Diphtheria, tetanus, whole-cell pertussis (DTwP) based pentavalent combination vaccine (liquid

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1 Study report: Quantitative determination of the saccharide and unconjugated saccharide content of Haemophilus influenzae type b conjugate component in liquid vaccine presentations Outcome of a small study Abstract Diphtheria, tetanus, whole-cell pertussis (DTwP) based pentavalent combination vaccine (liquid DTwP-HepB-Hib presentation) is a high-priority vaccine as indicated by the WHO vaccines prequalification priority list WHO therefore receives many applications for prequalification of this product. The various vaccine combinations differ according to Haemophilus influenzae type b (Hib) carrier protein, antigen combinations, adjuvant, and preservatives and other excipients. Each of these factors can interfere in the determination of the polyribosyl-ribitol-phosphate (PRP) content, the active ingredient of a vaccine that prevents infection by H. influenzae type b. Laboratories performing tests on behalf of the WHO Prequalification of Vaccines Programme must establish and validate various methodologies to determine the PRP content of the individual products. This is very time-consuming and slows the testing process. This study consisted of testing of five selected vaccines using two different test protocols and two reference standards: the WHO PRP 1st International Standard and a ribitol reference standard. Keywords Haemophilus influenzae type b (Hib); polysaccharide analysis; acid hydrolysis; high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD); liquid vaccine combinations; polyribosyl-ribitol-phosphate (PRP); Prequalification of Vaccines Programme (PQV) Introduction Diphtheria, tetanus, whole-cell pertussis (DTwP) based pentavalent combination vaccine (liquid DTwP-HepB-Hib presentation) is a high-priority vaccine as indicated by the WHO vaccines prequalification priority list [1]. WHO therefore receives many applications for prequalification of this type of product [2]. WHO prequalified vaccines are assessed and monitored for their quality by an independent test programme. The testing of vaccines containing various antigens is time consuming. In Hib vaccines, the polyribosyl-ribitol-phosphate protects children against invasive Hib infection [3]. By linking this antigen covalently to a carrier protein, a T-cell-dependent immunogenicity is induced [4]. The most widely-used used carrier proteins are tetanus toxoid (TT) or a non-toxic variant of the diphtheria toxin, the cross-reacting material 197 (CRM 197). Laboratories performing tests on behalf of the WHO prequalification of vaccines programme must establish and validate various methodologies to determine the PRP content of the individual products. This is very time-consuming and slows the testing process. WHO-contracted laboratories therefore usually apply their own methods for performing independent re-testing of products since application of a manufacturer-specific methodology is even more time-consuming and would represent an additional challenge. The quality of the Hib component in vaccine combinations is usually controlled by determining the total polysaccharide content and the free unconjugated saccharide content. This determination is 1

2 critical in a fully liquid vaccine matrix that is characterized by differences in the Hib carrier proteins, antigen combinations, adjuvants, and preservatives and other excipients of the different products. During evaluation of a new product, a WHO-contracted laboratory obtained non-compliant results following testing of the Hib content of a pentavalent vaccine lot. They were discussed by a group of experts who were of the opinion that the conflicting results were due to the difference in the methodology used by the laboratories rather than an indication of deficient product quality. The group recommended that further efforts be made to standardize the methodologies used to assess PRP in liquid vaccine combinations. As a result, WHO PQV initiated a Hib project to establish a methodology with a single test protocol to be applied for the quantitative determination of the total saccharide and free saccharide content of the H. influenzae type b conjugate component of liquid vaccine presentations (suspension). WHO selected five vaccine samples to be tested by using two different HPAEC-PAD test protocols and two Reference Standards: the WHO PRP 1 st International Standard (code no. 02/208) and a ribitol reference standard. Materials and Methods Vaccines Panel of 5 vaccine samples (the order does not reflect the vaccine sample numbers in the discussion of the results 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 (i.e. low content of total PRP and high content of free PRP). Reference standards Ribitol reference standard (Fluka, lot number BCBJ6567V). The WHO PRP 1 st International Standard [5, 6] was already available at the laboratories. Analytical conditions The laboratories were requested to apply their own test protocol and validity criteria. They were also requested to use the two reference standards (since both standards are used by manufacturer) to estimate the total and the free saccharide content of the five vaccine samples. The analysis of the acid hydrolyzed vaccine samples was performed as described in both test protocols using the HPAEC-PAD [7]. A Dionex DX-500 chromatography system was equipped with a CarboPac MA1 analytical column in combination with a CarboPac MA1 guard column. The PRP was hydrolyzed with 0.3 M HCl for 2h at 100 C. A pre-treatment of the vaccine samples was performed for determining the free PRP content. The pre-treatment differed according to the protocols with respect to dilution of the samples and separation of the free PRP through the use of C4 SPE cartridges (Laboratory 1) and 30 or 100 kda pore size Microcon ultrafiltration membranes (Laboratory 2). 2

