A method for evaluating the quality of the HPV vaccine production process

Abstract

The present disclosure relates to a method for evaluating the quality of the production process of HPV vaccines. By using the method of infrared detection of target proteins and simultaneously establishing a quality evaluation model for HPV vaccine bulk solutions based on the infrared spectra of HPV vaccine target proteins by means of chemometric methods, the present disclosure can achieve the quality evaluation of the intermediate product - the vaccine bulk solution for preparing HPV vaccines, and can also evaluate the batch-to-batch consistency. It can be applied to the quality control of the production process of HPV vaccines, with strong specificity, low cost, simple operation, and high sensitivity to quality variability.

Description

Technical Field

[0001] The present disclosure relates to the technical field of infrared detection, and particularly to a method for evaluating the quality of the production process of HPV vaccines. Background Art

[0002] HPV vaccines mainly utilize virus-like particles assembled from internal proteins of the HPV virus to induce an immune response in the body, especially a humoral immune response, generating specific antibodies and activated T cells, thereby bringing about an immune defense effect. Currently, there are 3 types of HPV prophylactic vaccines on the global market, namely bivalent vaccines, quadrivalent vaccines, and nonavalent vaccines. The intermediate products involved in the bivalent vaccine are HPV bulk solutions of types 16 and 18; the intermediate products involved in the quadrivalent vaccine are HPV bulk solutions of types 6, 11, 16, and 18; the intermediate products involved in the nonavalent vaccine are bulk solutions of types 6, 11, 16, 18, 31, 33, 45, 52, and 58.

[0003] The above-mentioned HPV vaccine products involve a total of 9 types of vaccine bulk solutions of different valences, all of which are obtained by extracting and purifying the products using protein separation technology. These bulk solutions are processed through a formulation process to become finished preparations that meet the conditions for human use, such as injections, oral preparations, etc. Therefore, the consistency of the quality of HPV vaccine bulk solutions during the production process greatly ensures the quality of the final products.

[0004] Currently, requirements for process control in the production of human vaccines have been put forward, which are divided into three levels: whole-process quality control, control of batch-to-batch consistency, and control of target components and non-target components, that is, effectively controlling the entire production process of vaccines from three aspects: raw materials, intermediate products, and products to ensure the quality of the final products.

[0005] Currently, the identification of gene engineering recombinant protein vaccine bulk solutions mainly uses methods with high specificity such as N-terminal amino acid sequence determination and anti-idiotypic immunity to identify the target protein, and it is stipulated that "it should be measured at least once a year". Although the method of amino acid sequencing 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. Therefore, the current evaluation methods cannot perform daily inspections and process quality control during the production process, and there is currently a lack of a rapid process quality evaluation method with strong specificity but low cost and simple operation.

[0006] Infrared spectroscopy can achieve qualitative analysis of proteins, with strong specificity but low sensitivity; the content of the target protein in the HPV vaccine bulk solution is low, and the water in the HPV vaccine bulk solution has a great influence on infrared spectroscopy; and the target proteins of each HPV genotype have small differences. Therefore, conventional infrared spectroscopy methods cannot be directly used for the process quality control of HPV vaccines.

[0007] Therefore, there is an urgent need for a rapid and accurate evaluation method for the quality and batch - to - batch consistency of HPV vaccine bulk solution. Summary of the Invention

[0008] To solve the above - mentioned technical problems, the present disclosure provides a method for evaluating the quality of the HPV vaccine production process.

[0009] In a first aspect, the present disclosure provides a method for evaluating the quality of the HPV vaccine production process, where the production process quality includes the quality of the HPV vaccine bulk solution and the batch - to - batch consistency of the HPV vaccine bulk solution. The method includes the following steps:

[0010] (1) Establish an HPV vaccine bulk solution quality evaluation model using the infrared spectrum of a control HPV vaccine bulk solution sample and determine the evaluation criteria through verification of the HPV vaccine bulk solution quality evaluation model. The HPV vaccine bulk solution quality evaluation model includes the evaluation models for each type of HPV vaccine bulk solution.

[0011] (2) Calculate statistical process control parameters using the matching values obtained from cross - validation of each type of HPV bulk solution in step (1) and establish a consistency evaluation model.

[0012] (3) Enrich the target protein in the test HPV vaccine bulk solution sample, prepare an infrared detection sample for infrared spectrum detection.

[0013] (4) According to the type of the test HPV vaccine bulk solution, select the corresponding HPV vaccine bulk solution quality evaluation model to analyze and calculate the infrared spectrum of the obtained test HPV vaccine bulk solution sample, obtain multiple matching values, and then compare the matching values with the evaluation criteria to achieve the quality evaluation of the test HPV vaccine bulk solution sample through the comparison results.

[0014] (5) According to the quality evaluation results, use the matching values obtained in step (4) and further use the consistency evaluation model to achieve the batch - to - batch consistency evaluation of the HPV vaccine bulk solution.

