Technological advances in analytics, as well as scientific and regulatory knowledge and experience gained from biosimilar development/approvals over the last decade, enabled the World Health Organization (WHO) in 2022 to revise its guidelines on the evaluation of biosimilars. Among the revisions, there is more reliance on analytical and functional aspects to prove similarity (and likely fewer clinical requirements). WHO international reference standards for biosimilars provide additional confidence to regulators looking for evidence- and data-based regulatory convergence in scientific and technical measures of quality attributes. These standards serve as a benchmark for harmonizing the bioactivity or potency of biosimilars, ensuring their future sustainability. This article discusses the availability and role of WHO international reference standards throughout the product life cycle of biosimilars.

Article Complexity of serological assays and misunderstandings of WHO International Units was published on September 1, 2022 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 60, issue 10).

The first WHO International Standard and International Reference Panel for anti-SARS-CoV-2 immunoglobulin were established by the WHO Expert Committee on Biological Standardization in December, 2020. The WHO International Antibody Standards are intended to serve as global reference reagents, against which national reference preparations or secondary standards can be calibrated. Calibration will facilitate comparison of results of assays (eg, of the neutralising antibody response to candidate COVID-19 vaccines) conducted in different countries. Use of these standards is expected to contribute to better understanding of the immune response, and particularly of the correlates of protection. This Personal View provides some technical details of the WHO Antibody Standards for SARS-CoV-2, focusing specifically on the use of these standards for the evaluation of the immune response to COVID-19 vaccines, rather than other applications (eg, diagnostic or therapeutic). The explanation with regard to why rapid adoption of the standards is crucial is also included, as well as how funders, journals, regulators, and ethics committees could drive adoption in the interest of public health.

Regulatory standards for rDNA derived medicinal products put in place over 40 years ago provided a framework for moving forward with novel biotechnologies and biosimilars leading to their success as highly effective medicines. As biologicals and biosimilars are increasingly developed, licensed and used worldwide less experienced manufacturers and regulatory agencies need support in dealing with these highly complex products. This Commentary highlights the need for regulatory convergence and support, notes the critical role of GMP and draws attention to the comprehensive review by Sia Chong Hock et al. which strongly advocates improving harmonization of regulatory efforts especially in the Association of South East Asian Nations (ASEAN).

Enterovirus A71 (EV71) is one of the major causative agents of hand, foot and mouth disease (HFMD), and is sometimes associated with severe central nervous system syndromes. Vaccines against EV71 infection have been developed or are in development in several countries and few have been licensed in China. In response to requests from some of these countries, WHO convened a working group meeting in Shanghai China from 11 to 12 September 2019 to develop WHO Recommendations to assure the quality, safety and efficacy of EV71 vaccines. Meeting participants included members of the drafting group, experts from vaccine developers, manufacturers, regulators and academia. The epidemiology of EV71, as well as the development, regulation and standardization of EV71 vaccines were reviewed in the meeting. Information on R&D, manufacturing, quality control and standardization of EV71 vaccines was presented by vaccine developers, manufacturers and regulators. Based on their experience, the working group discussed the main principles that would determine WHO’s position on quality, safety and efficacy of EV71 vaccines. The working group agreed to develop WHO Recommendations to assure the quality, safety and efficacy of inactivated EV71 vaccines with a scope covering only whole virus inactivated vaccines. Other type of vaccines, such as EV71 virus-like particles (VLPs) will not be covered as they are still at the developmental stage. The outline of the document was agreed and will follow the usual style of WHO recommendations. It was also agreed to submit the draft Recommendations for review and adoption to the WHO ECBS in 2020 following discussion at a WHO informal consultation, which will include NRAs and vaccine manufacturers.

Biosimilar medicines are being increasingly developed and used worldwide. Many regulatory agencies across the globe are assessing how policy and regulation can be adapted and developed to ensure that biosimilars can enter markets successfully, without affecting the safety and efficacy of treatment. To discuss biosimilar regulation and approval across Southeast Asia, GaBI held the First ASEAN Educational Workshop on Regulation and Approval of Biosimilars/Similar Biotherapeutic Products in July 2017, in Thailand. This provided a forum to exchange knowledge on best practice and quality assessment related to biosimilar approval.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

A major environmental signal for invading pathogens is the extremely low availability of iron in mammalian tissue fluids. Normal body fluids contain high-affinity, iron-binding glycoproteins, transferrin or lactoferrin, or both. These proteins bind iron extremely tightly and ensure that no free iron is available to invading pathogens. Bacteria that multiply under these conditions to establish an infection are able to adapt to this iron-restricted environment and express mechanisms for assimilating protein-bound iron, or for acquiring it from liberated hemoglobin or heme. The ability to grow pathogenic bacteria under iron-restricted conditions is an essential step toward being able to study the metabolism of such bacteria as they multiply in vivo during infection. Growth of bacteria in an iron-restricted environment can result in considerable phenotypic changes. The best understood iron-uptake system employed is that known as siderophore-mediated iron uptake, in which the bacteria, in response to iron restriction, secrete soluble, low molecular weight, high-affinity iron-chelating compounds known as siderophores.

