Maintaining the quality of vaccines through the use of standards: Current challenges and future opportunities.

An international hybrid meeting held 21-22 June 2023 in Ottawa, Canada brought together regulators, scientists, and industry experts to discuss a set of principles and best practices in the development and implementation of standards. Although the use of international standards (ISs) and international units (IUs) has been an essential part of ensuring human and animal vaccine quality in the past decades, the types and uses of standards have expanded with technological advances in manufacture and testing of vaccines. The needs of stakeholders are evolving in response to the ever-increasing complexity, diversity, and number of vaccine products as well as increasing efforts to replace animal-based potency tests with in vitro assays that measure relevant quality attributes. As such, there must be a concomitant evolution in the design and implementation of both international and in-house standards. Concomitantly, greater harmonization of regulatory expectations must be achieved through collaboration with standard-setting organizations, national control laboratories and manufacturers. Stakeholders provided perspectives on challenges and several recommendations emerged as essential to advancing agreed upon objectives.

Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals.

Next-generation sequencing (NGS) has been proven to address some of the limitations of the current testing methods for adventitious virus detection in biologics. The International Alliance for Biological Standardization (IABS), the U.S. Food and Drug Administration (FDA), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) co-organized the "3rd Conference on Next-generation Sequencing for Adventitious Virus Detection in Biologics for Humans and Animals", which was held on September 27-28, 2022, in Rockville, Maryland, U.S.A. The meeting gathered international representatives from regulatory and public health authorities and other government agencies, industry, contract research organizations, and academia to present the current status of NGS applications and the progress on NGS standardization and validation for detection of viral adventitious agents in biologics, including human and animal vaccines, gene therapies, and biotherapeutics. Current regulatory expectations were discussed for developing a scientific consensus regarding using NGS for detection of adventitious viruses. Although there are ongoing improvements in the NGS workflow, the development of reference materials for facilitating method qualification and validation support the current use of NGS for adventitious virus detection.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

Independent control of COVID-19 vaccines by EU Official Control Authority Batch Release: challenges, strengths and successes.

Vaccines have been a key tool in stemming the tide of the COVID-19 pandemic. The rapid development of effective vaccines against COVID-19, together with their regulatory approval and wide scale distribution has been achieved in an impressively short period thanks to the intense efforts of many. In parallel to vaccine development, the EU considered it important to prepare for the independent control of the COVID-19 vaccines, including testing, to help ensure that only vaccines that comply with the approved quality requirements reach the public and to help improve/increase public confidence in the vaccines. The existing EU Official Control Authority Batch Release (OCABR) system, co-ordinated by the European Directorate for the Quality of Medicines and HealthCare (EDQM), was able to effectively respond to the need, through rapid co-ordination, work-sharing, advance planning and early interaction with manufacturers, the Coalition for Epidemic Preparedness Innovation (CEPI) and regulatory authorities. The Official Medicines Control Laboratories (OMCLs) involved in the OCABR activity, using the strength of the established system in the OCABR network and adaptations to the crisis conditions, were ready to release the first COVID-19 vaccine batches, after protocol review and testing, at the time of the conditional marketing authorisation for each of the COVID-19 vaccines, with no delay for batches reaching the public. Thanks to the dedication of resources by the EU and national authorities as well as by the EDQM, this was done without impacting the release of the other vaccines and human blood and plasma derived medicinal products, essential for public health. Transparency and communication of practices were important factors to support reliance on the OCABR outcome in non-EU countries, with the goal to improve access to vaccines in Europe and beyond. An overview of the process, legal background, challenges and successes of OCABR for COVID-19 vaccines as well as a look at the international perspective and lessons learned is provided.

IABS/DCVMN webinar on next generation sequencing.

Next Generation Sequencing (NGS) is a new technology that could overcome some of the limitations of the current viral testing methods for demonstrating the absence of adventitious agents in biologics. This report is for the webinar that was organized by the International Alliance for Biological Standardization (IABS) and the Developing Countries Vaccine Manufacturers Network (DCVMN), held on July 20, 2022, as an introduction to the technical and bioinformatics concepts of NGS and to some of the strengths and limitations of using the technology for those working in vaccine production or development. The current state of scientific knowledge and readiness of NGS to replace or supplement the current viral tests was further discussed in the 3rd Conference on NGS for Adventitious Virus Detection in Biologics for Humans and Animals that was held in Rockville, Maryland, USA, on September 27-28, 2022. The application of NGS to supplement or replace current in vivo and in vitro assays in adventitious virus testing during vaccine production is promising; however, assay performance (sensitivity, specificity, and reproducibility) needs to be demonstrated, which may include laboratory and bioinformatics work. Efforts from regulatory authorities, industry, and researchers are ongoing to facilitate validation and establishment of NGS as a new method for virus detection.

