2022 Volume 4 Issue 3 Pages 61-67
In the past decade, broad virus detection methods, as represented by next-generation sequencing (NGS) technology, have gained more recognition as an effective approach to assessing the virus safety of biologics such as antibody drugs, vaccines, and gene/cell therapy products. A global group was organized as a joint effort of regulatory and industry scientists from the United States and Europe to further discuss this regulatory issue and to facilitate the implementation of NGS testing in the virus safety assessment of biologics. This global activity has proactively promoted practical studies for performance evaluations of virus detection by NGS, as well as the development of analytical tools such as reference viral reagents and a virus database. However, in Japan, it is ambiguous whether this regulatory issue concerns domestic pharmaceutical companies or the regulatory body. Therefore, we conducted a questionnaire survey to gain a picture of the Japanese pharmaceutical industry’s views regarding the utilization of NGS for virus safety assessment for biologics. The survey results indicate that most respondents have little or limited experience with NGS and a passive attitude towards NGS utilization for virus safety assessments. With the ongoing revision of the relevant guideline, ICH Q5A, concerned parties in Japanese industry and regulatory body must urgently catch up with global discussions on NGS utilization for the virus safety assessment of biologics and join the international movement.
Although regulatory and industry scientists from Europe and the United States have actively facilitated discussions on implementation of the NGS-based virus safety assessment of biologics, the questionnaire survey conducted by the authors suggests that many of pharmaceutical companies in Japan have little or limited experience of NGS and passive attitude to NGS utilization for the virus safety assessment.
Most biologics, such as biopharmaceuticals, vaccines, or gene and cell therapy products, are manufactured using human- and animal-derived cell substrates and/or materials. Although various viruses are known or suspected to exist in biological tissues and fluids, it is difficult to characterize the virome precisely, for example in terms of the quantity, infectivity, or pathogenicity of each viral component. Therefore, virus safety should be ensured to protect patients from unexpected virus infection by mitigating the risk of viral contamination with an appropriate strategy based on scientific evidence. The internationally harmonized guideline, ICH Q5A, provides basic concepts for ensuring viral safety in biopharmaceuticals [1]. In Japan, gene and cell therapy products are also required to comply with the ICH Q5A concept to prevent virus contamination.
More than 2 decades have already passed since the release of ICH Q5A. Since 2019, to reflect new technologies and knowledges that have been developed over the past 2 decades, the expert working group has been discussing a revision of ICH Q5A. In this context, the use of molecular-based comprehensive virus detection techniques, such as NGS, has been adopted as one of the topics of discussion for the ICH Q5A revision. As one of the background factors raising this topic of discussion, the advent of NGS technology enabled the dramatic advancement of broad virus detection using an agnostic approach. Notably, in the case of an NGS-based characterization survey with commercially available attenuated live virus vaccines, it was unexpectedly revealed that a rotavirus vaccine was contaminated with porcine circovirus type-1 (which was presumed to be due to the porcine-derived trypsin used in the cell bank preparation by subsequent inquests) [2], and consequently, this report prompted many concerned individuals to reaffirm the usefulness of the NGS technology for virus safety assessment. In addition, risk assessment of virus contamination using an agnostic approach has become more important under the circumstances of the diversification of manufacturing platforms and increased use of biological materials without adequate use experience or characterization. Moreover, discussion about NGS implementation as an alternative to the in vivo virus test has been supported by an international pharmaceutical industry trend in which compliance with the 3Rs principle of animal experiments (Replacement, Reduction, and Refinement) is becoming increasingly important. Particularly in Europe, regulatory efforts toward 3Rs compliance are active and included in one of the regulatory science efforts by the European Medicines Agency [3]. The requirements for replacing the in vivo virus test with NGS or a related molecular-based method are also included in the European Pharmacopoeia [4, 5].