[0015] For the application of the HPV vaccine bulk solution quality evaluation model, when analyzing a test sample, it is necessary to select according to the specific test product. For example, in the production process control of the bulk solution of a bivalent HPV vaccine product, only the HPV16 quality evaluation model and the HPV18 quality evaluation model need to be selected respectively.

[0016] The detection method provided by the present disclosure has strong specificity, low cost, and is easy to operate. 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.

[0017] The present disclosure adopts an infrared detection method and simultaneously uses a chemometric method to establish an HPV vaccine bulk solution quality evaluation model based on the infrared spectrum of the target protein in the HPV vaccine bulk solution. This can solve the evaluation difficulties caused by the small differences in target proteins of various HPV genotypes and the difficulty in characterizing the batch-to-batch consistency of bulk solutions of multiple HPV genotypes, and can achieve accurate evaluation of HPV vaccine bulk solutions of different genotypes (or manufacturers).

[0018] As a preferred technical solution of the present disclosure, the method for establishing the consistency evaluation model in step (2) includes: using the matching values output by the cross-validation of the quality evaluation models of each genotype of HPV vaccine bulk solution, and applying statistical process control methods to establish a consistency evaluation model.

[0019] As a preferred technical solution of the present disclosure, the parameters of the consistency evaluation model include a quality target and an upper limit of quality variability;

[0020] As a preferred technical solution of the present disclosure, the method for determining the parameters of the consistency evaluation model includes:

[0021] (A) Group and statistically analyze the matching values of 9 genotypes of HPV bulk solution samples, and calculate the median of the matching values of each group respectively, representing the quality level of each genotype;

[0022] (B) Take the average of the quality levels of 9 genotypes as the quality target;

[0023] (C) Estimate the quality discreteness using the median of the mean absolute deviation;

[0024] (D) Calculate the upper limit of quality variability with the quality target and 3 times the quality discreteness.

[0025] For the method for determining the quality target and the upper limit of quality variability of the present disclosure, the present disclosure provides the following determination method:

[0026] (A) Group and statistically analyze the matching values of 9 genotypes of HPV bulk solution samples, and use the formula shown in Formula I to calculate the median of the matching values of each group respectively, representing the quality level of each genotype;

[0027]

[0028] Among them, X i med represents the median of each group of matching values, and x ij represents the jth matching value;

[0029] (B) Take the average of the quality levels of 9 genotypes as the quality target, and the specific calculation method is shown in Formula II;

[0030]

[0031] Among them, represents the average quality level; X i med represents the median of each group of matching values, and m represents there are m matching values;

[0032] (C) Estimate the quality discreteness using the median of the mean absolute deviation, and the specific calculation method is shown in Equation III;

[0033]

[0034] Among them, represents the quality discreteness, and x ij represents the j-th matching value of the i-th group (HPV genotype), represents the average matching value of the i-th group;

[0035] (D) Calculate the upper limit (UCL) of the statistical process control interval using the quality target and 3 times the quality discreteness, and the specific calculation method is shown in Equation IV;

[0036]

[0037] In the present disclosure, the average quality level finally determined by the above determination method is the quality target, and the upper limit (UCL) of the statistical process control interval is the upper limit of quality variability.

[0038] As a preferred technical solution of the present disclosure, the method for evaluating the between-batch consistency of the HPV vaccine bulk solution in step (5) includes:

[0039] According to the quality target and the upper limit of quality variability included in the HPV vaccine bulk solution consistency evaluation model, compare the relationship between the distribution characteristics of the matching values of the HPV vaccine bulk solution sample to be tested and the quality target and the upper limit of quality variability, and evaluate the between-batch consistency and its corresponding quality risk.

[0040] In the present disclosure, the between-batch consistency actually refers to the quality variability. If the quality variability is small and within the limit range, the quality risk is low; if the quality variability is large and exceeds the limit, it indicates that there is a high quality risk in the production process.

[0041] As a preferred technical solution of the present disclosure, the method for establishing the HPV vaccine bulk solution quality evaluation model in step (1) includes:

[0042] (i) Enrich the target protein in the control HPV vaccine bulk solution samples of 9 genotypes, and prepare infrared detection samples for infrared detection. Multiple infrared detection samples are prepared for each control HPV vaccine bulk solution sample, and all infrared spectra are screened to obtain infrared spectra with qualified quality;

[0043] (ii) Divide the qualified infrared spectra into a training set and a validation set, and perform first derivative preprocessing on each infrared spectrum respectively;

[0044] (iii) According to all the infrared spectra included in the training set, select at least one continuous spectral segment in the target protein specific spectral region with small differences among the same HPV genotypes and large differences among different HPV genotypes as the evaluation spectral region;

[0045] (iv) Perform vector normalization processing on each evaluation spectral region of all the infrared spectra included in the training set, and calculate the reproducibility level of each evaluation spectral region to obtain the primary HPV vaccine bulk quality evaluation model;

[0046] (v) First, use the cross - validation mode. Utilize the primary HPV vaccine bulk quality evaluation model to calculate the matching values between any spectral pattern in the training set and other spectral patterns. Then, optimize the parameters of the primary HPV vaccine bulk evaluation model based on the calculated matching values. Next, further verify and optimize the parameters of the HPV vaccine bulk quality evaluation model using the validation set to obtain the optimized HPV vaccine bulk quality evaluation model;

[0047] (vi) After determining the parameters of the HPV vaccine bulk quality evaluation model, incorporate the infrared spectra of other control HPV vaccine bulk samples included in the validation set into the model to obtain the final HPV vaccine bulk quality evaluation model;

[0048] (vii) Separate the infrared spectra of the control HPV vaccine bulk samples of each genotype in the model, and establish the quality evaluation models for different genotypes of HPV vaccine bulk respectively.