Patient blood management in elective bypass cardiac surgery: A 2-step single-centre interventional trial to analyse the impact of an educational programme and erythropoiesis stimulation on red blood cell transfusion.

Anaemia and iron deficiency are frequent in patients scheduled for cardiac surgery. Perioperative patient blood management (PBM) is widely recommended in current practice guidelines. The aim of this protocol is to analyse the effect of a global perioperative PBM programme on the red blood cell (RBC) transfusion ratio, morbidities and rehabilitation score in elective cardiac surgery.This study is a prospective, single-centre trial with a 2-step protocol, A and B, as follows: A: non-drug intervention: the caregiver is given a blood management educational programme; B: drug intervention: systematic correction of perioperative iron, vitamin deficiencies, and anaemia. This study was designed to enrol 900 patients (500 in group A and 400 in group B) in a rolling period starting at anaesthesia consultation and ending 3 months after surgery. The primary objective was a 20% reduction in RBC transfusion after implementation of PBM programmes (protocol A + B) when compared to our previous transfusion ratio in the first half of 2018 (30.4% vs 38%). The secondary objectives were to evaluate the impact for each step of the study on the RBC transfusion rate, morbidity and the quality of postoperative rehabilitation.The strength of this study is its evaluation of the effect of a global PBM programme on RBC transfusion in cardiac surgery through a 2-step protocol. We aim to assess for the first time the impact of non-drug and drug interventions on RBC transfusion, comorbidities and delayed rehabilitation parameters. TRIALS REGISTRATIONS: ClinicalTrials.gov, NCT04040023: registered 29 July 2019.

Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked with 22 viruses. Specificity was demonstrated for all 22 viruses at the species level. Our method was capable of detecting and identifying adventitious viruses spiked at 104 genome copies per milliliter in a viral vaccine crude harvest and 0.01 viral genome copies spiked per cell in a cell substrate matrix. Moreover, 9 of the 11 NIH model viruses with published in vivo data were detected by HTS with an equivalent or better sensitivity (in a viral vaccine crude harvest). Our general-purpose HTS method is unbiased and highly sensitive for the detection of adventitious viruses, and has a large breadth of detection, which may obviate the need to perform in vivo testing.

Report of the second international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals.

The IABS-EU, in association with PROVAXS and Ghent University, hosted the "2nd Conference on Next Generation Sequencing (NGS) for Adventitious Virus Detection in Human and Veterinary Biologics" held on November 13th and 14th 2019, in Ghent, Belgium. The meeting brought together international experts from regulatory agencies, the biotherapeutics and biologics industries, contract research organizations, and academia, with the goal to develop a scientific consensus on the readiness of NGS for detecting adventitious viruses, and on the use of this technology to supplement or replace/substitute the currently used assays. Participants discussed the progress on the standardization and validation of the technical and bioinformatics steps in NGS for characterization and safety evaluation of biologics, including human and animal vaccines. It was concluded that NGS can be used for the detection of a broad range of viruses, including novel viruses, and therefore can complement, supplement or even replace some of the conventional adventitious virus detection assays. Furthermore, the development of reference viral standards, complete and correctly annotated viral databases, and protocols for the validation and follow-up investigations of NGS signals is necessary to enable broader use of NGS. An international collaborative effort, involving regulatory authorities, industry, academia, and other stakeholders is ongoing toward this goal.

Viral contamination in biologic manufacture and implications for emerging therapies.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

A next-generation sequencing based method for determining genetic stability in Clostridium tetani vaccine strains.

Production of tetanus and other clostridial vaccines highly depends on the stable and reproducible production of high toxin levels. This creates a need to ensure the genetic stability of seed strains. We developed a two-stage method for improved assessment of the genetic stability of Clostridium seed strains. This method is based on next-generation sequencing (NGS) of strain DNA and mapping the sequence reads to a reference sequence. The output allows analysis of global genome consistency followed, if necessary, by detailed expert judgement of potential deviations at the gene level. The limit of detection of our method is an order of magnitude better than that of the currently established pulsed-field gel electrophoresis (PFGE). Improved genetic characterization of bacterial seed lots will have a positive impact on the characterization of the production process. This will be a first step towards applying the consistency approach to vaccine batch release of established vaccines. This can contribute to the reduction and ultimately replacement of routinely used animal tests in vaccine production. This work was carried out as part of the Innovative Medicines Initiative 2 (IMI2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing).

Achieving scientific and regulatory success in implementing non-animal approaches to human and veterinary rabies vaccine testing: A NICEATM and IABS workshop report.