In this context, since around 2012, mainly in Europe and the United States, industry, academia, and regulatory agencies have collaborated to implement NGS-based virus safety assessments [5,6,7,8,9,10]. In these activities, participants discussed technical considerations [11, 12], conducted multicenter studies to assess the performance of NGS-based testing [5, 13], and developed a reference viral genome database and standard reference viral reagents for spiking tests [5, 14, 15], all of which could provide specific images of the NGS-based virus safety assessment in discussions for ICH Q5A revision. However, it is currently unclear whether Japanese pharmaceutical companies are participating in such international initiatives, and it is also unclear whether they intend to use NGS for virus safety assessment. Therefore, in order to determine the needs of promoting related regulatory science activities, we conducted a questionnaire survey with the Japanese pharmaceutical industry to understand the domestic situation and the extent to which Japanese pharmaceutical companies have a positive attitude towards the implementation of NGS-based virus safety assessment.
This questionnaire survey was conducted as part of the research project “Research and development of core technologies for gene and cell therapy”, which is funded by the Japan Agency for Medical Research and Development (AMED) and implemented by the National Institute of Health Sciences (NIHS), which is one of the participating institutions in the project. The survey’s target audience was members of the Biopharmaceutical Committee (the name and number of participating companies are not disclosed) of the Japanese Pharmaceutical Manufacturers Association (JPMA, http://www.jpma.or.jp). The response period was from August 5 to October 1, 2021. The questionnaire was divided into 6 sections, with a total of 52 multiple-choice questions [Supplementary Information]. All questions were made by the authors with the help from experts in virus safety and NGS technology.
We received responses from a total of 19 companies. Among them, 17 of them were adopted, and the other 2 were excluded for the following reasons: one company declined to answer because it is not involved in the manufacturing and/or development of biologic products requiring virus safety assessment in Japan, and the other responded only to section 1. The summary of each section is described below, and the results of individual questions are shown in Supplementary Information.
Involvement in virus safety assuranceIn section 1, we determined the extent to which each company surveyed is involved in virus safety within its own domestic operations. As per this survey, 15 of the 17 companies responded that they have authorized product (s) in Japan that requires virus safety assurance (Question 1-1), where biopharmaceuticals, blood-derived products, and vaccines were chosen as product modality (none of the companies have authorization of gene and cell therapy products) (Question 1-2). The other 2 companies responded “already have a specific product under development or planning to develop” and “consider potential developments in the future”, respectively (Questions 1-3), and eventually all 17 companies may have experience with virus safety-related studies.
When asked about the location (in Japan or overseas) of the testing facility where virus safety-related tests are conducted primarily, most companies responded “frequently use a facility overseas” (Questions 1-4, 1-5). Even among the 5 companies that responded “have the facility in Japan” to the question of whether they own a facility where commercial-scale cell or viral culture can be conducted, 3 companies responded “frequently use a facility overseas” and 2 companies responded “use both in Japan and overseas facilities depending on the type of product and evaluation items” (Questions 1-4, 1-5, and 1-6). These findings indicate that Japanese companies tend to use overseas facilities for virus safety-related studies. Based on this situation, it is inferred that facilities in Japan have less testing capacity and experience than overseas facilities, in addition to the fact that the testing facilities of major global outsourcing companies are in Europe and the United States. 7 companies responded that they have facilities in Japan where they can evaluate virus infectivity and/or infectivity titers, and 3 of the 7 companies responded “have a facility in Japan” (Question 1-7). However, note that some of these facilities may be used only for characterization/analysis of virus vaccines, because 4 of the 7 companies answered that they manufacture vaccines in Question 1-2.
Regarding the recruitment and/or development of human resources specialized in virus safety, 13 companies responded “providing existing employees with opportunities to learn related knowledge and skills on-job training”, while no company chose the option “recruiting and developing human resources internally specialized in virus safety”, and 2 companies chose the option “no need for human resources specialized in virus safety” (Question 1-8). In addition, one respondent commented that outsourcing virus safety-related studies was more cost-effective than conducting them in-house. From the results of section 1, it is inferred that domestic employees are not given enough opportunities to participate in discussions and/or academic investigations on virus safety, even in companies that have experience in the development of biologics such as biopharmaceuticals, vaccines, etc.