[0049] The method for establishing the HPV vaccine bulk quality evaluation model provided by the present disclosure can not only realize the establishment of the HPV vaccine bulk quality evaluation model, but also be maintained through verification (forward verification and reverse verification). At the same time, when evaluating the quality of the HPV vaccine bulk sample to be tested, on the one hand, it can realize the evaluation of the HPV vaccine bulk sample to be tested, and on the other hand, it is also the maintenance of the HPV vaccine bulk quality evaluation model. That is, the method provided by the present disclosure can achieve rapid and accurate evaluation of the HPV vaccine bulk by continuously optimizing the parameters.

[0050] The evaluation model described in the present disclosure includes the quality evaluation of the HPV vaccine bulk. At this time, the infrared spectra in the model are multiple infrared spectra of 6 - type HPV vaccine bulk, 11 - type HPV vaccine bulk, 16 - type HPV vaccine bulk, 18 - type HPV vaccine bulk, 31 - type HPV vaccine bulk, 33 - type HPV vaccine bulk, 45 - type HPV vaccine bulk, 52 - type HPV vaccine bulk, and 58 - type HPV vaccine bulk, which can realize the quality evaluation of different HPV vaccine bulks, being convenient, fast, and highly accurate.

[0051] As a preferred technical solution of the present disclosure, for the same reference substance, such as the bulk solution of the HPV type 6 vaccine, when sampling for infrared detection, in order to exclude accidental errors as much as possible, usually, at least three batches of the bulk solution of the HPV type 6 vaccine are taken, and at least six infrared detection samples are prepared for one sample. That is, for the same reference sample, at least 18 infrared detection spectra can be obtained.

[0052] As a preferred technical solution of the present disclosure, the infrared spectrum quality inspection method for qualified quality satisfies the following conditions:

[0053] In the range of 1485 - 1800 cm -1 , the minimum spectral intensity ≥ 0.1, and the maximum spectral intensity ≤ 1.0;

[0054] In the range of 1585 - 1800 cm -1 , the signal-to-noise ratio is 30, and the signal / water is 80;

[0055] In the range of 1485 - 1585 cm -1 , the signal-to-noise ratio is 10, and the signal / water is 20;

[0056] In the range of 2000 - 2100 cm -1 , the maximum noise is below 0.00023;

[0057] In the range of 1837 - 1847 cm -1 , the maximum value of the water signal is below 0.0003;

[0058] In the range of 2000 - 2300 cm -1 , the interference fringes are 0.0002.

[0059] Specifically, as shown in Table 1:

[0060] Table 1

[0061]

[0062] For the calculation of the reproducibility level described in the present disclosure, the present disclosure provides the following calculation method:

[0063] (i) Calculate the Pearson correlation factor r of two infrared spectra in each modeling spectral region using the formula shown in Equation V:

[0064]

[0065] where k is the frequency of the evaluation spectral region, cm -1 ; a m and b m are the average spectral intensities, a n(k) and b n (k) is the spectral intensity corresponding to the evaluated spectral region after vector normalization.

[0066] In the present disclosure, the value range of r is [-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;

[0067] (ii) Calculate the spectral distance D using the formula shown in Equation VI:

[0068] D = (1 - r) × 1000;

[0069] Equation VI;

[0070] In the present disclosure, the range of D is [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;

[0071] (iii) Calculate the reproducibility level reprolevel using the formula shown in Equation VII:

[0072]

[0073] where and σ are the statistics of the pairwise correlated spectral distances of the same kind (or the same batch) of HPV vaccine stock solutions included in the training set - the mean value and the standard deviation;

[0074] For the HPV vaccine stock solution evaluation model, its reproducibility level corresponds to the evaluated spectral region. The reproducibility level of each evaluated spectral region is the maximum value of the reproducibility levels calculated for several different vaccine stock solutions. If the infrared detection spectra in the training set include multi - batch samples of different HPV vaccine stock solutions, the reproducibility level corresponding to each evaluated spectral region is the maximum value of the reproducibility levels calculated for each batch of samples.

[0075] For the matching value D NR The calculation is referred to the formula shown in Equation VIII:

[0076]

[0077] where reprolevel i is the reproducibility level corresponding to the i - th evaluated spectral region, D i is the spectral distance corresponding to the i - th evaluated spectral region, and n is the number of evaluated spectral regions.

