresponding to the recombinant hepatitis B vaccine bulk with a CHO matrix needs to be selected, as well as the overall mean and control upper limit corresponding to the CHO matrix.

[0112] The technical solutions provided by the embodiments of the present disclosure have the following advantages compared with the prior art:

[0113] The evaluation method for the quality of the production process of the recombinant hepatitis B vaccine based on infrared spectroscopy provided by the present disclosure can solve the problems of high cost, cumbersome operation, and inability to monitor the process quality in the prior art, and at the same time fill the blank of the method for characterizing the batch-to-batch consistency of the vaccine bulk in the prior art; moreover, the method provided by the present disclosure has good specificity and high accuracy, and can quickly and accurately evaluate the quality risk and batch-to-batch consistency of the recombinant hepatitis B vaccine bulk. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The drawings here are incorporated into the specification and form a part of this specification, showing the embodiments consistent with the present disclosure, and are used together with the specification to explain the principles of the present disclosure.

[0115] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0116] Figure 1 is a flowchart of the method described in the present disclosure;

[0117] Figure 2 is an infrared spectrogram of four kinds of recombinant hepatitis B vaccine bulk obtained in Example 1 of the present disclosure;

[0118] Figure 3 is a model verification diagram obtained by forward verification and reverse verification of the recombinant hepatitis B vaccine bulk quality evaluation model provided in Example 1 of the present disclosure;

[0119] wherein, the abscissa is the sample number;

[0120] Figure 4 is a product consistency evaluation result diagram of the recombinant hepatitis B vaccine bulk of four different manufacturers.

[0121] Figure 5 is a batch-to-batch consistency evaluation result diagram of the recombinant hepatitis B vaccine bulk of different batches of manufacturer 1. Detailed implementation mode

[0122] In order to more clearly understand the above-mentioned objects, features and advantages of the present disclosure, the solutions of the present disclosure will be further described below. It should be noted that, without conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other.

[0123] Many specific details are set forth in the following description in order to fully understand the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present disclosure, rather than all of the embodiments.

[0124] Embodiment 1

[0125] This embodiment provides a method for evaluating the quality of recombinant hepatitis B vaccine bulk solution, as Figure 1 shown, including the following steps:

[0126] (1) Prepare a control sample of recombinant hepatitis B vaccine bulk solution, enrichment (select a 50 kD cut-off centrifuge tube, take 0.5 mL of the sample in the cut-off centrifuge tube, and centrifuge in a centrifuge for 1 minute), sample preparation (take 10 μL of the retentate and spot it on a 96-well silica plate, put the silica plate into an oven, select a drying temperature of 40 °C in the oven, and dry for 10 minutes), collect the infrared spectrum and screen to obtain an available infrared spectrum;

[0127] Figure 2 are the infrared spectra of recombinant hepatitis B vaccine bulk solution of 3 matrices from four manufacturers. It can be seen from the figure that the infrared spectra of recombinant hepatitis B vaccine bulk solution from different manufacturers are relatively similar.

[0128] (2) Establish a quality evaluation model for recombinant hepatitis B vaccine bulk solution and determine the quality evaluation criteria:

[0129] Divide the available infrared spectra of the recombinant hepatitis B vaccine bulk solution control sample into a training set and a validation set according to spectral representativeness, and at the same time perform first derivative preprocessing on all infrared spectra;

[0130] According to all the infrared spectra included in the training set, select at least one continuous spectral segment with small differences between the same products and large differences between different products as the quality evaluation spectral region;

[0131] Perform vector normalization processing on each quality evaluation spectral region of all the infrared spectra included in the training set, and calculate the reproducibility levels of the recombinant hepatitis B vaccine bulk solution from different manufacturers in each quality evaluation spectral region according to Formulas I to III respectively, and then take the maximum value corresponding to each quality evaluation spectral region to obtain the reproducibility level of the spectral region;

[0132] Using any one spectrum in the training set for cyclic verification, and using the parameters (quality evaluation spectral region and reproducibility level) calculated above, while calculating the matching values with other spectra in the training set according to Formula IV, a preliminary recombinant hepatitis B vaccine bulk quality evaluation model is obtained through cross-verification of the recombinant hepatitis B vaccine bulk quality evaluation model; using the validation set (positive verification) to further optimize the model parameters;

[0133] Calculating the matching values between the spectra included in the reverse verification spectral set and the spectra included in the recombinant hepatitis B vaccine bulk quality evaluation model, and determining the critical matching value and quality evaluation criteria according to the distribution of the matching values;

[0134] Figure 3 The model verification diagram obtained by positive verification and reverse verification for the recombinant hepatitis B vaccine bulk quality evaluation model. It can be confirmed from the figure that the critical matching value is 0.2, and the quality evaluation criteria confirmed thereby are:

[0135] Arrange the multiple matching values described in step (2) from small to large. If the first three matching values are all greater than 0.2, the quality risk of the vaccine bulk sample to be tested is high, and an alarm should be triggered during production;

[0136] On the contrary, further use the consistency evaluation model to evaluate the batch-to-batch consistency of the vaccine bulk sample to be tested and its corresponding quality risk;

[0137] Separate the infrared spectra of the recombinant hepatitis B vaccine bulk samples of different manufacturers and different matrices in the model, and establish a quality evaluation model for the recombinant hepatitis B vaccine bulk for different manufacturers respectively. The parameters included in the quality evaluation models corresponding to different manufacturers (matrices) are shown in Table 2.