The most advanced regulatory processes for complex biological products have been put in place in many countries to provide appropriate regulatory oversight of biotherapeutic products in general, and similar biotherapeutics in particular. This process is still ongoing and requires regular updates to national regulatory requirements in line with scientific developments and up-to-date standards. For this purpose, strong knowledge of and expertise in evaluating biotherapeutics in general and similar biotherapeutic products, also called biosimilars, in particular is essential. Here, we discuss the World Health Organization's international standard-setting role in the regulatory evaluation of recombinant DNA–derived biotherapeutic products, including biosimilars, and provide examples that may serve as models for moving forward with nonbiological complex medicinal products. A number of scientific challenges and regulatory considerations imposed by the advent of biosimilars are described, together with the lessons learned, to stimulate future discussions on this topic. In addition, the experiences of facilitating the implementation of guiding principles for evaluation of similar biotherapeutic products into regulatory and manufacturers’ practices in various countries over the past 10 years are briefly explained, with the aim of promoting further developments and regulatory convergence of complex biological and nonbiological products.

Biotechnology and nanotechnology provide a growing number of innovator-driven complex drug products and their copy versions. Biologics exemplify one category of complex drugs, but there are also nonbiological complex drug products, including many nanomedicines, such as iron–carbohydrate complexes, drug-carrying liposomes or emulsions, and glatiramoids. In this white paper, which stems from a 1-day conference at the New York Academy of Sciences, we discuss regulatory frameworks in use worldwide (e.g., the U.S. Food and Drug Administration, the European Medicines Agency, the World Health Organization) to approve these complex drug products and their follow-on versions. One of the key questions remains how to assess equivalence of these complex products. We identify a number of points for which consensus was found among the stakeholders who were present: scientists from innovator and generic/follow-on companies, academia, and regulatory bodies from different parts of the world. A number of topics requiring follow-up were identified: (1) assessment of critical attributes to establish equivalence for follow-on versions, (2) the need to publish scientific findings in the public domain to further progress in the field, (3) the necessity to develop worldwide consensus regarding nomenclature and labeling of these complex products, and (4) regulatory actions when substandard complex drug products are identified.

The most advanced regulatory processes for complex biological products have been put in place in many countries to provide appropriate regulatory oversight of biotherapeutic products in general, and similar biotherapeutics in particular. This process is still ongoing and requires regular updates to national regulatory requirements in line with scientific developments and up-to-date standards. For this purpose, strong knowledge of and expertise in evaluating biotherapeutics in general and similar biotherapeutic products, also called biosimilars, in particular is essential. Here, we discuss the World Health Organization's international standard-setting role in the regulatory evaluation of recombinant DNA-derived biotherapeutic products, including biosimilars, and provide examples that may serve as models for moving forward with nonbiological complex medicinal products. A number of scientific challenges and regulatory considerations imposed by the advent of biosimilars are described, together with the lessons learned, to stimulate future discussions on this topic. In addition, the experiences of facilitating the implementation of guiding principles for evaluation of similar biotherapeutic products into regulatory and manufacturers' practices in various countries over the past 10 years are briefly explained, with the aim of promoting further developments and regulatory convergence of complex biological and nonbiological products.

Biotechnology and nanotechnology provide a growing number of innovator-driven complex drug products and their copy versions. Biologics exemplify one category of complex drugs, but there are also nonbiological complex drug products, including many nanomedicines, such as iron-carbohydrate complexes, drug-carrying liposomes or emulsions, and glatiramoids. In this white paper, which stems from a 1-day conference at the New York Academy of Sciences, we discuss regulatory frameworks in use worldwide (e.g., the U.S. Food and Drug Administration, the European Medicines Agency, the World Health Organization) to approve these complex drug products and their follow-on versions. One of the key questions remains how to assess equivalence of these complex products. We identify a number of points for which consensus was found among the stakeholders who were present: scientists from innovator and generic/follow-on companies, academia, and regulatory bodies from different parts of the world. A number of topics requiring follow-up were identified: (1) assessment of critical attributes to establish equivalence for follow-on versions, (2) the need to publish scientific findings in the public domain to further progress in the field, (3) the necessity to develop worldwide consensus regarding nomenclature and labeling of these complex products, and (4) regulatory actions when substandard complex drug products are identified.