[0078] As a preferred technical solution of the present disclosure, the parameters in the HPV vaccine stock solution evaluation model include: the evaluated spectral region and the reproducibility level corresponding to the evaluated spectral region.

[0079] As a preferred technical solution of the present disclosure, the specific parameters included in the HPV vaccine bulk solution evaluation model and the quality evaluation models for HPV vaccine bulk solutions of different genotypes are shown in Table 2:

[0080] Table 2

[0081]

[0082]

[0083] The present disclosure combines chemometric methods with the infrared detection method of the present disclosure to establish a rapid quality evaluation model for HPV vaccine bulk solutions. Model parameters such as the evaluation spectral region and the reproducibility level corresponding to the evaluation spectral region are stored in the model. At the same time, in cooperation with the infrared spectrum set of the control HPV vaccine bulk solution provided by the present disclosure, the method provided by the present disclosure can achieve rapid and accurate evaluation of the HPV vaccine bulk solution sample to be tested.

[0084] As a preferred technical solution of the present disclosure, the method for determining the evaluation criteria includes: verifying the HPV vaccine bulk solution evaluation model using the infrared spectra of reverse verification samples, and determining the critical matching value and the evaluation criteria according to the output matching value.

[0085] As a preferred technical solution of the present disclosure, the reverse verification samples include placental polypeptide injection, recombinant human interleukin-2 injection, and human albumin injection.

[0086] In the present disclosure, in fact, the verification of the HPV vaccine bulk solution evaluation model includes forward verification and reverse verification. For forward verification, when establishing the HPV vaccine bulk solution evaluation model, 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, samples such as water for injection, sodium chloride injection, HPV vaccine preparation excipient solution, 18AA compound amino acid injection, and human albumin injection are taken, and the obtained infrared spectra are used for reverse verification.

[0087] The present disclosure determines the critical matching value in a way that combines forward verification and reverse verification, which can prevent the defects of false positives or false negatives in the results output by the HPV vaccine bulk solution quality evaluation model. Among them, forward verification is to verify the established model using the infrared spectra of true HPV vaccine bulk solutions, and determine the critical matching value by examining the variation range of the HPV vaccine bulk solution through the matching value and the matching object; reverse verification is to verify the model using non-HPV vaccine bulk solutions not involved in the model, and examine the rationality of the critical matching value through the matching value and the matching object.

[0088] When enriching and preparing samples for reverse-phase verification or samples to be tested, when the composition of the sample does not include macromolecular substances above 30 kD, take the filtrate to prepare an infrared detection sample.

[0089] As a preferred technical solution of the present disclosure, the critical matching value is 0.9.

[0090] As a preferred technical solution of the present disclosure, the evaluation criteria are as follows:

[0091] The output matching values are arranged from small to large. If the first three matching values are all greater than 0.9, the quality risk of the HPV vaccine bulk sample to be tested is high, and an alarm should be triggered during production;

[0092] On the contrary, further use the consistency evaluation model to evaluate the batch-to-batch consistency of the HPV vaccine bulk and its corresponding quality risk.

[0093] As a preferred technical solution of the present disclosure, the control HPV vaccine bulk samples include HPV type 6 vaccine bulk, HPV type 11 vaccine bulk, HPV type 16 vaccine bulk, HPV type 18 vaccine bulk, HPV type 31 vaccine bulk, HPV type 33 vaccine bulk, HPV type 45 vaccine bulk, HPV type 52 vaccine bulk, and HPV type 58 vaccine bulk that have been confirmed as "true" and have qualified quality by legal methods.

[0094] As a preferred technical solution of the present disclosure, the enrichment method includes centrifuging using a cut-off centrifuge tube to obtain a retentate containing the target protein. Preferably, the cut-off centrifuge tube is a 30 kD cut-off centrifuge tube. Preferably, take the retentate to prepare an infrared detection sample of the target protein.

[0095] As a preferred technical solution of the present disclosure, the preparation method of the infrared detection sample of the target protein 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 in a constant temperature drying oven to obtain a dry film as the infrared detection sample.

[0096] For the HPV vaccine bulk that the present disclosure wants to evaluate, if the vaccine is true, the target protein it contains is a macromolecular protein, and the remaining excipients are small molecule components. Therefore, using a 30 kD cut-off centrifuge tube can achieve the enrichment of the target protein, solve the problem of low sample content and inability to perform infrared detection; at the same time, adopting the dry film-making method can avoid the influence of water on the infrared spectrum.

[0097] As a preferred technical solution of the present disclosure, the method for analyzing and calculating the infrared spectrum of the HPV vaccine bulk sample to be tested obtained in step (4) using the HPV vaccine bulk quality evaluation model includes:

[0098] According to the type of the HPV vaccine bulk solution to be measured, select the evaluation spectral regions and the corresponding reproducibility levels included in the corresponding quality evaluation model of the HPV vaccine bulk solution, calculate the matching values between the infrared spectrum of the HPV vaccine bulk solution sample to be measured and each infrared spectrum in the model, and obtain multiple matching values.