[0138] (3) Enrich, prepare samples, and perform infrared detection on the vaccine bulk sample to be tested using the same method to obtain an infrared detection sample;

[0139] (4) According to the reproducibility level included in the recombinant hepatitis B vaccine bulk quality evaluation model, calculate the matching values between the infrared spectrum of the vaccine to be tested and each infrared spectrum in the recombinant hepatitis B vaccine bulk quality evaluation model to obtain multiple matching values;

[0140] (5) Arrange the matching values from small to large, and conduct quality evaluation of the vaccine to be tested according to the quality evaluation criteria.

[0141] Example 2

[0142] This example provides a method for evaluating the batch-to-batch consistency of vaccine bulk.

[0143] (1) On the basis of Example 1, establish a consistency evaluation model:

[0144] Using the matching values output by cross-verification of the quality evaluation models of vaccine bulk from different manufacturers, group and statistically analyze the matching values;

[0145] Calculate the above statistics according to Formulas V - VIII respectively, and establish a consistency evaluation model by applying statistical process control methods. The model parameters include quality objectives and upper limits of quality variability. The specific parameter values are shown in Table 1.

[0146] (2) Evaluate the interval consistency:

[0147] Based on the matching values output by the sample to be tested, draw a quality control chart, and obtain the relationship between its distribution characteristics and quality objectives and upper limits of quality variability to evaluate the quality risk (batch - to - batch consistency).

[0148] Verification of the evaluation method:

[0149] (1) Take 10 confirmed recombinant hepatitis B vaccine bulk solutions with different matrices, and use the method provided in Example 1 for verification. The verification results are shown in Table 4:

[0150] Table 4

[0151] Sample True / % Suspicious / % False / % Recombinant hepatitis B vaccine bulk (Saccharomyces cerevisiae) 100 0 0 Recombinant hepatitis B vaccine bulk (Hansenula polymorpha) 100 0 0 Recombinant hepatitis B vaccine bulk (CHO) 100 0 0

[0152] (2) Use human albumin solution, compound amino acid injection, hepatitis A vaccine, EV71 vaccine, HPV vaccine, glucose injection, sodium chloride injection, etc. as reverse - verification vaccine bulk solutions, and use the recombinant hepatitis B vaccine bulk solution quality evaluation model and method provided in Example 1 for verification. The verification result accuracy is shown in Table 5:

[0153] Table 5

[0154]

[0155]

[0156] As can be seen from Tables 4 - 5, the recombinant hepatitis B vaccine bulk solution quality evaluation model and quality evaluation method provided by the present disclosure can accurately evaluate the quality of the vaccine bulk solution to be tested. The evaluation method is simple, fast, and has extremely high accuracy.

[0157] (3) Use the method provided in Example 2 to evaluate the consistency of products of vaccine bulk solutions from four different manufacturers. The results are as follows:

[0158] Figure 4 is the product consistency evaluation diagram of vaccine bulk solutions from four different manufacturers. As can be seen from the figure, the batch - to - batch consistency of hepatitis B vaccine bulk solutions from Manufacturer 1 and Manufacturer 2 is significantly better than that of Manufacturer 3 and Manufacturer 4. It is known that both Manufacturer 1 and Manufacturer 2 use Saccharomyces cerevisiae matrix. From the result analysis, it is possible that producing recombinant hepatitis B vaccine bulk solution using this matrix is easier or better to control than using other matrices, and thus relatively better product consistency can be obtained.

[0159] (4) Evaluate the batch consistency of the vaccine bulk provided by Manufacturer 1 using the method provided in Example 2. The results are as follows:

[0160] Figure 5 It is a product consistency evaluation diagram of the vaccine bulk provided by Manufacturer 1. From the diagram, violation products can be immediately detected through trend changes, realizing the process quality control of recombinant hepatitis B bulk. Among them, although the batches in the circles did not trigger alarms, they can prompt timely attention during production.

[0161] It should be noted that in this disclosure, "triggering an alarm" samples should be sent for inspection in a timely manner; for new batches to be tested, if they do not trigger an alarm, their infrared spectra can also be incorporated into the consistency evaluation model to readjust the parameters of the recombinant hepatitis B vaccine bulk consistency evaluation model, further improving the representativeness and accuracy of the model.

[0162] It should be noted that in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article or device including the said element.