This two-day workshop, co-sponsored by NICEATM and IABS-NA, brought together over 60 international scientists from government, academia, and industry to advance alternative methods for human and veterinary Rabies Virus Vaccine (RVV) potency testing. On day one, workshop presentations focused on regulatory perspectives related to in vitro potency testing, including recent additions to the European Pharmacopoeia (5.2.14) that provide a scientific rationale for why in vivo methods may be less suitable for vaccine quality control than appropriately designed in vitro methods. Further presentations reviewed the role of the consistency approach to manufacturing and vaccine batch comparison to provide supportive data for the substitution of existing animal-based methods with in vitro assays. In addition, updates from research programs evaluating and validating RVV glycoprotein (G) quantitation by ELISA as an in vitro potency test were presented. On the second day, RVV stakeholders participated in separate human and veterinary vaccine discussion groups focused on identifying potential obstacles or additional requirements for successful implementation of non-animal alternatives to the in vivo potency test. Workshop outcomes and proposed follow up activities are discussed herein.

Comparative analysis of the performance of residual host cell DNA assays for viral vaccines produced in Vero cells.

Residual host cell DNA (rcDNA) from continuous cell lines used for manufacturing of biological medicinal products has been considered as safety risk. Historically, several analytical methods have been used for rcDNA quantitation including hybridization assay, Threshold® assay and quantitative polymerase chain reaction (qPCR). Sanofi Pasteur has a wealth of experience in the development of methods quantifying rcDNA in vaccines. Here, we compared the performance of our in-house assays for quantifying rcDNA in viral vaccines produced in Vero cells. Vero alpha-satellite sequence qPCR was compared with the hybridization and Threshold® assays in terms of specificity, sensitivity and precision. The impact of viral inactivation with ß-propiolactone (BPL) on rcDNA, within the vaccine production process, was also assessed. We demonstrate that the quantity of rcDNA measured is influenced by the analytical method used. Vero cell DNA-specific qPCR assay was shown to be robust with a large dynamic range and no matrix interference on a range of products. The qPCR assay demonstrated greater sensitivity and specificity versus the hybridization and Threshold® methods. Vero alpha-satellite sequence qPCR is a specific and sensitive method for the assessment of the quantity of Vero rcDNA in the highly purified vaccines.

Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation.

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG) The topics include: use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

Report of the international conference on next generation sequencing for adventitious virus detection in biologicals.

A fundamental aspect of biological product safety is to assure absence of adventitious agents in the final product. Next-generation or high-throughput sequencing (NGS/HTS) has recently demonstrated detection of viruses that were previously missed using the recommended routine assays for adventitious agent testing of biological products. This meeting was co-organized by the International Alliance for Biological Standardization (IABS) and the U.S. Food and Drug Administration (FDA) to assess the current status and discuss the readiness of NGS for adventitious virus detection in biologics. The presentations included efforts for standardization, case studies on applications in biologics, comparison with routine virus detection assays, and current regulatory thinking. Participants identified the need for standard reference reagents, well-annotated databases, large data storage and transfer capacity, personnel with relevant expertise, particularly in bioinformatics; and harmonization of international regulations for testing biologic products and reagents used for their manufacturing. We hope this meeting summary will be of value to regulators and industry for considerations of NGS applications for adventitious virus detection in biologics.

Replacement of primary monkey kidney cells by L20B cell line in the test for effective inactivation of inactivated poliovirus vaccine.

Absence of 'live' residual poliovirus in Inactivated Poliovirus Vaccine (IPV) is routinely checked using Primary Monkey Kidney Cells (PMKC). However, the increasing demand for IPV and the ethical, technical and safety issues associated with the use of non-human primates in research and quality control, has made the replacement of primary cells with an established cell line a priority, in line with the principles of the 3Rs (Replacement, Reduction and Refinement in animal testing). As an alternative to PMKC, we evaluated the L20B cell line; a mouse cell-line genetically engineered to express human poliovirus receptor, CD155. L20B is already used for the detection and diagnosis of poliovirus in clinical samples. We demonstrate the stability of L20B cells in terms of CD155 gene and receptor expression, and permissivity to polioviruses for at least 16 sequential passages. In addition, the L20B cell line was found to be at least as sensitive as PMKC in detecting the presence of 'live' poliovirus in IPV samples. Equivalence or superiority of L20B cells versus PMKCs was demonstrated for assessing the presence of residual 'live' poliovirus in formaldehyde-inactivated preparations for the three poliovirus serotypes. These results demonstrate that the L20B cell line is a suitable alternative to PMKC in IPV inactivation testing.

Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid.

Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RT-qPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an R2 from the linear regression analysis of >0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.

Validation of a PCR coupled to a microarray method for detection of mycoplasma in vaccines.