[0099] The technical solutions provided by the embodiments of the present disclosure have the following advantages compared with the prior art:

[0100] The evaluation method for the quality of the HPV vaccine production process based on infrared spectrum 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 solution in the prior art; moreover, the method provided by the present disclosure has good specificity and high accuracy, and can realize the rapid and accurate evaluation of the quality risk and batch-to-batch consistency of the HPV vaccine bulk solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The accompanying 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.

[0102] 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 also be obtained based on these drawings without creative efforts.

[0103] Figure 1 is a flowchart of the method described in the present disclosure;

[0104] Figure 2 is an infrared spectrogram of 9 different typed vaccine bulk solutions obtained in Embodiment 1 of the present disclosure;

[0105] Figure 3 is a model verification diagram obtained by positive verification and negative verification of the HPV vaccine bulk solution evaluation model provided in Embodiment 1 of the present disclosure;

[0106] wherein, the abscissa is the sample number;

[0107] Figure 4 is a diagram of the evaluation results of the batch-to-batch consistency of 9 typed HPV vaccine bulk solutions;

[0108] In the figure, 1-9 correspond to the 6-type HPV vaccine bulk solution, 11-type HPV vaccine bulk solution, 16-type HPV vaccine bulk solution, 18-type HPV vaccine bulk solution, 31-type HPV vaccine bulk solution, 33-type HPV vaccine bulk solution, 45-type HPV vaccine bulk solution, 52-type HPV vaccine bulk solution and 58-type HPV vaccine bulk solution in sequence. Detailed implementation manners

[0109] 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 may be combined with each other.

[0110] Many specific details are set forth in the following description in order to provide a thorough understanding of 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.

[0111] Figure 1 is a flowchart of the method provided for the present disclosure, wherein the method includes:

[0112] I. Establishment of an evaluation model for HPV vaccine bulk solution and confirmation of evaluation criteria

[0113] (i) Prepare control samples and reverse verification samples of HPV vaccine bulk solution:

[0114] Provide a variety of control samples, including HPV type 6 vaccine bulk solution, HPV type 11 vaccine bulk solution, HPV type 16 vaccine bulk solution, HPV type 18 vaccine bulk solution, HPV type 31 vaccine bulk solution, HPV type 33 vaccine bulk solution, HPV type 45 vaccine bulk solution, HPV type 52 vaccine bulk solution and HPV type 58 vaccine bulk solution that are confirmed to be genuine by legal methods; the reverse verification samples include placental polypeptide injection, recombinant human interleukin-2 injection and human albumin injection;

[0115] (ii) Enrichment and sample preparation:

[0116] The control samples of HPV vaccine bulk solution are centrifuged using a 30 kD cut-off centrifuge tube, and the retentate is spotted on a silica plate and dried; for the reverse verification samples, if there are macromolecular substances, the operation is the same as that of the control samples of HPV vaccine bulk solution to obtain infrared detection samples, and if there are no macromolecular substances, the samples are directly prepared by the dry film-forming method. At least three batches are taken for each sample, and each sample is spotted on the plate 6 times for sample preparation, that is, at least 18 infrared detection samples are prepared for each control sample;

[0117] (iii) Infrared detection and screening:

[0118] Collect the infrared spectra of the infrared detection samples by the transmission method; among them, the collection conditions of the infrared spectra are in the mid-infrared spectral range with a wavelength of 4000 - 400 cm -1 , the number of scans is 64 times, and the resolution is 4 cm -1 , and the obtained infrared spectra are screened using the screening conditions in Table 1 to obtain infrared spectra with qualified quality;

[0119] (iv) Establish a quality evaluation model for HPV vaccine bulk solution and determine evaluation criteria:

[0120] Divide the infrared spectra with qualified quality into a training set and a validation set according to the HPV vaccine bulk solution evaluation model to be established. Then, use chemometric methods to establish a quality evaluation model for HPV vaccine bulk solution from the spectra of the training set. At the same time, use the infrared spectra obtained from the validation set and reverse-phase validation samples to conduct forward and reverse validations on it, optimize the model, and obtain the quality evaluation model and evaluation criteria for HPV vaccine bulk solution;

[0121] (v) Put the infrared spectra of other HPV vaccine control samples in the validation set into the quality evaluation model of HPV vaccine bulk solution to establish the subsequent consistency model;

[0122] (vi) Separate the infrared spectra of the HPV vaccine bulk solution samples of each genotype control in the model, and establish a quality evaluation model for HPV vaccine bulk solution of different genotypes respectively.