Experience in NGS utilizationIn section 2, we surveyed the experience of NGS analyses (not limited to virus safety assessment) and the facility/laboratory possession situation for NGS analyses (Fig. 1). As for the frequency of NGS utilization in operations, 7 companies selected either “constantly” or “irregularly”, while 9 companies selected either “rarely” or “never” (1 company left unanswered), grouped approximately equally in terms of experience in NGS analysis (Question 2-1). As for the purpose of NGS utilization, many respondents selected “basic research/drug discovery research” (Question 2-2), which is presumed to reflect the extent of each company’s efforts in basic research/drug discovery research. Regarding the future opportunity of NGS utilization in the respondents’ business, 8 companies responded “it will increase” (Question 2-3), and 3 of them were respondents who selected “rarely” or “never” in Question 2-1 (Fig. 1). Regarding the expected purpose of using NGS in the future, multiple companies selected not only “basic research/drug discovery research”, but also “non-clinical study”, “clinical development”, “quality assessment”, and “manufacturing process development” (Question 2-4), implying broader utilization of NGS in the future.
Frequency of conducting NGS analysis in each respondent company. The distribution of respondents for options (a) through (d) is shown: The horizontal axis represents Question 2-1 (current frequency of NGS analysis) and the vertical axis represents Question 2-3 (expectation on future opportunities of NGS analysis). The gray-shaded circles represent companies that responded “own in-house facility/laboratory where the sequencing process (wet-lab process) can be conducted” (option (a) in Question 2-5) and the bold-lined circles represent “own in-house facility/laboratory where bioinformatics analysis (dry-lab process) can be conducted” (option (a) in Question 2-8).
Regarding the possession status of the NGS analysis environment, 6 of the 9 companies with NGS analysis experience responded that they have their own facilities in which NGS analysis can be conducted, and 5 of them have facilities that are available for both sequencing (wet-lab process) and bioinformatics analysis (dry-lab process), while the other 1 company has a facility that is only available for data analysis (Questions 2-5, 2-8) (Fig. 1). Regarding NGS analysis outsourcing, 3 of 6 companies possessing in-house facilities responded “use both internal and outsourcing facilities depending on the purposes and methods”, while only 2 companies responded that both sequencing and data analysis are primarily conducted in the in-house facility (Questions 2-6 and 2-9). Regarding the type of outsourcing facilities, respondents prefer commercial companies to non-profit organizations such as academic laboratories (Questions 2-7 and 2-10). These findings indicate that Japanese companies often outsource NGS analysis. The rationale for outsourcing NGS analysis is that companies find internal NGS operations to be cost-ineffective because sequencing systems are likely to become ‘vintage’ due to the rapid update of NGS technology, and recruiting bioinformaticians and NGS system operators is expensive, given that the primary purpose of NGS is currently basic research and drug discovery. If the cost of installing and operating an NGS system decreases in the future, more companies may set up an in-house analysis environment when considering NGS for quality assessment or manufacturing process development.
