Evaluation of the Efficacy of the Vaccine Production Process in Removing Residual Host Cell DNA from the Vero Cell Rabies Vaccine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study reports This study investigated the effectiveness of key production steps in antigen recovery and DNA removal . However, the work need to be revised as a whole:

 

1.        Figure 5. Batch no get out of order

2.        The author considers using ion-exchange chromatography removed residual HCD，Whether to consider ultrafiltration to improve recovery rate in the future

3.        Whether there are differences in purification between the two batches, whether the antigens are consistent, and whether the two batches can explain the problem

Author Response

Comments 1:Figure 5. Batch no get out of order

Response 1:Thank you for pointing this out.The present study aims to assess the quality of continuous batches and perform a trend analysis to ascertain the stability of the process. The seven batches included in the study were obtained from continuous production at scale during the established cycle of this study, and the relevant tests were conducted exclusively for this study. The other batches produced during the same large-scale continuous production cycle were for other research projects and were not included in this study. Consequently, the batch no. listed in this study are accurate.

 

Comments 2:The author considers using ion-exchange chromatography removed residual HCD，Whether to consider ultrafiltration to improve recovery rate in the future.

Response 2:Thank you for pointing this out.Despite the relatively low antigen recovery rate, the production process developed in this study is suitable for large-scale and continuous production of rabies vaccines, with good stability of product quality and compliance with quality standards. Consequently, there are no immediate plans to modify the process.

 

Comments 3:Whether there are differences in purification between the two batches, whether the antigens are consistent, and whether the two batches can explain the problem

Response 3:Thank you for pointing this out.In order to ascertain the viability of the production process, two batches  were initially evaluated for glycoprotein antigen content, residual HCD content, and residual HCD fragment size distribution. The results of the two batches were found to be essentially identical and met the anticipated outcomes. Secondly, in order to confirm the large-scale and continuous production of rabies vaccines, the relevant tests were performed on several batches of products. The test results were in alignment with the established quality standards, and the dynamic trend analysis did not reveal any aberrations, which were in accordance with the anticipated outcomes. Consequently, the vaccine purification process delineated in this study is stable and amenable to control, and is capable of continuously and stably producing high-quality vaccines with optimal safety and efficacy. Additionally, it offers technical support for the optimization of vaccine production processes.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Vero cell DNA with high passage of potential oncogenes. Therefore, the concentration of residual host cell DNA (HCD) in the final product should be as low as possible. To achieve this, several purification steps are used. In this study, the effect of purification steps on antigen and HCD concentration was investigated.

The authors demonstrated the importance of the ion exchange chromatography step in obtaining a purer product. As a result, the amount of residual HCD in the final product was well below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The following comments should be corrected:

 

1. Lanes 37-38. Please provide references to support this statement.

 

2. All tables are missing the standard deviation of the data obtained. How many tests were performed on each sample?

 

3. Please combine sections 2.2.1 - 2.2.5.

 

4. Section 2.3.1. Please provide primer and probe sequences.

 

5. Please provide the ion exchange chromatography and gel filtration profiles in section 3.1. This would make the purification process more visual. In this case, was DNA quantification performed by PCR? Please specify the assay method and provide the raw data, e.g. ∆Ct.

 

6. Table 1. Please indicate in the table heading which method was used to obtain these results.

 

7. Figures 4 and 5. A line graph is not appropriate in this case. Please use a box plot for each batch.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the manuscript has been revised accordingly.

 

Comments 1: Lanes 37-38. Please provide references to support this statement.

Response 1:Thank you for pointing this out.The final vaccine product is prepared by diluting the purified solution by appropriate multiples and adding complex excipients. The residual HCD and HCD fragment distribution cannot be detected because the final product contains a large amount of carbohydrates. Therefore, this study uses the purified solution as the research object to characterize the quality of the final product, and the test data obtained in the study are also purified solution data. When preparing the final product, it is prepared according to the fixed antigen content (i.e., antigen preparation point). The purified solution determines the corresponding dilution multiple based on the antigen content test results, which is usually several times the antigen preparation point. The amount of residual HCD in the final vaccine product is calculated according to the corresponding dilution multiple. Therefore, the test results of each batch of purified solution described in this paper are converted by the dilution multiple of the corresponding batch. The results of each batch of vaccine final products are all lower than 2ng/dose. In addition, during the preparation of the final vaccine product, the HCD fragment will only cause a decrease in the concentration of each component after dilution, and the proportion of each component will not change. Therefore, the distribution results of HCD fragments in the final vaccine product are consistent with those of the purified solution.

