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Downloaded from orbit.dtu.dk on: Oct 02, 2021 Analysis of vaccine Structure, storage, moisture, and classification by infrared technology Zheng, Yiwu Publication date: 2006 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Zheng, Y. (2006). Analysis of vaccine: Structure, storage, moisture, and classification by infrared technology. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Analysis of vaccine: Structure, storage, moisture, and classification by infrared technology Ph.D. Thesis by Yiwu Zheng 2006 BioCentrum-DTU Section of Biochemistry and Nutrition Technical University of Denmark Building 224, Sølvtofts Plads 2800 Kgs. Lyngby Denmark Preface This thesis has been submitted as partial fulfilment of the requirement for a Ph.D. degree at the Technical University of Denmark. The work has been done at Biochemistry and Nutrition Group, BioCentrum-DTU and ALK-Abelló A/S. I would like to thank my supervisors Susanne Jacobsen and Henning Løwenstein for giving me the opportunity to do this project and for all the work they have done for me. I specially wish to thank Ib Søndergaard, Henrik Ipsen, and Jørgen Nedergaard Larsen for enthusiastic discussions and the comments on the project and the thesis. If I were not with them, I would need a couple of more years to finish this Ph.D. project. I also would like to thank Charlotte Hejl for bridging the communication between the company and me when I worked at DTU. And also thank her for the helpful suggestion on the project. I want to thank Helene Henmar for the introduction of CD technique and the help on the experiments and data analysis. Mercedes Ferreras are greatly appreciated for the great discussion and references. I want to thank Marianne K. Pertersen, Ljljana Nesic, Gitte Nordskov Hansen, and Lotte Friberg for the help in the labs. Susanne Warng Bruun is greatly appreciated for discussions of the same interest—infrared and that we have been able to support each other. I would like to thank all the people in the research department, ALK-Ablleó A/S and at Biochemistry and Nutrition Group, BioCentrum-DTU for kind help and the beautiful working environment. And also thank all the Danish and Chinese friends for your help during these years. At last, but not the least, I would like to thank my pretty wife Xuxin and my lovely daughter Xinyi for all your love and supports. Yiwu Zheng June 2006, Denmark i Summary Aims of this thesis The research described in this thesis is to extend our knowledge on the properties of vaccines and antigens using fast and cheap infrared techniques. FTIR-ATR spectroscopy is applied to elucidate the structural changes when antigen is adsorbed by aluminium hydroxide and when it is subsequently released. The structural stability during the storage and heating process is investigated as well. The alterations of other parameters, such as adsorption, release, and pH are also investigated during the storage. FT-NIR is applied as a fast and non-destructive method to determine the residual moisture content in lyophilized vaccines and to classify and identify different vaccine products. Organization of this thesis In Chapter I, the background of the project and the introductions to infrared spectroscopy as well as chemometrics are given respectively. In Chapter II, the current techniques for protein structure analysis have been discussed, especially the FTIR-ATR method. In Chapter III, the structural changes of two model antigens are investigated when they are adsorbed by aluminium hydroxide and the structures refold to their native state when they are released from adjuvant. The mechanism of the adjuvant enhancing the immune response is discussed in this chapter too. Chapter IV describes the structural stability of adsorbed model antigens in thermal and aging process in comparison to the antigens in solutions. The results indicate that aluminium hydroxide can protect the structure of antigens against aggregation at high temperature and/or after long period storage. It is more pronounced for BSA. Further study is required to confirm if the protection is protein specific. Chapter V gives the alterations of model vaccines during storage at two different temperatures. It demonstrates that the change of adsorption capacity by aluminium hydroxide during the storage is protein specific. The release of adsorbed protein from adjuvant decreases as a function