[0123] II. Evaluate the HPV vaccine bulk solution to be tested

[0124] (vi) Enrich, prepare samples, and perform infrared detection on the HPV vaccine bulk solution samples to be tested with reference to the methods in (ii)-(iii) to obtain infrared detection samples;

[0125] (vii) Calculate the matching values between the infrared spectra of the HPV vaccine bulk solution samples to be tested and each infrared spectrum in the HPV vaccine bulk solution evaluation model according to the reproducibility level included in the quality evaluation model of each genotype of HPV vaccine bulk solution, and obtain multiple matching values and the corresponding matching objects;

[0126] (viii) Arrange the matching values from small to large. If the first three matching values are all greater than 0.9, the quality risk of the HPV vaccine bulk solution samples to be tested is high, and an alarm should be triggered during production;

[0127] On the contrary, the consistency evaluation model can be further applied in production to continuously monitor the quality risk (batch-to-batch consistency) of HPV bulk solution.

[0128] Example 1

[0129] This example provides a method for evaluating the quality of HPV vaccine bulk solution, including the following steps:

[0130] (1) Prepare HPV vaccine bulk solution control samples, enrich (select a 30kD cut-off centrifuge tube, take 0.5 ml of the sample in the centrifuge tube, and centrifuge for 1 minute in a centrifuge), prepare samples (take 10 μL of the retentate and spot it on a 96-well silicon plate, put the silicon plate into an oven, select a drying temperature of 40 °C in the oven, and dry for 10 minutes), collect and screen infrared spectra to obtain infrared spectra with qualified quality;

[0131] Figure 2 This is the infrared spectra of 9 different types of vaccine concentrates. It can be seen from the figure that the infrared spectra of the nine HPV vaccine concentrates are relatively similar.

[0132] (2) Establishing a quality evaluation model for HPV vaccine stock solution and determining evaluation criteria:

[0133] The infrared spectra of the control samples with qualified quality were divided into a training set and a validation set according to the spectral representativeness, and all the infrared spectra were preprocessed by first-order derivatives;

[0134] According to all infrared spectra included in the training set, at least one continuous spectrum segment with small differences for the same product and large differences for different products is selected as the evaluation spectrum region;

[0135] Each evaluation spectral region of all infrared spectra included in the training set is subjected to vector normalization processing, and the reproducibility level of nine different types of vaccine stock solutions in each evaluation spectral region is calculated respectively according to Formula V to Formula VII, and then the maximum value corresponding to each evaluation spectral region is taken to obtain the reproducibility level of the spectral region;

[0136] Any spectrum in the training set is used for cyclic verification, and the parameters obtained by the above calculation (evaluation spectral area and reproducibility level) are used. At the same time, the matching values ​​with other spectra in the training set are calculated according to Formula VIII, and the HPV vaccine bulk solution evaluation model is cross-validated to obtain a preliminary HPV vaccine bulk solution evaluation model; the model parameters are further optimized using the validation set (forward validation); the infrared spectra of the control HPV samples in the validation set are added to the model to obtain the HPV vaccine bulk solution quality evaluation model; the infrared spectra of the control HPV vaccine bulk solution samples of each type in the model are separated, and HPV vaccine bulk solution quality evaluation models of different types are established respectively.

[0137] Calculating the matching value of the spectra included in the reverse validation spectrum set with the spectra included in the HPV vaccine stock solution evaluation model, and determining the critical matching value and evaluation criteria according to the distribution of the matching values;

[0138] Among them, the parameters included in the HPV vaccine concentrate quality evaluation model provided in this embodiment are shown in Table 2 above.

[0139] Figure 3 The model validation diagram obtained by forward validation and reverse validation of the HPV vaccine stock solution quality evaluation model. It can be confirmed from the figure that the critical matching value is 0.9, and the evaluation criteria thus confirmed are:

[0140] Arrange the matching values ​​from small to large. If the first three matching values ​​are all greater than 0.9, the quality risk of the HPV vaccine stock solution sample to be tested is high, and an alarm should be triggered during production;

[0141] Otherwise, the consistency evaluation model can be further applied in production to continuously monitor the quality risk (batch - to - batch consistency) of HPV bulk vaccine;

[0142] (3) The HPV bulk vaccine sample to be tested is enriched, sampled, and infrared - detected using the same method to obtain an infrared - detected sample;

[0143] (4) Select the quality evaluation model corresponding to the specific type according to the product. According to the reproducibility level included in the model, calculate the matching values between the infrared spectrum of the HPV bulk vaccine sample to be tested and each infrared spectrum in the model to obtain multiple matching values;

[0144] (5) Arrange the matching values from small to large, and conduct the quality evaluation of the HPV bulk vaccine sample to be tested according to the evaluation criteria.

[0145] Example 2

[0146] This example provides a method for evaluating the batch - to - batch consistency of vaccine bulk.

[0147] (1) On the basis of Example 1, establish a consistency evaluation model:

[0148] Use the matching values output by the cross - validation of the quality evaluation models of each type of HPV vaccine bulk to statistically group the matching values of 9 types of HPV bulk samples;

[0149] Calculate the statistics of the above 9 groups respectively according to Formulas I - IV, and apply the statistical process control method to establish a consistency evaluation model. The model parameters include quality target and upper limit of quality variability;

[0150] (2) Evaluate the interval consistency:

[0151] According to the matching values output by the sample to be tested, draw a quality control chart, and obtain the relationship between its distribution characteristics and the quality target and upper limit of quality variability to evaluate the quality risk (batch - to - batch consistency).

