Quality testing of vaccines: hands-on training on a standardized protocol to test saccharide content of priority vaccines

176 WHO Drug Information Vol. 30, No. 2, 2016 Quality testing of vaccines Hands-on training on a standardized protocol to test saccharide content of priority vaccines To facilitate access to good-quality vaccines, the WHO Prequalification Team (PQT) promotes standardized testing protocols that can be used to test vaccines produced by different manufacturers. A harmonized test method has been identified to determine polyribosyl-ribitol-phosphate (PRP) content in liquid vaccine combinations containing a whole-cell pertussis component. The use of this method saves significant time and resources at quality control laboratories. Hands-on training courses for quality control laboratory technicians from 13 countries have been organized, enabling them to implement this protocol at their institutions. Introduction Diphtheria, tetanus, whole-cell pertussis (DTwP)-based pentavalent vaccine (liquid presentation) is a globally important vaccine. This combination “five-in-one” vaccine protects children from diphtheria, pertussis (whooping cough), tetanus, hepatitis B and Haemophilus influenzae type b (Hib), which causes pneumonia and meningitis. It is less traumatic for infants to receive and easier for programmes to administer than previous formulations. In recent years, global demand for this vaccine has increased rapidly (1). The WHO Prequalification Team (PQT) prequalifies this type of vaccine for use in national immunization programmes (2). The active ingredient of a Hib vaccine that prevents infection by H. influenzae type b is polyribosyl-ribitol-phosphate (PRP), a saccharide. Total and free (unconjugated) saccharide content is the single critical parameter indicative of Hib conjugate vaccine quality. However, quantitative determination of PRP in different liquid formulations is challenging for laboratories and manufacturers because differences in Hib carrier protein, antigen combinations, adjuvant, preservatives and other excipients interfere with the testing. Specific methodologies need to be established and validated for each individual product, which is time-consuming. Laboratories that test Hib vaccines on behalf of WHO PQT during the prequalification process usually use their own testing methods, since applying the manufacturer’s methodology The content of this article was contributed by Dr Ute Rosskopf of the WHO Prequalification Team. The study to identify a harmonized assay was organized in the framework of the WHO Vaccines Prequalification Programme, with funding from the United States Agency for International Development (USAID). WHO thanks Dr Christina von Hunolstein, Bacterial Vaccine Unit, National Centre for Immunobiologicals Research and Evaluation (CRIVIB), Istituto Superiore di Sanità (ISS), Italy, and Dr Barbara Bolgiano, Division of Bacteriology, National Institute of Biological Standards and Control (NIBSC), United Kingdom, for performing the tests in the study and co-organizing the subsequent hands-on training courses. WHO is grateful to the following manufacturers for their donations of vaccine samples (in alphabetical order): Berna Biotech Korea Corp., Yeonsu-gu, Incheon, Republic of Korea; Biological E. Limited, Hyderabad, India; Novartis Vaccines and Diagnostics S.r.I., Siena, Italy; Serum Institute of India Limited, Hadapsar, Pune, India. The preparation of a pentavalent vaccine exclusively for the purpose of this study was much appreciated. 177 WHO Drug Information Vol. 30, No. 2, 2016 Quality testing of vaccines would take even more time and would represent an additional challenge. A harmonized assay During evaluation of a new product for prequalification, a WHO-contracted laboratory obtained non-compliant results when testing the PRP content of the Hib component of a pentavalent vaccine lot. A group of experts convened by WHO concluded that the non- compliance was due to differences in testing methodologies rather than a deficiency in the product itself. The experts recommended that WHO focus on standardization of a test protocol to assess PRP in liquid vaccine preparations. In 2013 WHO PQT initiated a small project to identify a protocol to reliably determine the total and free PRP content of the Hib conjugate component of liquid vaccine presentations (3). Two laboratories were requested to test samples of five selected vaccines according to their own test protocols, using two different reference standards. The data demonstrated that one of the protocols was successful in providing accurate measurements of total and unconjugated PRP in complex matrices of immunogens, adjuvants and excipients (Box 1). The test protocol identified in the study gives laboratories an efficient means of determining Hib component in liquid vaccine presentations produced by different manufacturers. Additional investigations showed that the test protocol is applicable to all WHO- prequalified vaccine combinations containing a whole-cell pertussis component (5) – which is used in most developing countries consistent with WHO recommendations (6) – using either of the two reference standards. To verify the method further, another study was subsequently conducted in collaboration with the Biological Standardisation Programme of the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the EU Commission, with participation of five manufacturers and five national control laboratories. Publication of the results is under way. Hands-on training courses Testing of Hib-combination vaccines To support countries in quality assurance for Hib-containing liquid combination vaccines, a hands-on training course was designed to enable correct implementation of the assay identified in the study. The trainees perform sample preparation and other critical steps for the chromatographic runs, and calculate their own test results using a dedicated Excel spreadsheet. Test outcomes, real-life experiences and challenges in using the test protocol are discussed extensively. During 2014 and 2015 four one-week courses were co-organized by WHO and the Bacterial Vaccine Unit of the Istituto Superiore di Sanità (ISS) in Rome, Italy. A total of 20 participants were trained, including four from India, three from the Republic of Korea, two