The revised section of the European, United States, and Japan Pharmacopeias on mycoplasma testing provided guidance for the set up and validation of a nucleic acid amplification technique (NAT) as an alternative method to agar culture and indicator cell culture compendial methods. The CytoInspect™ method, based on Polymerase Chain Reaction (PCR) coupled to microarray analysis, has been selected for detection and identification of mycoplasma in vaccines. To replace compendial methods, the alternative method must demonstrate equivalence in both limit of detection (LOD) and specificity compared with compendial methods. Here, we summarize the validation of the CytoInspect™ method according to current pharmacopeia requirements. Validation of the robustness, sensitivity (at least 10 colony forming units/ml) and specificity of the CytoInspect™ method are demonstrated. Likewise, a comparability study was performed to compare the LOD for CytoInspect™ compared with the previously validated LOD for compendial culture tests.

Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing.

Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC. The linearity of NGS was demonstrated at revertant concentrations equivalent to the study range of 0.25%-1.5%. NGS repeatability and intermediate precision were comparable across all tested samples, and NGS was highly reproducible, irrespective of sequencing platform or analysis software used. NGS was performed on OPV serotype 3 working seed lots and monovalent bulks (n=21) that were previously tested using MAPREC, and which covered the representative range of vaccine production. Percentages of 472-C revertants identified by NGS and MAPREC were comparable and highly correlated (r≥0.80), with a Pearson correlation coefficient of 0.95585 (p<0.0001). NGS demonstrated statistically equivalent performance to that of MAPREC for quantifying low-frequency OPV serotype 3 revertants, and offers a valid alternative to MAPREC.

Vesivirus 2117: Cell line infectivity range and effectiveness of amplification of a potential adventitious agent in cell culture used for biological production.

Vesivirus 2117 (VV-2117) has been recently described as a contaminant of cell culture operations in several biologics manufacturing facilities. VV-2117 has been poorly studied and little information exists about its biology, pathogenicity and infectivity range in cell culture settings. In this study we evaluated the potential in vitro viral infectivity of VV-2117 using a range of established mammalian cell lines from various species, the effectiveness of virus amplification in CHO-K1 cells at differing infection levels, and the relative sensitivity of two test methods (cytopathic effect [CPE] and polymerase chain reaction [PCR]) to detect infection and viral amplification. Of eight cell culture systems studied, two originating from hamster (CHO-K1 and BHK-21) and one from canine (MDCK) were positive for CPE and also showed a marked increase of viral RNA in a reverse transcriptase quantitative PCR (RT-qPCR) test. CHO-K1 cell cultures inoculated at 10, 1 and 0.1 genome copies per cell (gc/cell) showed both CPE and amplification of VV-2117 RNA, indicating that infection had occurred in these cultures. CHO-K1 cultures inoculated at 0.01, 0.001, 0.0001 and 0.00001 gc per cell showed neither CPE nor VV-2117, indicating that infection had not occurred. Therefore, the minimum dose necessary for infection of CHO-K1 cells was approximately 0.1 genome copies per cell. At any infection level where VV-2117 amplification was observed by RT-qPCR, the cultures also showed CPE. There was no low-level infection that could be detected by RT-qPCR without developing signs of CPE. However, the RT-qPCR assay appeared more sensitive in that it detected VV-2117 infection earlier than the onset of observable CPE.

Preliminary Evaluation of Next-Generation Sequencing Performance Relative to qPCR and In Vitro Cell Culture Tests for Human Cytomegalovirus.

To compare the performances of conventional in vitro indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS. When using NGS, the detection of HCMV is 10 fold more sensitive than observed using the conventional CPE endpoint method. It may be possible for qPCR to achieve the detection level demonstrated by NGS, but further optimization of the technique would be required. In addition, NGS was able to detect HCMV in the initial inoculum when it was spiked in at 10 CCID50/mL, suggesting the potential to eliminate cell culture amplification with an NGS-based assay. This study highlights the advantage of NGS as a surrogate broad-spectrum technology for the detection of adventitious agents in biologics. LAY ABSTRACT: Human cytomeglovirus (HCMV) is highly prevalent in the general population and can lead to serious health issues in both immumocompromised individuals and/or newborns. The testing of HCMV in biological materials is stipulated by multiple regulatory agencies where HCMV is a potential risk. This test involves inoculating cell lines that are susceptible to HCMV infection, incubating the cultures for 28 days, and observing the cells for signs of viral infection. Next-generation sequencing and quantitative polymerase chain reaction (qPCR) are two technologies that can potentially shorten the extended 28 day cell culture incubation. In this study, we compared the sensitivity of the compendial cell culture assay with NGS and qPCR for the detection of HCMV. Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assay. In addition, NGS was able to detect HCMV in the initial inoculum, potentially eliminating the need for cell culture amplification of the virus. Finally, sequence data generated by NGS directly demonstrate the presence of HCMV, and such information can serve as the foundation for any follow-up investigation.