[0152] Verification of the evaluation method:

[0153] (1) Take 10 vials each of the confirmed 6 - type HPV vaccine bulk, 11 - type HPV vaccine bulk, 16 - type HPV vaccine bulk, 18 - type HPV vaccine bulk, 31 - type HPV vaccine bulk, 33 - type HPV vaccine bulk, 45 - type HPV vaccine bulk, 52 - type HPV vaccine bulk, and 58 - type HPV vaccine bulk that are not included in the HPV vaccine bulk evaluation model, and use the method provided in Example 1 for verification. The verification results are shown in Table 3:

[0154] Table 3

[0155] Sample Matching value is less than 0.9 / % 6-valent HPV vaccine bulk solution 100 11-valent HPV vaccine bulk solution 100 16-valent HPV vaccine bulk solution 100 18-valent HPV vaccine bulk solution 100 31-valent HPV vaccine bulk solution 100 33-valent HPV vaccine bulk solution 100 45-valent HPV vaccine bulk solution 100 52-valent HPV vaccine bulk solution 100 58-valent HPV vaccine bulk solution 100

[0156] (2) Using placental polypeptide injection, recombinant human interleukin-2 injection, and human albumin injection as reverse verification vaccines, the HPV vaccine bulk solution evaluation model and method provided in Example 1 were used for verification, and the verification result accuracy is shown in Table 4:

[0157] Table 4

[0158] Sample Matching value is greater than 0.9 / % Placenta polypeptide injection 100 Recombinant human interleukin-2 injection 100 Human albumin injection 100

[0159] As can be seen from Tables 3 - 4, the HPV vaccine bulk solution evaluation model and evaluation method provided in the present disclosure can achieve rapid and accurate evaluation of the quality of HPV vaccine bulk solution.

[0160] (2) The above samples were evaluated for batch - to - batch consistency using the method provided in Example 2, and the results are as follows:

[0161] Figure 4 It is a batch - to - batch consistency evaluation diagram for 9 - type HPV vaccine bulk solutions. As can be seen from the figure, the distribution characteristics, quality targets, and positions of the upper limits of quality variability of each type of HPV vaccine bulk solution, as well as the quality risk situations of each batch; among them, although the batches in the circles did not trigger an alarm, they can prompt timely attention during production.

[0162] It should be noted that for the present disclosure, "triggering an alarm" samples should be sent for inspection in a timely manner; for new batches to be tested that do not trigger an alarm, their infrared spectra can also be incorporated into the consistency evaluation model to re - adjust the parameters of the HPV vaccine bulk solution consistency evaluation model, further improving the representativeness and accuracy of the model.

[0163] 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 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 explicitly listed, or also includes elements inherent to such process, method, article, or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of another identical element in the process, method, article, or device including the said element.

[0164] The above are only specific embodiments of the present disclosure, enabling those skilled in the art to understand or implement the present disclosure. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for evaluating the quality of the HPV vaccine production process, characterized in that, The quality of the production process includes the quality of the HPV vaccine bulk solution and the between-batch consistency of the HPV vaccine bulk solution. The method includes the following steps: (1) Establish a quality evaluation model for the HPV vaccine bulk solution using the infrared spectrum of the control HPV vaccine bulk solution sample and determine the evaluation criteria through the verification of the quality evaluation model for the HPV vaccine bulk solution. The quality evaluation model for the HPV vaccine bulk solution includes the evaluation models for the HPV vaccine bulk solution of each genotype; (2) Calculate the statistical process control parameters using the matching values obtained from the cross-validation of the HPV bulk solution of each genotype in step (1) and establish a consistency evaluation model; (3) Enrich the target protein in the HPV vaccine bulk solution sample to be tested, prepare an infrared detection sample for infrared spectrum detection; (4) According to the type of the HPV vaccine bulk solution to be tested, select the corresponding quality evaluation model for the HPV vaccine bulk solution to analyze and calculate the infrared spectrum of the HPV vaccine bulk solution sample to be tested, obtain multiple matching values, and then compare the matching values with the evaluation criteria to achieve the quality evaluation of the HPV vaccine bulk solution sample to be tested; (5) According to the quality evaluation results, use the matching values obtained in step (4) and further use the consistency evaluation model to achieve the between-batch consistency evaluation of the HPV vaccine bulk solution.

2. The method according to claim 1, characterized in that The method for establishing the consistency evaluation model in step (2) includes: using the matching values output from the cross-validation of the quality evaluation models for the HPV vaccine bulk solution of each genotype, applying the statistical process control method, and establishing a consistency evaluation model.

3. The method according to claim 2, wherein The parameters of the consistency evaluation model include the quality target and the upper limit of quality variability.

4. The method according to claim 3, characterized in that, The method for determining the parameters of the consistency evaluation model includes: (A) Group and statistically analyze the matching values of the 9 genotype HPV bulk solution samples, and calculate the median of the matching values of each group to represent the quality level of each genotype; (B) Take the average of the quality levels of the 9 genotypes as the quality target; (C) Estimate the quality discreteness using the median of the mean absolute deviation; (D) Calculate the upper limit of quality variability using the quality target and 3 times the quality discreteness.