3 Results and discussion Laboratory 1 performed two independent test runs. Four of the five lots satisfied the specifications for the total (Fig. 1) and free PRP content (Fig. 2). The sub-potent vaccine lot was identified: it did not meet the specification for the total PRP (Fig. 1), having a value lower than 8.8 µg/shd and a relatively high percentage of free PRP (Fig. 2). The variance between the two independent test runs was low for both reference standards used. The generated results were very similar: the highest and lowest coefficient of variation were 11.61% and 0.39% respectively. The variance between the WHO PRP standard and the ribitol reference standard had a variation coefficient ranging between from 4.79% to 0.01%, which was therefore below the variance between the two independent test runs. Laboratory 2 completed 25 test runs in total: Five tests were repeated since the assay outcome of the first test indicated a failed specification. three of the five vaccine lots met the specification for total PRP content (Fig.1) and five of five met the specification for free PRP content (Fig.2). The sub-potent vaccine was identified as such, failing the specification, with values below 8.8 µg/shd for total PRP content (Fig. 1) and a higher percentage of free PRP content compared to the other lots tested (Fig. 2). The variance between the independent runs of laboratory 2, performed for five tests, showed a highest and lowest coefficient of variation of 18.33% and 0.14% respectively. The variance in using the WHO PRP standard or the ribitol reference standard had a highest and lowest coefficient of variation of 13.69% and 0% respectively. That is, the variance between standards was lower than that of the variance between the two test runs. Figures 3 and 4 compare the results of the participating laboratories with those obtained by the manufacturers at lot release. The manufacturers used either a ribitol reference standard, the WHO PRP International Standard or an in-house PRP reference standard. The test results between manufacturers and laboratory 1 correlated very well for both reference standards used. The comparison of the test data of laboratory 2 showed a difference with respect to vaccines 2, 3 and 5. For these vaccines, a lower PRP content was determined compared to that determined by the manufacturer. Additionally, vaccine 3 was observed to be out of specification when the results of two independent test runs were combined. Figure 4 compares the results relating to unconjugated, free PRP content. The results of both laboratories showed a higher percentage of free PRP for vaccines 1, 2, 3 and 4 than did the results of the manufacturer. Since the percentage of unconjugated PRP is a time-dependent parameter i.e. the percentage increases over time, these results were to be expected. A vaccine lacking the requested quality (specification of the manufacturer) could be clearly identified by the protocols of both laboratories. However, the data of laboratory 1 showed better agreement with the manufacturers data for the free PRP content. Conclusion The data demonstrated that, for one of the test protocols applied (Laboratory 1), precise measurement of the critical vaccine parameter total and unconjugated PRP was possible for complex matrices of immunogens, adjuvants and excipients for five different vaccines. The agreement of the data of one laboratory with the manufacturers results, might be related to less critical differences between its method and the operating procedures of the different manufacturers. 3

4 Furthermore, use of the WHO PRP 1 st International Standard or the ribitol reference standard has been demonstrated to give the same results. WHO PQV will conduct an international collaborative study, with a higher number of participating laboratories, and in cooperation with the European Directorate for the Quality of Medicines & HealthCare, to confirm the reliability of the aforementioned test protocol. Acknowledgments The study was organized in the framework of the WHO Vaccines Prequalification Programme. The programme is very grateful to the manufacturers for their donation of the vaccine samples (in alphabetical order): Berna Biotech Korea Corp., 13-42, Songdo-dong 23, Harmony-ro 303beon-gil, Yeonsu-gu, Incheon, Republic of Korea Biological E. Limited, 18/1&3, Azamabad, Hyderabad, India Novartis Vaccines and Diagnostics S.r.I., Via Fiorentina 1, Siena, Italy Serum Institute of India Limited, 212 /2 Hadapsar, Pune, India The preparation of a pentavalent vaccine exclusively for the purpose of this study was very much appreciated. Special acknowledgment is due to the participating laboratories that performed the tests based on specific contracts for this study: the Bacterial Vaccine Unit of the Instituto Superiore di Sanità (Italy) and the Division of Bacteriology of the National Institute of Biological Standards and Control (UK). Abbreviations Al: aluminium CRM: cross-reacting material DTwP: diphtheria, tetanus, whole-cell pertussis DTwP-HepB-Hib: diphtheria, tetanus, whole-cell pertussis, Hepatitis-B, Haemophilus influenzae type b PQV: prequalification of vaccines PRP: polyribosyl-ribitol-phosphate Shd: single human dose WHO: World Health Organization References [1] WHO vaccines prequalification priority list [2] WHO Technical Report Series 978, Annex 6: Procedure for assessing the acceptability, in principle, of vaccines for purchase by United Nations agencies 6_PQ_vaccine_procedure.pdf [3] Crisel RM, Baker RS, Dorman DE. Capsular polymer of Haemophilus influenzae type b. I. Structural characterization of the capsular polymer of strain Eagan. Journal of Biological Chemistry 1975; 250: [4] World Health Organization, Recommendations for the production and control of Haemophilus influenzae type b conjugate vaccines. In WHO Expert Committee on Biological Standardization. Forty-ninth report. WHO Technical Report Series, No. 897, Annex 1;

5 [5] Mawas F et al. Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide. Biologicals 35 (2007); [6] WHO/BS/ International collaborative study to evaluate a candidate International Standard for Haemophilus influenzae type b capsular polysaccharide. [7] 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):

6 Figure 1: Test results of the total PRP content determination laboratories 1 and 2 Figure 2: Test results of the free (unconjugated) PRP content determination laboratories 1 and 2 6

7 Figure 3: Comparison of test results between laboratories and manufacturers total PRP content Figure 4: Comparison of test results between laboratories and manufacturers free PRP content 7