Involvement in international trendsIn section 3, we surveyed each company’s understanding of international trends and intentions to use NGS for virus safety assessment (Fig. 2). Regarding the fact that the ongoing discussion for the revision of ICH Q5A includes NGS utilization for virus safety assessment, 13 companies selected either “some persons concerned in our company know that revision of the guideline is ongoing, but they may not follow the contents of the discussions” or “most persons concerned in our company may not even know that the discussion for the guideline revision is ongoing” (Question 3-1). Similarly, regarding the global activity toward the implementation of the NGS-based virus safety assessment, 14 companies selected either “some persons concerned in our company know that the activity is taking place, but they may not follow the contents of the activity” or “most persons concerned in our company may not even know that the activity is taking place” (Question 3-2). In addition, for the internal situation in the responding companies, no company responded that employees have engaged in specific discussion/consideration regarding the implementation of NGS-based virus safety assessment of biologics, and 10 companies responded that persons concerned in the company are not currently engaged in any discussion/consideration (Question 3-3). According to this survey, many domestic pharmaceutical companies are not much concerned about the regulatory issues of NGS utilization for viral safety assessment and related international trends. Although 7 companies responded “have participated in specific discussions/considerations” or “have been collecting information to begin discussions/considerations” regarding the utilization of NGS for virus safety assessment, there appears to be no relation between the status of internal discussions/considerations and understanding of international trends (Fig. 2). Moreover, 13 companies selected “not sure/difficult to answer” in response to the question of whether Japan lags behind Europe and the United States (Question 3-4). These responses may be a result of domestic pharmaceutical companies’ lack of interest in NGS utilization. As a member of the Expert Working Group, the JPMA has participated in the discussion for the revision of the ICH Q5A guideline, and it is believed that the outline of the discussion is shared with the industry through a regular debriefing seminar. However, the Japanese pharmaceutical industry is still unaware of the potential benefits of actively incorporating NGS technology into virus safety assessment.
The acceptance rate of the international discussion toward the implementation of NGS-based virus safety assessment. The distribution of respondents for options (a) through (d) is shown: The horizontal axis represents Question 3-1 (recognition rate of the discussion topics for the revision of ICH Q5A) and the vertical axis represents Question 3-2 (recognition rate of the international activity toward implementation of NGS-based virus safety assessment). The gray-shaded circles represent companies that responded that persons concerned in our company have engaged in specific discussion/consideration regarding the implementation of NGS testing, or have been gathering information to begin discussion/consideration (options (b) or (c) in Question 3-3), and the bold-lined circles represent companies that responded that they would like to have an in-house facility (or already own one), or they may consider having one depending on the situation (option (a) or (b) in the Question 3-9).
When asked about the likelihood of participation if an opportunity for open discussion was arranged in Japan, 8 companies responded “would like to participate” or “would like to participate depending on discussion topics” (Questions 3-5, 3-7). On the other hand, 2 companies responded “do not feel a need to participate”, and 7 companies responded “not sure/difficult to answer”. When it came to who should lead the discussion, 7 companies chose “Regulatory agency or regulatory science experts”. Concerning consultation with regulatory authorities, 13 companies responded “would like to consult with regulatory authorities” or “may consider consulting with regulatory authorities depending on the situation” in regard to NGS use for virus safety assessment (Questions 3-6 and 3-8). It is suggested that the domestic pharmaceutical industry anticipates the involvement of regulatory agencies.
Although there were no correlations between companies who positively consider owning an in-house facility for NGS analysis and those who understand international trends (Fig. 2), many respondents with experience in NGS analysis responded positively to the question of an in-house facility, whereas companies without experience in NGS do not appear to feel a necessity for the same (Question 3-9). When asked about the likelihood of developing an in-house reference virus database for NGS analysis, 15 companies responded “do not feel a need of in-house database/would prefer to use an external or public database” or “not sure/difficult to answer” (Question 3-10). Since the result of the NGS analysis may vary depending on the database used, respondents may be more inclined to use a well-established public database rather than assure the reliability of an in-house database.
Regarding the future demand for contract NGS testing business in Japan, 10 companies responded “a certain level of demand can be expected” (Question 3-11). Although most virus safety-related studies are outsourced to overseas facilities, respondents may begin to consider the use of NGS-based testing if domestic outsourcing companies are able to provide packaged testing services.
Virus safety assessment of cell substratesIn section 4, we asked respondents about their intention to adopt NGS for virus safety assessment of cell substrates used to manufacture biologics such as biopharmaceuticals or vaccines. We surveyed respondents’ opinions on the necessity of virus-detection assays using animals, such as in vivo virus tests and antibody-production tests, and their replacement with molecular-based methods (Fig. 3), in light of the growing international discussion on the replacement of animal tests with NGS [5, 16]. Currently, none of the respondents have any experience with NGS-based virus safety assessment with cell banks (Question 4-1). Regarding the possibility of future use, 12 companies responded either “do not have any plan to use NGS-based assessment at present” or “not sure/difficult to answer” (Question 4-2). In contrast, 5 companies responded “may consider depending on the trend of the pharmaceutical industry” (Question 4-2) and assumed that NGS would be used as an alternative to conventional tests (Question 4-3).