Since the earliest days of biological product manufacture, there have been a number of instances where laboratory studies provided evidence for the presence of adventitious agents in a marketed product. Lessons learned from such events can be used to strengthen regulatory preparedness for the future. We have therefore selected four instances where an adventitious agent, or a signal suggesting the presence of an agent, was found in a viral vaccine, and have developed a case study for each. The four cases are: a) SV40 in polio vaccines; b) bacteriophage in measles and polio vaccines; c) reverse transcriptase in measles and mumps vaccines; and d) porcine circovirus and porcine circovirus DNA sequences in rotavirus vaccines. The lessons learned from each event are discussed. Based in part on those experiences, certain scientific principles have been identified by WHO that should be considered in regulatory risk evaluation if an adventitious agent is found in a marketed vaccine in the future.

The Decade of Vaccines Collaboration and development of the Global Vaccine Action Plan provides a catalyst and unique opportunity for regulators worldwide to develop and propose a global regulatory science agenda for vaccines. Regulatory oversight is critical to allow access to vaccines that are safe, effective, and of assured quality. Methods used by regulators need to constantly evolve so that scientific and technological advances are applied to address challenges such as new products and technologies, and also to provide an increased understanding of benefits and risks of existing products. Regulatory science builds on high-quality basic research, and encompasses at least two broad categories. First, there is laboratory-based regulatory science. Illustrative examples include development of correlates of immunity; or correlates of safety; or of improved product characterization and potency assays. Included in such science would be tools to standardize assays used for regulatory purposes. Second, there is science to develop regulatory processes. Illustrative examples include adaptive clinical trial designs; or tools to analyze the benefit-risk decision-making process of regulators; or novel pharmacovigilance methodologies. Included in such science would be initiatives to standardize regulatory processes (e.g., definitions of terms for adverse events [AEs] following immunization). The aim of a global regulatory science agenda is to transform current national efforts, mainly by well-resourced regulatory agencies, into a coordinated action plan to support global immunization goals. This article provides examples of how regulatory science has, in the past, contributed to improved access to vaccines, and identifies gaps that could be addressed through a global regulatory science agenda. The article also identifies challenges to implementing a regulatory science agenda and proposes strategies and actions to fill these gaps. A global regulatory science agenda will enable regulators, academics, and other stakeholders to converge around transformative actions for innovation in the regulatory process to support global immunization goals.

WHO Collaborating Centres (CCs) form part of an international collaborative network set up by WHO in support of its mandated programme at the country, intercountry, regional, interregional and global levels, as appropriate. As part of its mandate in the area of biologicals, WHO has broadened the scope of its work and has expanded the range of activities devoted to the establishment of international standards for vaccines. In line with global immunization goals, the need for standards for evaluation of quality, safety and efficacy of new vaccines, as well as those that have been in use for a long time, has significantly increased. Furthermore, complex issues related to new production methodologies, more sophisticated techniques for characterization and laboratory testing, and for nonclinical and clinical evaluation of vaccines have raised a number of regulatory challenges for WHO when requested to assist its Member States. In this context, CCs in the area of standardization of vaccines and biotherapeutics (excluding blood products) have provided technical assistance and have broadened the scope of their work over time. In the area of standardization and regulatory evaluation of vaccines, WHO currently has six CCs as well as one candidate centre for which the designation process has been initiated and a further three candidate centres with great potential. The purpose of the meeting held on 24-26 April 2012 was to improve understanding of WHO's priorities in setting standards, to facilitate their implementation, and to increase transparency of the roles and responsibilities of CCs. The meeting was also an excellent opportunity to explore possibilities for improving collaboration between WHO and CCs, as well as among CCs themselves by working as a CC network. All CCs expressed a wish for increased interaction, information-sharing, collaboration and other ways of working together that may lead to cross-fertilization between the CCs. Synergy was recognized as a significant mechanism for leveraging existing resources in responding to global public health challenges and in addressing WHO's priorities. Agreement was reached for operating as a network of CCs.

Serogroup B Neisseria meningitides (MenB) is a significant cause of endemic and epidemic outbreaks of the disease worldwide. Although polysaccharide and conjugate vaccines are available against other meningococcal serogroups, the poor immunogenicity of MenB polysaccharide has led to the development of protein-based vaccines. However, the diversity and antigenic variability of MenB strains has been a major challenge. Recently a new generation of MenB vaccines that contain conserved antigens has been developed to provide broader coverage and they are in an advanced stage of development and regulatory consideration. In October 2011, the World Health Organization and Health Canada jointly organized a consultation on regulatory considerations for the evaluation and licensing of new MenB vaccines. The aim was to seek consensus on key regulatory issues relevant to the evaluation of candidate MenB vaccines and on approaches to the standardisation of in vitro assays used in the evaluation process. Participants agreed that functional antibodies as measured in the Serum Bactericidal Activity (SBA) assay could be used to evaluate MenB vaccine efficacy and ways of improving assay standardization proposed. Approaches to bridging SBA data to large collections of strains in order to give an indication of the prospective breadth of vaccine coverage were discussed.