of storing time. The pH and NIR absorbance also change. The aging effects undergo faster modification at 37 ºC than 4 ºC during the storage. The rotation effects when preparing vaccines are also discussed in this chapter as well. NIR method for determination of residual moisture content of lyophilized allergen vaccines is ii introduced in Chapter VI. Five different allergen vaccines are analyzed. Acceptable calibration model (RMESEP 10.20 μg H2O / vial with 2 PLS factors) is obtained for multi-products data. The models based on product-specific data are slightly better (Appendix B). However, a general multi- products model is more attractive since one model can predict all samples. The reference data which are measured by Karl Fischer titration are presented in Appendix A. NIR spectroscopy is applied to classify and identify five different allergen vaccines (the information of samples in Appendix C) at different concentrations of antigens and different batches in Chapter VII. The results show that NIR spectroscopy can be used to classify different allergen vaccines at highest antigen concentration (100,000 SQ) without api. The highest concentration products can be separated from other low concentrations. The discrimination of different batches may also be possible but need further experiments to confirm this. All SIMCA results are given in Appendix C to Appendix S. iii Resumé Hensigt med afhandlingen Forskningsarbejdet, beskrevet i denne afhandling, skal øge vores viden om vacciners og antigeners egenskaber ved hjælp af hurtige og billige infrarød-teknikker. FTIR-ATR spektroskopi benyttes til bestemmelse af strukturændringer, når antigen adsorberer til aluminium hydroxid, og når det efterfølgende frigives. Den strukturelle stabilitet under opbevaringen og under opvarmningsprocessen undersøges ligeledes. Desuden undersøges ændringer i andre parametre, såsom adsorption, frigivelse og pH, under opbevaringen. FT-NIR benyttes som en hurtig og ikke- destruktiv metode til at bestemme resterende fugtindhold i frysetørrede vacciner og til at klassificere og identificere forskellige vaccineprodukter. Organisering af afhandlingen I kapitel I er baggrunden for projektet samt introduktion til infrarød spektroskopi og kemometri beskrevet. I kapitel II er de nuværende teknikker til protein-struktur-analyse, især FTIR-ATR metoden, blevet diskuteret. I kapitel III er undersøgt strukturændringer af to model antigener ved adsorption til aluminium hydroxid samt deres foldning tilbage til nativ tilstand ved frigivelse fra adjuvanten. Mekanismen for den immunfremmende effekt af adjuvanten er ligeledes diskuteret. Kapitel IV beskriver strukturel stabilitet af adsorberet modelantigen i termiske- og aldringsprocesser sammenlignet med antigener i opløsning. Resultaterne indikerer at aluminium hydroxid kan beskytte strukturen af antigenet mod aggregeringen ved høj temperatur og/eller lang tids opbevaring. Dette er mest udtalt for BSA. Yderligere studier kræves for at bekræfte om denne beskyttelse er protein-specifik. Kapitel V omhandler ændringen af modelvacciner under opbevaring ved to forskellige temperaturer. Det demonstrerer at ændringen i aluminium hydroxids adsorptionskapacitet under opbevaring er protein-specifik. Frigivelse af adsorberet protein fra adjuvanten nedsættes som funktion af opbevaringstid. Ligeledes ændres pH og NIR-absorbansen. Aldringseffekterne gennemgår hurtigere modifikationer med opbevaringstiden ved 37 oC end ved 4 oC. Rotationseffekter under fremstilling af vacciner diskuteres også i kapitlet. NIR-metoden til bestemmelse af resterende fugtindhold i frysetørrede allergenvacciner introduceres i kapitel VI. Fem forskellige allergenvacciner analyseres. Acceptable kalibreringsmodeller (RMSEP iv 10.20 μg H2O/ flaske med 2 PLS faktorer) er opnået for multi-produkt data. Modellerne baseret på produktspecifikke data er lidt bedre (Appendiks B). Imidlertid er en generel multiprodukt-model mere attraktiv idet en enkelt model kan forudsige alle prøverne. Referencedataene som måles ved Karl Fischer-titrering præsenteres i Appendiks A. NIR spektroskopi benyttes til at klassificere og identificere fem forskellige allergenvacciner (information om prøverne ses i Appendiks C) ved forskellige koncentrationer af antigener og forskellige batcher i kapitel VII. Resultaterne viser at NIR spektroskopi kan benyttes til at klassificere
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