5. The method according to claim 2, wherein The method for the between-batch consistency evaluation of the HPV vaccine bulk solution in step (5) includes: According to the quality target and the upper limit of quality variability included in the HPV vaccine bulk solution consistency evaluation model, compare the distribution characteristics of the matching values of the HPV vaccine bulk solution sample to be tested with the quality target and the upper limit of quality variability, and evaluate the between-batch consistency and its corresponding quality risk.

6. The method according to claim 1, characterized in that, The method for establishing the quality evaluation model for the HPV vaccine bulk solution in step (1) includes: (i) Enrich the target protein in the control HPV vaccine bulk solution samples of 9 genotypes, prepare infrared detection samples for infrared detection, prepare multiple infrared detection samples for each control HPV vaccine bulk solution sample, screen all infrared spectra, and obtain qualified infrared spectra; (ii) Divide the qualified infrared spectra into a training set and a validation set, and perform first derivative preprocessing on each infrared spectrum respectively; (iii) selecting at least one continuous spectral segment in the target protein-specific spectral region where the differences between the same HPV typing are small and the differences between different HPV typing are large as the evaluation spectral region based on all infrared spectra included in the training set; (iv) performing vector normalization processing on each evaluation spectral region of all infrared spectra included in the training set, and calculating the reproducibility level of each evaluation spectral region to obtain a quality evaluation model for primary HPV vaccine stock solution; (v) first using the interactive verification mode, using the primary HPV vaccine stock solution quality evaluation model to calculate the matching value between any spectrum in the training set and other spectra, then optimizing the parameters of the primary HPV vaccine stock solution evaluation model based on the calculated matching value, and then using the verification set to further verify and optimize the parameters of the HPV vaccine stock solution quality evaluation model to obtain an optimized HPV vaccine stock solution quality evaluation model; (vi) after determining the parameters of the HPV vaccine stock solution quality evaluation model, the infrared spectra of other control HPV vaccine stock solution samples included in the validation set are incorporated into the model to obtain the final HPV vaccine stock solution quality evaluation model; (vii) Separate the infrared spectra of the HPV vaccine bulk samples of each genotype in the model, and establish quality evaluation models for HPV vaccine bulk samples of different genotypes.

7. The method according to claim 6, wherein The parameters in the HPV vaccine stock solution quality evaluation model include: the quality evaluation spectrum area and the reproducibility level corresponding to the quality evaluation spectrum area.

8. The method according to claim 1, characterized in that, The method for determining the evaluation standard includes: verifying the HPV vaccine stock solution quality evaluation model using the infrared spectrum of the reverse verification sample, determining the critical matching value according to the output matching value and determining the evaluation standard.

9. The method according to claim 8, wherein The reverse verification samples include placental polypeptide injection, recombinant human interleukin 2 injection and human serum albumin injection.

10. The method according to claim 8, wherein The critical matching value is 0.

9.

11. The method according to claim 10, wherein The evaluation criteria are: the output matching values ​​are arranged from small to large, and if the first three matching values ​​are all greater than 0.9, the quality risk of the HPV vaccine stock solution sample to be tested is high, and an alarm should be triggered during production; Conversely, the consistency evaluation model is further used to evaluate the batch consistency of the HPV vaccine concentrate to be tested and its corresponding quality risks.

12. The method according to any one of claims 1-11, characterized in that, The control HPV vaccine stock samples include HPV vaccine type 6 stock, HPV vaccine type 11 stock, HPV vaccine type 16 stock, HPV vaccine type 18 stock, HPV vaccine type 31 stock, HPV vaccine type 33 stock, HPV vaccine type 45 stock, HPV vaccine type 52 stock and HPV vaccine type 58 stock, which have been confirmed as "authentic" and of qualified quality by legal methods.

13. The method according to any one of claims 1-11, characterized in that The enrichment method comprises using a cut-off centrifuge tube to perform centrifugation to obtain a retentate containing the target protein; And / or, the preparation method of the infrared detection sample of the target protein is a drying film forming method.

14. The method according to claim 13, wherein The cut-off centrifuge tube is a 30kD cut-off centrifuge tube.

15. The method according to claim 14, wherein The retentate was taken to prepare infrared detection samples of the target protein.

16. The method according to any one of claims 1-11, characterized in that, The method of analyzing and calculating the infrared spectrum of the HPV vaccine stock solution sample to be tested using the HPV vaccine stock solution quality evaluation model in step (4) comprises: According to the type of the HPV vaccine bulk solution to be tested, select the evaluation spectral regions and the corresponding reproducibility levels included in the corresponding HPV vaccine bulk solution quality evaluation model, calculate the matching values between the infrared spectrum of the HPV vaccine bulk solution sample to be tested and each infrared spectrum in the model, and obtain multiple matching values.

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