Influenza and Other Respiratory VirusesVolume 5, Issue 6 p. 438-442 MEETING REPORTOpen Access Confronting the next pandemic—Workshop on lessons learned from potency testing of pandemic (H1N1) 2009 influenza vaccines and considerations for future potency tests, Ottawa, Canada, July 27–29, 2010 Stephanie Hardy, Stephanie Hardy Health Canada, Biologics and Genetic Therapies Directorate, Ottawa, ON, Canada.Search for more papers by this authorMaryna Eichelberger, Maryna Eichelberger US Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD, USA.Search for more papers by this authorElwyn Griffiths, Elwyn Griffiths Health Canada, Biologics and Genetic Therapies Directorate, Ottawa, ON, Canada.Search for more papers by this authorJerry P. Weir, Jerry P. Weir US Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD, USA.Search for more papers by this authorDavid Wood, David Wood World Health Organization, Geneva, Switzerland.Search for more papers by this authorClaudia Alfonso, Claudia Alfonso World Health Organization, Geneva, Switzerland.Search for more papers by this author Stephanie Hardy, Stephanie Hardy Health Canada, Biologics and Genetic Therapies Directorate, Ottawa, ON, Canada.Search for more papers by this authorMaryna Eichelberger, Maryna Eichelberger US Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD, USA.Search for more papers by this authorElwyn Griffiths, Elwyn Griffiths Health Canada, Biologics and Genetic Therapies Directorate, Ottawa, ON, Canada.Search for more papers by this authorJerry P. Weir, Jerry P. Weir US Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD, USA.Search for more papers by this authorDavid Wood, David Wood World Health Organization, Geneva, Switzerland.Search for more papers by this authorClaudia Alfonso, Claudia Alfonso World Health Organization, Geneva, Switzerland.Search for more papers by this author First published: 07 April 2011 https://doi.org/10.1111/j.1750-2659.2011.00250.xCitations: 27AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Please cite this paper as: Hardy et al. (2011) Confronting the next pandemic—Workshop on lessons learned from potency testing of pandemic (H1N1) 2009 influenza vaccines and considerations for future potency tests, Ottawa, Canada, July 27–29, 2010. Influenza and Other Respiratory Viruses 5(6), 438–442. Introduction The 2009 H1N1 influenza pandemic created an urgent need to expedite vaccine manufacture, bringing to light some of the challenges associated with the methods and processes used in the regulatory release of influenza vaccines. The release of vaccines on a lot-by-lot basis is required by most national regulatory authorities to confirm that distributed product meets pre-approved specifications. The single radial immunodiffusion (SRID) assay has been the standard assay to measure the potency of influenza vaccines for decades. In the context of a public health emergency, the suitability of the SRID assay has been questioned, and during the 2009 H1N1 influenza pandemic, alternative approaches were used in some cases at the beginning of the campaign for product release. A workshop was jointly organized by Health Canada, the United States Food and Drug Administration (FDA), and the World Health Organization (WHO) in July 2010 with the specific goals to exchange knowledge and experience gained in vaccine release around the world throughout the 2009 influenza pandemic; formulate plans to address gaps in our knowledge about the use of alternative approaches to assess the potency of influenza vaccines; and identify ways forward on possible incorporation of such assays into pandemic and seasonal influenza vaccine regulatory decision making. Scientists from national regulatory agencies, national control laboratories, research institutes, academia, WHO, and the vaccine industry were invited to attend. The meeting was structured as a series of short presentations followed by open discussion. Day three consisted of a closed session that excluded individuals with conflicts of interest. This publication contains the collective views of meeting participants and does not necessarily represent policies or recommendations of the WHO. Lessons learned from the pandemic (H1N1) 2009 experience Testing for potency Dr. Robert Webster opened the session with an overview of the advantages and shortcomings of influenza vaccine characterization, highlighting the need for surveillance of influenza in swine to identify new strains with pandemic potential. Dr. Othmar Engelhardt (National Institute for Biological Standards and Control), Dr. Rajesh Gupta (FDA), Dr. Michael Pfleiderer (European Medicines Agency), Dr. Junzhi Wang (National Institute for the Control of Pharmaceutical and Biological Products (NICPBP)), and Mr. Tony Colegate (International Federation of Pharmaceutical Manufacturers and Associations) then presented on lessons learned from potency testing of H1N1 2009 pandemic influenza vaccines using SRID and alternative methods. The SRID assay is based on diffusion of virus antigen through agarose gel containing hemagglutinin (HA)-specific antibodies and subsequent formation of an antigen–antibody precipitate, with the area of the zone of precipitation proportional to antigen concentration. An overview of this method and its history is included in a review by Williams.1 In 1979, the WHO recommended that SRID be used to standardize influenza vaccine potency and it has been used ever since. Advantages of the SRID assay are that it measures the quantity of antigenic HA and has been shown to