each from Cuba and Indonesia and one each from Bangladesh, Brazil, Canada, China, Iran, Mexico, Poland, South Africa and Thailand. A fifth course will take place in June 2016 at the national control laboratory in China. Feedback from course participants has been extremely positive. Suggestions were made for WHO to offer similar training on other glycoconjugated or polysaccharide vaccines, as well as additional theoretical training on issues such as validation of High Performance Quality testing of vaccines WHO Drug Information Vol. 30, No. 2, 2016 178 Box 1: Identification of a harmonized assay Study aim: To compare total and free polyribosyl-ribitol-phosphate (PRP) content of the H. influenzae type b conjugate component of liquid vaccine presentations as determined at two independent laboratories with the values obtained by the manufacturer at lot release. Materials and methods: High-performance anion exchange chromatography pulsed amperometric detection (HPAEC-PAD) (4) was performed at two laboratories using their own HPAEC-PAD protocols and validity criteria. Vaccine sample panel (the order does not reflect the sample numbers in the Figures below): • DTPwHepB-Hib: Hib-TT, Thiomersal (0.01 %), Al phosphate • DTPwHepB-Hib: Hib-TT, Thiomersal (0.005 %), Al phosphate • DTPwHepB-Hib: Hib-CRM, Al phosphate • DTPw-Hib: Hib-CRM, Thiomersal, Al phosphate • DTPwHepB-Hib: Thiomersal, Al phosphate, sub-potent Hib-TT component (low total PRP content and high free PRP content) prepared for the purpose of the study. Reference standards (RS): • Ribitol RS (Fluka, lot number BCBJ6567V). • WHO PRP 1st International Standard (code no. 02/208). Analytical conditions: Samples were analyzed as described in the laboratories’ HPAEC-PAD test protocols. A Dionex DX-500 chromatography system equipped with a CarboPac MA1 analytical column was used in combination with a CarboPac MA1 guard column. PRP was hydrolyzed with 0.3 M HCl for 2 hours at 100°C. Samples were pre-treated to determine free PRP content, using C4 SPE cartridges (Laboratory 1) or 30 or 100 kDa pore size Microcon ultrafiltration membranes (Laboratory 2). Two independent runs were performed to determine total and free PRP content. Results: Figures 1 and 2 show the total and free PRP content determined by the manufacturer at lot release (Mf) and the geometrical means of the two values obtained at each of the two laboratories (Lab1, Lab2). Figure 1: Total PRP content (µg per single human dose) WHO RS; manufacturer in-house RS; Ribitol RS; ..... Specification Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Figure 2: Free PRP content (percentage per single human dose) WHO RS; manufacturer in-house RS; Ribitol RS; ..... Specification Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Conclusions • Both laboratories identified the sub-potent vaccine (Sample 5). • The two reference standards gave similar results at both laboratories. • The test protocol of Laboratory 1 showed better agreement with the manufacturers’ data for the free PRP content than the test protocol of Laboratory 2. 179 WHO Drug Information Vol. 30, No. 2, 2016 Quality testing of vaccines Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC- PAD) analysis, statistical analysis, creation of validity criteria and qualification of chromatographic equipment. Testing of meningococcal vaccines Course participants had expressed interest in trainings on meningococcal vaccines, which – like Hib combination vaccines – are on the priority list for WHO prequalification (7). In February 2016 WHO and the Division of Bacteriology of the U.K. National Institute of Biological Standards and Control (NIBSC) organized a two- week course on the use of HPAEC-PAD for the quantitative determination of the saccharide content of the meningococcal serotypes A, C, W and Y. Participants from the national control laboratories of Brazil, China, Cuba, India, Indonesia and South Africa attended the training. Conclusions The harmonized assay for testing of saccharide content in liquid combination vaccine preparations enables national quality control laboratories and manufacturers to reduce the time spent on developing and transferring test methods. There are currently eight liquid Hib combination vaccines with a whole cell pertussis component on the WHO prequalification list (5), originating from Italy, the Republic of Korea, India and Indonesia. Technicians from these and ten additional countries participated in hands-on training courses on the use of the harmonized test protocol. Training on HPAEC-PAD testing of meningococcal vaccines has also started. Further courses will follow. Successful implementation of the harmonized assay, based on proper validation, will enable quality control laboratories to obtain accurate and reliable testing outcomes. This will benefit the prequalification process and thereby help to ensure a sustainable supply of good quality vaccines to national immunization programmes. Considering that immunization is one of the most cost-effective public health interventions (8), the value for WHO Member States is significant. References 1 UNICEF Supply Division. Pentavalent vaccine (DTwP-HepB-Hib): Market & Supply Update. July 2015. 2 WHO. Procedure for assessing the acceptability, in principle, of vaccines for purchase by United Nations agencies. Annex 6. In: WHO Technical Report Series 978. Geneva: World Health Organization, 2010. 3 Study report: Quantitative determination of the saccharide and unconjugated saccharide content of Haemophilus influenzae type b conjugate component in liquid vaccine presentations. Available at: www.who.int/immunization_standards/ vaccine_quality/Study_Report_Vaccines_ May2014.pdf 4 Bardotti A et al. Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection. Vaccine 2000;(18):1982‒93. 5 WHO. WHO prequalified vaccines_List of prequalified vaccines [web page]. Available at: https://extranet.who.int/gavi/PQ_Web/ 6 WHO. Pertussis vaccines: WHO position paper. Weekly epidemiological record. 40, 2010, 85, 385–400. 7 WHO Immunization. Priority setting for WHO vaccine prequalification [web page]. Available at: www.who.int/immunization_ standards/vaccine_quality/pq_priorities/en/. 8 WHO, UNICEF, World Bank. State of the world’s vaccines and immunization. Third Edition. Geneva: World Health Organization, 2009. å