Opinions on substituting animal-based virus detection tests with NGS-based test. The distribution of respondents for options (a) through (d) is shown: The horizontal axis represents Question 4-5 (attitude toward consideration of alternative method) and the vertical axis represents Question 4-6 (opinions on possibility to substitute the animal tests with NGS-based test). The gray-shaded circles represent companies that responded that they may consider using NGS-based virus safety assessment for the cell bank of the products depending on the trend of the pharmaceutical industry (option (b) in Question 4-2), and the bold lined circles represent companies that responded that tests using animals should be helpful/effective to ensure virus safety or may have some significance (options (a) or (b) in Question 4-4).
Regarding the significance of animal tests, 2 companies responded “should be helpful/effective to assure virus safety” while 6 responded “may have a certain significance, though not sure about the helpfulness/effectiveness” (Question 4-4). However, no company responded “would like to continue using the animal tests required by the current ICH Q5A guideline.” 11 companies responded that they would like to consider alternative methods (Question 4-5), and many of them assumed that NGS-based method could be a viable alternative (Question 4-6) (Fig. 3). In addition, 11 companies expressed interest in participating in open discussions about NGS replacement in light of technical requirements and methods of implementation (Question 4-7).
The results of section 4 indicate that many companies are in favor of replacing animal testing with NGS. If Japan is well-informed about the ICH Q5A revision and related international discussion, the discussion environment should be more conducive to the implementation of NGS-based virus safety assessment.
Acquisition of host-cell genome sequenceIn section 5, we asked each respondent their opinion on the necessity of genome sequencing of cell substrates, and whether virus safety assessments for cell substrates would be performed by NGS. It is helpful for reliable virus detection when detecting viral contamination in cells by NGS to distinguish precisely between host cell-derived and viral sequences with consideration of non-coding regions of the cellular genome, because virus-like sequences existing in the human/animal genome may cause false positive signals. Therefore, we surveyed whether respondents viewed the acquisition of host-cell genome sequences as basic information for distinguishing cellular sequences.
When asked whether they would consider acquiring whole-genome sequences by NGS when using newly established cell substrates, many respondents responded “not sure/difficult to answer” and no company indicated an intention to do so (Questions 5-1, 5-2, and 5-6). In addition, many companies responded “not sure/difficult to answer” when asked if the sequence of non-coding region or phase information is useful for quality control of cell substrates (Questions 5-3, 5-4, 5-7, 5-8). Since these questions are difficult to answer without actual experience of virus testing with cellular samples using agnostic approaches, it can be inferred that there were few companies capable of providing sufficient scientific evidence to answer these questions.
Regarding the cost of NGS to acquire whole-genome sequence, 12 companies responded “not sure/difficult to answer” and none responded “more than 10 million JPY” (Question 5-5). Generally, outsourcing sequencing and data analysis to acquire a new precise whole-genome sequence can cost 10 millions of JPY, and this disparity in cost perception may reflect a lack of experience in NGS analysis.
NGS testing data integrityIn section 6, we surveyed the current perceptions of NGS testing data integrity. The extent to which reliability should be assured can vary depending on the intended use of NGS; however, data integrity may be an important concern when using NGS under GMP manufacturing.
Regarding the opportunity to discuss the reliability of NGS testing used for virus safety assessment, 11 companies responded, “reliability of conventional tests can basically be diverted to NGS-based testing, although discussion about NGS-specific issues is required” (Question 6-1). Additionally, for the intention to participate in an open discussion, while 2 companies responded “do not feel a need to participate”, 2 companies responded “would like to participate”, and 8 companies responded “would like to participate depending on the discussion topics” (Question 6-2). In response to the question of who should lead the discussion, 3 companies selected “pharmaceutical companies”, while 6 selected “regulatory agencies and regulatory science experts” (Question 6-3).