correlate with HAI titer and vaccine efficacy, although it is unclear whether correlation is maintained with all strains.2–4 The assay is simple and cheap and is type and subtype specific. Disadvantages of SRID include the time required to produce the reference reagents (6–8 weeks). In addition, the assay is unsuitable for certain adjuvanted vaccines, is not sensitive enough for very low–dose vaccines, and does not lend itself to automation. Generation and calibration of SRID reference reagents is a complex and time-consuming process and was a challenge to accomplish in a short time frame during the H1N1 2009 pandemic. In addition, there were difficulties in the purification of HA from the 2009 H1N1 virus that is used to immunize sheep as a source of reference antiserum. This was further complicated because the initial candidate vaccine virus had a low yield. A new strain had higher yield; however, strain-specific reference reagents were not immediately available. While homologous reference antigen is important for specificity of the assay,5 the FDA allowed the use of heterologous reagents that were appropriately re-calibrated for the new strain to expedite vaccine availability. When homologous antigen became available, it was demonstrated that similar results were obtained with this standard and the re-calibrated reagents. In some countries, H1N1 vaccine clinical trial materials were produced by the end of June 2009, while the H1N1 virus-specific reference reagents only became available at the end of July 2009. Alternative methods such as reverse-phase (RP) HPLC or SDS-PAGE were used in some cases to quantify HA for early lot release and immunogenicity studies. SRID is indicative of native HA structure6 and preliminary studies show that methods which measure the absolute quantity of HA, such as HPLC or SDS-PAGE, do not accurately measure antigenicity and stability and do not distinguish between native and denatured forms of HA. European Medicines Agency (EMA) guidelines for pandemic influenza vaccines encourage the development of alternative methods for antigen standardization to bridge the phase when no such reagents are available. During the development of H5N1 mock-up vaccines, several companies presented concepts for alternative potency testing. However, the availability of SRID reagents, absence of guidance, and lack of coordination to develop new methods decelerated or stopped these research initiatives, and all pivotal H5N1 trials have been performed based on SRID potency data. Only one clinical trial of monovalent pandemic H1N1 vaccine was initiated based on potency determination using HPLC as an alternative method in Europe. Retesting with standard reagents demonstrated an underestimation of HA content, so there were doubts whether these data should be used to recommend a single dose regimen. All other trials started after SRID reference reagents became available. In China, the State Food and Drug Administration (SFDA) first granted regulatory registration to pandemic (H1N1) 2009 vaccines in August 2009. This was approximately 1 month earlier than any other country. Early approval was because of the use of alternative methods that were established as part of H5N1 vaccine development efforts, including virus strain evaluation using RT-PCR and determination of HA content by SDS-PAGE and total protein measurement. All ten manufacturers of pandemic (H1N1) 2009 influenza vaccine in China applied SDS-PAGE to measure HA content. Results generated from SDS-PAGE achieved 88–120% consistency with those from traditional SRID methods.7 China may use SDS-PAGE in a future emergency situation, although it is recognized this method has limitations because it is not indicative of native protein structure. While SDS-PAGE was proven valuable in the initial assessment of monovalent pandemic vaccine lots, it is not suitable for trivalent vaccines because of the inability of the method to distinguish between types and subtypes. Testing for stability Observations from multiple manufacturers and regulators suggest that the pandemic (H1N1) 2009 influenza strain is less stable than most seasonal strains. The root cause of the poor stability is unknown and investigations are ongoing. Potential contributing factors include the inherent structural properties of the A/California/07/2009-like HA[8]* the formation of aggregates unique to this strain, or the potential effect of total protein concentration, inactivating agents, or vaccine preservatives on stability. In Canada, real-time stability data supporting an 18-month shelf life for an H5N1 mock-up vaccine (GSK) were used to justify a preliminary 18-month shelf life for the Arepanrix H1N1 antigen, with a post-market commitment to provide ongoing accelerated and real-time testing data to confirm the shelf life. Analysis of field samples showed HA levels below the level predicted for an 18-month shelf life. No irregularities in cold chain management or sample handling were detected and the issue did not appear to be lot-specific. The impact of transportation has not been ruled out as a potential cause of the loss of potency. Based on data collected post-authorization, Health Canada determined that the shelf life should be revised to 6 months. Collection of real-time stability data is ongoing. In the United States, strain change supplements to existing seasonal influenza vaccine licenses were approved for the pandemic (H1N1) 2009 monovalent formulations. Data to support vaccine stability were not required for approval, and shelf life