Since the antigen preparation point involves the key process and is non-public data, this paper does not mention the specific data.

 

Comments 2: All tables are missing the standard deviation of the data obtained. How many tests were performed on each sample?

Response 2: Thank you for pointing this out.All the test results described in this paper are single test results. The ELISA test for glycoprotein antigen and the qPCR test for HCD residue are both Chinese Pharmacopoeia methods, which are also approved routine test methods for product release and have been fully validated.As for part of HCD distribution detection by capillary gel electrophoresis, this method is precedents for HCD analysis of other viral products (attached paper1), and the conditions of capillary gel electrophoresis method are exactly the same as those of plasmid purity analysis, and this methodology has also been validated under ICH Guiding Principles, and it will become the Chinese Pharmacopoeia method (attached paper 2 and 3).

 

Comments 3: Please combine sections 2.2.1 - 2.2.5.

Response 3:Thank you for pointing this out. Already combined.

 

Comments 4: Section 2.3.1. Please provide primer and probe sequences

Response 4:Thank you for pointing this out.

Probe: 5'FAM-CCTTCAAGAAGCCTTTCGCTAAGT AMRA3'

Forward primer: 5'-GCTTTCTGAGAAACTGCTCTGTGT-3'

Reverse primer: 5'-GGAAGATATTTCCTTTTTCACCATAGC-3'

 

Comments 5: Please provide the ion exchange chromatography and gel filtration profiles in section 3.1. This would make the purification process more visual. In this case, was DNA quantification performed by PCR? Please specify the assay method and provide the raw data, e.g. ∆Ct.

Response 5: Thank you for pointing this out.The purpose of this paper is to evaluate the effects of each production process link on the effective recovery of antigens and the removal of HCD, to prove the scientificity, effectiveness and stability of the production process described in this paper, and to prove its suitability for large-scale rabies vaccine production. The purification process discussed in this paper mainly involves ion exchange and gel chromatography. The relevant ion exchange and gel chromatography diagrams are attached in the uploaded attachments.

The quantitative test method for HCD is qPCR. The following is an overview of the test method. The original data package is in the uploaded attachment.

Take 10μL of nucleic acid and add 20μL of qPCRMIX. During the PCR reaction, the amount of PCR product can be detected by incorporating fluorescently labeled specific probes or fluorescent dyes. By continuously monitoring the changes in the fluorescence value in the reaction system, the changes in the amount of specific amplification products can be immediately reflected. When the fluorescence intensity released during the reaction reaches the preset threshold, the PCR cycle number (Ct value) of the system is linearly related to the logarithmic value of the amount of the starting DNA template contained in the system. Using a DNA standard with a known concentration of 10 times serial dilution, a standard curve is constructed based on the above relationship to quantitatively analyze the specific template and determine the amount of DNA residue in the sample.

 

Comments 6: Table 1. Please indicate in the table heading which method was used to obtain these results.

Response 6: Thank you for pointing this out.Already revised.

 

Comments 7: Figures 4 and 5. A line graph is not appropriate in this case. Please use a box plot for eachbatch.

Response 7: Thank you for pointing this out.

According to the WHO recommendation document WHO Guidelines for independent lot release of vaccines by regulatory authorities-TRS_978_Annex_2(attached document), trend analysis is generally drawn as a line graph, which can more intuitively show the relationship between sample test results and action limit and alert limit.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept

Reviewer 2 Report

Comments and Suggestions for Authors

The new additions to the manuscript made a big difference. The quality of the paper had improved, and all my questions were addressed. No more comments.