When asked if well-experienced outsourcing company with reliable NGS analysis can be found in Japan, 9 respondents chose “not sure/difficult to answer”, and 3 respondents responded “do not think there is one” (Question 6-4). Similarly, when asked if well-experienced outsourcing company with the purpose and technical requirements of viral safety assessment of biologics can be found in Japan, 11 respondents chose “not sure/difficult to answer”, and 5 respondents responded “do not think there is one” (Question 6-5). Currently, it seems that the Japanese pharmaceutical industry believes there are no reliable outsourcing companies for NGS-based virus safety assessment. As a possible reflection of this situation, 10 companies responded “very helpful” or “possibly helpful” to the question of whether the certification system for contracted testing companies is useful from a reliability standpoint (Question 6-6). In addition to serving as a guide for selecting outsourcing companies, the certification system may also assist NGS testing companies in acquiring regulatory literacy.
Regarding information sharing about reagents and devices that could cause background noise, 12 companies selected either “very helpful” or “possibly helpful” (Question 6-7). As several reports showed that NGS detected contaminated nucleotides derived from the reagents used in the library preparation process [17,18,19,20,21], detection of unexpected sequences may occur frequently in agnostic NGS analysis. Disclosure and sharing of the information would be beneficial in terms of reducing the time required for follow-up investigation.
As for the source material for quality assurance, it should be the “original record” initially outputted from testing instruments, but when asked about NGS, half of the respondents responded “not sure/difficult to answer” (Question 6-8). The options “dataset of unprocessed sequence reads” and “electrical signals or graphical image obtained directly from sensors in the sequencer” were selected by 5 and 1 company(ies), respectively, for responses from the other half, and the option “dataset of trimmed sequence reads” was not selected (Question 6-8). It seems impractical to keep raw electric signal or image data for the long term due to their enormous storage volume. Though we anticipate that a consensus or common knowledge on what should be considered raw data in NGS testing will emerge gradually in the future, it would be appropriate to consider unprocessed sequence reads as original raw data for the time being, assuming data integrity is maintained. Further discussion on the reliability of NGS data is required, including whether processed sequence data such as trimmed reads are considered raw data for NGS testing.
Overview and future perspectivesFrom the results of this questionnaire survey, it can be concluded that the industry as a whole is less concerned with understanding the global discussions on the ICH Q5A amendment and NGS testing implementation, although it appears that some companies are interested in using NGS for virus safety assessment.
For the 3 questions that asked about the possibility of joining an open place to discuss the use of NGS for virus safety, technical requirements, and the concept of quality assurance (Questions 3-5, 4-7, and 6-2), 3 companies responded “would like to participate positively” in at least one of the 3 questions, and 13 companies responded positively if those who chose the option of “would like to join depending on topics” were included. Awareness of NGS usage can be increased by providing open discussion opportunities with an understanding of the issues Japanese pharmaceutical companies are interested in. In addition, it is necessary to raise awareness among regulators in the field, as many companies expect regulatory authorities and/or regulatory science experts to take the lead in discussions (Questions 3-6 and 6-3).
The authors declare no conflicts of interest associated with this manuscript.
Since this article is highly useful in Japan in terms of content, a Japanese translation is available separately from the TRS. The Japanese translation is available separately from the TRS listed in the Catalyst Unit Home Page (https://www.id3catalyst.jp/e-jornal/hirasawa.pdf). The views and opinions described in this article are those of the authors and should not be attributed to any organization with which the author is employed or affiliated.
We thank the staff of the JPMA Biopharmaceutical Committee and Prof. Kazuhisa Uchida (Kyowa Kirin Co., Ltd./Graduate School of Science, Technology and Innovation, Kobe University) for their assistance with this questionnaire survey. We also thank the program supervisor and program officers of the AMED project “Research and development of core technologies for gene and cell therapy” for their professional advice.