was expected to be the same as the seasonal licensed vaccine. However, problems with vaccine stability resulted in various recalls or field corrections of some inactivated as well as live attenuated pandemic (H1N1) 2009 influenza vaccine lots between December 2009 and February 2010. Potential instability of pandemic (H1N1) 2009 monovalent vaccine raises concern regarding stockpiled H5N1 vaccines. Long-term stability results for stockpiled H5N1 pandemic influenza vaccines in the United States suggest that there are product- and strain-specific differences in stability. A study to compare an enzyme-linked immunosorbent assay (ELISA) with SRID as potency assay is ongoing in the laboratory of Dr Jerry Weir (FDA) and will evaluate whether these methods correlate with in vivo measures of immunogenicity. Alternative assays for evaluation of quality Biological/immunological assays Antisera used to evaluate vaccine composition are usually strain and subtype specific to facilitate the analysis of trivalent vaccine formulations. In contrast, pandemic vaccines are likely to be monovalent, as was the case for the (H1N1) 2009 vaccine. Evaluation of the quality and total concentration of HA or NA in these vaccines may be expedited through the use of antibodies to conserved regions of these antigens. A collaborative study between Health Canada, Canada's National Microbiology Laboratory, and China's SFDA/NICPBP was conducted to generate antibodies to conserved HA and NA peptides that can easily be applied to analyze vaccine content. Antisera and monoclonal antibodies generated in response to immunization with conserved peptides bound to virtually all subtypes of HA and NA and could be used in a variety of immunoassays, such as ELISA, slot blot, and Western blot.9,10 The immunoassays allow the quantification of the total amount of either HA or NA in a vaccine sample but do not distinguish between different subtypes nor necessarily distinguish between biologically active and inactive antigen. Assays with these cross-reactive antibodies have practical value for quality control testing of in-process and monovalent bulk samples because there is no need to generate new strain-specific reagents. Antibodies that inhibit NA activity reduce virus titers and disease symptoms in animal models of highly pathogenic viruses.11,12 Even though clinical studies have demonstrated NA-inhibiting (NI) titers correlate with vaccine efficacy,13,14 routine measurement of antibodies with this specificity is often not performed as the traditional NA inhibition assay, the thiobarbituric acid (TBA) method,15 is not practical for routine use. To consider NA as a relevant vaccine antigen from a regulatory perspective, a practical assay to measure responses to NA has to be developed. A miniaturized TBA method has been developed as well as an enzyme-linked lectin assay (ELLA). Currently, there is no requirement to determine the amount of NA in vaccines. While licensed vaccines contain NA, recent data show that the amount present in vaccines from different manufacturers using the same vaccine seeds may not be the same and can even vary among different lots from the same manufacturer.16 Multiple factors may account for low levels of NA in split-virion-inactivated vaccine preparations, including the viral strain, and different manufacturing processes. A method to measure the potency of NA is therefore also needed. This method should quantify native conformation of NA because correct tertiary structure is required for the induction of NA-inhibiting antibodies. Potency assays that are being considered include the measurement of enzyme activity or quantitation of NA with native conformation by antibody-independent mass spectrometry (MS)-based or antibody-dependent ELISA methods. Biophysical analysis At Health Canada, work is ongoing to further evaluate the potential of HPLC for the assessment of influenza vaccines and to provide qualitative and quantitative measurements for specific constituents. Size-exclusion (SE) HPLC provides a profile of protein components in vaccines according to protein size and has been used by Health Canada for research purposes to analyze seasonal vaccines. Size-exclusion-HPLC of multiple lots provides an indication of the production process reproducibility. The method is highly reproducible and can be applied to bulks and vaccines directly without modification of samples. RP-HPLC provides a profile of protein components in vaccines according to hydrophobicity. This method has been used to provide accurate absolute amounts of HA in both monovalent and trivalent formulations. RP-HPLC still needs a reference antigen to quantitate HA. RP-HPLC can distinguish HA1 from different strains as these have different elution profiles. Two-dimensional (2D) HPLC combines SE and RP chromatography, providing the highest level of selectivity and quantification of HA1.17 During the 2009 H1N1 pandemic, Health Canada monitored total HA concentrations in vaccine using a combination of SE-HPLC and RP-HPLC in conjunction with SRID during investigations of product instability. MS-based methods can be used for characterization and quality control of influenza vaccines. Health Canada is working on the development of proteomics-based MS methodology to identify proteins present in influenza vaccines. Although standard HPLC and MS-based methods do not provide assurance of correct folding or antigenic structure of HA, these tests provide important analysis of product identity and quality18,19 but do not indicate