[0163] The above are only specific implementation manners of this disclosure, enabling those skilled in the art to understand or implement this disclosure. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of this disclosure. Therefore, this disclosure will not be limited to these embodiments described herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for evaluating the quality of the production process of recombinant hepatitis B vaccine, characterized in that, the evaluation of the production process quality includes the quality evaluation of the recombinant hepatitis B vaccine bulk solution and the batch - to - batch consistency evaluation of the recombinant hepatitis B vaccine bulk solution, and the evaluation method includes the following steps: (1) Enrich the vaccine bulk solution to be tested, prepare an infrared detection sample for infrared spectrum detection; (2) Analyze and calculate the infrared spectrum of the vaccine bulk solution to be tested obtained by using the established quality evaluation model of the recombinant hepatitis B vaccine bulk solution to obtain multiple matching values, and then compare the matching values with the quality evaluation criteria, and realize the quality evaluation of the vaccine bulk solution to be tested through the comparison results; (3) Use the consistency evaluation model to evaluate the batch - to - batch consistency of the vaccine bulk solution to be tested; wherein: the establishment method of the quality evaluation model of the recombinant hepatitis B vaccine bulk solution in step (2) includes: (A) Enrich the control recombinant hepatitis B vaccine bulk solution samples, prepare infrared detection samples for infrared detection, and prepare multiple infrared detection samples for each control recombinant hepatitis B vaccine bulk solution sample. Screen all the infrared spectra to obtain available infrared spectra. The control recombinant hepatitis B vaccine bulk solution samples are recombinant hepatitis B vaccine bulk solution samples that have been detected as genuine by legal methods; (B) Divide the available infrared spectra into a training set and a validation set, and perform first - derivative pre - processing on each infrared spectrum respectively; (C) According to all the infrared spectra included in the training set, select at least one continuous spectral segment with small differences in the same matrix and large differences in different matrices as the quality evaluation spectral region; (D) Perform vector normalization processing on each quality evaluation spectral region of all the infrared spectra included in the training set, and calculate the reproducibility level of each quality evaluation spectral region to obtain a primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution; (E) First, use the cross - validation mode, use the primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution to calculate the matching values with other spectra in the training set, then optimize the primary quality evaluation model of the recombinant hepatitis B vaccine bulk solution according to the calculated matching values, and then use the validation set to further verify and optimize the quality evaluation model of the recombinant hepatitis B vaccine bulk solution to obtain the final quality evaluation model of the recombinant hepatitis B vaccine bulk solution; (F) Use the reverse - validation light set to verify the quality evaluation model of the recombinant hepatitis B vaccine bulk solution, determine the critical matching value according to the output matching value and determine the quality evaluation criteria; (G) Separate the infrared spectra of the recombinant hepatitis B vaccine bulk solution samples of different manufacturers and different matrices in the model, and establish a quality evaluation model for the recombinant hepatitis B vaccine bulk solution for different manufacturers respectively; the consistency evaluation model in step (3) includes a quality target and a quality variability upper limit, and the determination method of the quality target and the quality variability upper limit includes: (A') Group and statistically analyze the matching values calculated in step (2), and calculate the median of each group of matching values to represent the quality levels of different manufacturers; (B') Take the average of the quality levels of different manufacturers and / or different matrices as the quality target; (C') Estimate the quality discreteness by using the median of the mean absolute deviation; (D') Calculate the quality variability upper limit by using the quality target and 3 times the quality discreteness; The evaluation method for batch - to - batch consistency described in step (3) includes: According to the quality objectives and upper limits of quality variability included in the consistency evaluation model for the vaccine bulk to be tested, compare the distribution characteristics of the matching values of the vaccine bulk to be tested samples with the quality objectives and upper limits of quality variability, and evaluate the batch - to - batch consistency and its corresponding quality risks.

2. According to the evaluation method described in claim 1, it is characterized in that in the evaluation method, if the quality evaluation of the vaccine bulk to be tested is a high quality risk, then step (3) does not need to be carried out.

3. According to the evaluation method described in claim 1, it is characterized in that The quality target is the overall mean in the matching value statistical process parameters The upper limit of the quality variability is the upper control limit UCL in the matching value statistical process parameters.

4. According to the evaluation method described in any one of claims 1 - 3, it is characterized in that in step (1), the enrichment method includes centrifuging using a cut - off centrifuge tube to obtain a retentate.

5. According to the evaluation method described in claim 4, it is characterized in that the cut - off centrifuge tube is a 50kD cut - off centrifuge tube.

6. According to the evaluation method described in claim 4, it is characterized in that in step (1), take the retentate to prepare an infrared detection sample.

7. According to the evaluation method described in claim 1, it is characterized in that the method for analyzing and calculating the infrared spectrum of the vaccine bulk to be tested using the recombinant hepatitis B vaccine bulk quality evaluation model described in step (2) includes: According to the reproducibility level included in the recombinant hepatitis B vaccine bulk quality evaluation model, calculate the matching values of the infrared spectrum of the vaccine to be tested and each infrared spectrum in the training set to obtain a plurality of matching values.

8. According to the evaluation method described in claim 1, it is characterized in that the method for comparing the matching values with the quality evaluation criteria described in step (2) includes: Arrange the plurality of matching values described in step (2) from small to large. If the first three matching values are all greater than 0.2, then the quality risk of the vaccine bulk to be tested sample is high, and an alarm should be triggered during production; On the contrary, further use the consistency evaluation model to evaluate the batch - to - batch consistency of the vaccine bulk to be tested and its corresponding quality risks.

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