whether the product is stable or potent. MS-based methods are also being investigated in the United States to improve the current reagent calibration process, which currently takes several weeks. This process requires the determination of the HA concentration in a primary liquid standard by determining total protein concentration and determination of the proportion of HA present in the standard by densitometry of protein bands separated by SDS-PAGE. Within the United States, a multi-center collaboration (National Center for Environmental Health, CDC, and FDA's Center for Biologics Evaluation and Research (CBER), and Center for Food Safety and Applied Nutrition) has been initiated to evaluate the use of isotope-dilution MS to determine the absolute quantity of HA in primary influenza standards.20 If correlated with current methods, the time necessary for calibration could be reduced from weeks to days. MS-based methods to evaluate vaccine stability or potency tests are also being developed. The US CDC has used reference antiserum bound to beads as a means to extract HA with native conformation from vaccine. The amount of HA bound to these complexes can then be measured by isotope-dilution MS. Other MS-based methods that do not require the use of HA-specific antiserum to determine the relative amount of folded and unfolded HA present in vaccine are being developed as potential stability-indicating assays. These methods as well as other platforms such as ELISA and surface plasmon resonance are being investigated as alternative potency assays at CBER, FDA, and other institutions. In addition to expediting calibration of reference antigen as described earlier, other approaches to improve the SRID assay were discussed during the workshop. This includes generating HA-specific antiserum by preparing antigen for immunization of sheep in a manner that does not require purification of the antigen from whole virus. Antiserum generated in response to DNA and recombinant vaccinia was recently tested.21 Alternatively, a panel of monoclonal antibodies could be used as a source of reference antibodies, with the expectation that a specific antibody combination would be relevant for several seasons. Overall, different methods may be useful for different purposes. Manufacturers encouraged the standardization/harmonization of SRID as a first step, urging regulators to agree to use one set of reagents and the same method regardless of the market. Conclusions and recommendations The workshop concluded with the formulation of several recommendations regarding improved assessment of vaccine potency by SRID, the development and use of alternative approaches to assess potency of influenza vaccines, and how new assays might be incorporated into influenza vaccine regulatory decision making. The influenza vaccine industry is looking to national regulatory authorities and Essential Regulatory Laboratories (ERLs) for direction in these areas. New methods are more likely to be adopted by industry if they are low cost, not labor intensive, high throughput (preferably automated), high specificity, stability indicating, and indicative of antigenic structure and vaccine efficacy. In some cases, alternative methods were used during the 2009 H1N1 pandemic until SRID reference reagents became available or as part of special investigations of product quality and stability. While knowledge and technologies are expanding, more research and confidence building are needed before these alternative approaches can be incorporated into routine decision making. As a first step, it was recommended that improvements be made to the SRID assay. This can potentially be accomplished by using one assay method and one set of reagents for each strain, by improving the method used to calibrate reagents, and by harmonizing SRID calibration across markets and continents. This may require generating larger amounts of reference antigen, potentially through a contract laboratory. It was recommended that at the next ERL meeting (scheduled for January 2011), the participants explore ways to improve reference antigen production, calibration, and quality control. Various promising alternative in vitro methods are being explored, including several types of HPLC, ELISA, SDS-PAGE, MS, and surface plasmon resonance. Continued research is needed to determine how these techniques are best used, for example, as alternatives to SRID, as supplemental techniques, as in-process controls or for stability testing. The advantage of some of these methods is that they are antibody independent and thus can be used without delays owing to reagent preparation. It was recommended that priority be given to continuing research and development of these and other promising approaches. In the context of supporting implementation of new potency assays, it may be possible to use SRID to bridge new assays that measure antigenicity of HA; however, novel assays may require testing in animal models and clinical trials to demonstrate that results correlate with immunogenicity. To keep momentum toward development of alternative assays and to better coordinate and monitor research and development efforts, participants recommended that venues be identified for the purpose of communicating progress on the evaluation of alternative methods. The focus of the workshop was on potency assays for licensed inactivated influenza vaccines; however, research and development of new assays should also address the need for the assessment of the potency of new generation vaccines, such as adjuvanted vaccines, cell culture–derived vaccines, recombinant protein-based vaccines, and virus-like particles. This workshop was an important step toward developing a global understanding of alternative methods for potency testing that could potentially expedite the availability of seasonal and pandemic influenza vaccines and for identifying ways to move forward collaboratively. Footnotes * Data published online January 2011. References 1 Williams MS. Single radial immunodiffusion as an in vitro potency assay for human inactivated viral vaccines. Vet Microbiol 1993; 37: 253– 262. 2 Cate TR, Couch RB, Parker D, Baxter B. Reactogenicity, immunogenicity, and antibody persistence in adults given inactivated influenza virus vaccines – 1978. Rev Infect Dis 1983; 5(4): 737– 747. 3 La Montagne JR, Noble GR, Quinnan GV et al. Summary of clinical trials of inactivated influenza vaccine – 1978. Rev Infect Dis 1983; 5: 723– 736. 4 Wright PF, Cherry JD, Hjordis FM et al. Antigenicity and reactogenicity of influenza A/USSR/77 virus vaccine in children – a multicentered evaluation of dosage and safety. Rev Infect Dis 1983; 5: 758– 764. 5 Rodda SJ, Gallichio HA, Hampson AW. The single radial immunodiffusion assay highlights small antigenic differences among influenza virus hemagglutinin. J Clin Microbiol 1981; 14: 479– 482. 6 Luykx DMAM, Casteleijn MG, Jiskoot W, Westdijk D, Jongen PMJM. Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material. Eur J Pharm Sci 2004; 23: 65– 75. 7 Li C, Shao M, Cui X et al. Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China. Biologicals 2010; 38(2): 284– 289. 8 Farnsworth A, Cyr TD, Li C, Wang J, Li X. Antigenic stability of H1N1 pandemic vaccines correlates with vaccine strain. Vaccine 2011; 29(8): 1529– 1533. 9 Li C, Jaentschke C, Song Y et al. A simple slot blot for the detection of virtually all subtypes of the influenza A viral hemagglutinins using universal antibodies targeting the fusion peptide. Nat Protoc 2009; 5(1): 14– 19. 10 Chun S, Li C, Van Domselaar G et al. Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins. Vaccine 2008; 26(48): 6068– 6076. 11 Webster RG, Reay PA, Laver WG. Protection against lethal influenza with neuraminidase. Virology 1988; 164(1): 230– 237. 12 Sandbulte MR, Jimenez GS, Boon AC, Smith LR, Treanor JJ, Webby RJ. Cross-reactive neuraminidase antibodies afford partial protection against H5N1 in mice and are present in unexposed humans. PLoS Med 2007; 4(2): e59. 13 Murphy BR, Kasel JA, Chanock RM. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N Engl J Med 1972; 286: 1329– 1332. 14 Clements ML, Betts RF, Tierney EL, Murphy BR. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J Clin Microbiol 1986; 24(1): 157– 160. 15 Aymard-Henry M, Coleman MT, Dowdle WR, Laver WG, Schild GC, Webster RG. Influenzavirus neuraminidase and neuraminidase-inhibition test procedures. Bull WHO 1973; 48: 199– 202. 16 Gravel C, Li C, Wang J et al. Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences. Vaccine 2010; 28(36): 5774– 5784. 17 García-Cañas V, Lorbetskie B, Bertrand D, Cyr T, Girard M. Selective and quantitative detection of influenza virus proteins in commercial vaccines using two-dimensional high-performance liquid chromatography and fluorescence detection. Anal Chem 2007; 79: 3164– 3172. 18 Creskey MC, Smith DG, Cyr TD. Strain identification of commercial influenza vaccines by mass spectrometry. Anal Biochem 2010; 406: 193– 203. 19 Getie-Kebtie M, Chen D, Eichelberger M, Alterman M. Proteomics-based characterization of hemagglutinins in different strains of influenza virus. Proteomics Clin Appl 2009; 3(8): 979– 988. 20 Williams TL, Luna L, Guo Z et al. Quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry. Vaccine 2008; 26: 2510– 2520. 21 Schmeisser F, Vodeiko GM, Lugovtsev VY, Stout RR, Weir JP. An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination. Vaccine 2010; 28: 2442– 2449. Citing Literature Volume5, Issue6November 2011Pages 438-442 ReferencesRelatedInformation

In August 2010, the World Health Organization and the Korea Food & Drug Administration jointly organized the first implementation workshop of WHO guidelines on evaluating similar biotherapeutic products (SBPs) at the global level. The objective of the Workshop was to facilitate implementation of the newly adopted WHO Guidelines into the practice of national regulatory authorities (NRAs). WHO Guidelines were recognized by the workshop participants as a tool for harmonizing regulatory requirements worldwide. By reviewing and practicing several case studies, better understanding and consensus on the principles of clinical trial designs were reached. However, variations in terms of the national requirements for quality, safety and efficacy of these products revealed diversity in the regulatory expectations in different countries and regions. In addition, lack of terminology for the products developed as copy products (so called "me too" products) with a partial comparability to an RBP, led to a great diversity in evaluating as well as naming these products. The workshop participants proposed the following actions: a) NRAs should make efforts to build their capacities for regulation of SBPs; b) WHO should revise WHO Guidelines for assuring the quality of products prepared by recombinant DNA technology (WHO TRS 814) and continue monitoring progress with the implementation of the Guidelines on evaluating SBPs. Publication of the outcome of the Workshop was recognized as another action that WHO should coordinate.