Use of immuno assays during the development of a Hemophilus influenzae type b vaccine for technology transfer to emerging vaccine manufacturers

Abstract

Quality control of Hemophilus Influenzae type b (Hib) conjugate vaccines is mainly dependent on physicochemical methods. Overcoming sample matrix interference when using physicochemical tests is very challenging, these tests are therefore only used to test purified samples of polysaccharide, protein, bulk conjugate, and final product. For successful development of a Hib conjugate vaccine, several ELISA (enzyme-linked immunosorbent assay) methods were needed as an additional tool to enable testing of in process (IP) samples.

In this paper, three of the ELISA's that have been very valuable during the process development, implementation and scaling up are highlighted. The PRP-ELISA, was a very efficient tool in testing in process (IP) samples generated during the development of the cultivation and purification process of the Hib-polysaccharide. The antigenicity ELISA, was used to confirm the covalent linkage of PRP and TTd in the conjugate. The anti-PRP IgG ELISA was developed as part of the immunogenicity test, used to demonstrate the ability of the Hib conjugate vaccine to elicit a T-cell dependent immune response in mice.

ELISA methods are relatively cheap and easy to implement and therefore very useful during the development of polysaccharide conjugate vaccines.

Keywords:

• Haemophilus Influenzae type b vaccine
• ELISA
• characterization
• polysaccharide
• conjugate
• PRP
• antigenicity
• immunogenicity
• cultivation
• purification

Abbreviations:

• Hib, Haemophilus Influenzae type b
• PRP, poly-ribosylribitol phosphate (Hib capsular polysaccharide)
• TTd, tetanus toxoid
• PRP-T, Hib vaccine (PRP conjugated to tetanus toxoid)
• BSA, bovine serum albumin
• PBS, phosphate buffered saline
• TMB, tetramethyl benzidine
• ELISA, enzyme-linked immuno sorbent assay
• NMR, nuclear magnetic resonance
• IPC, in process control
• QC, quality control
• IgG, immunoglobulin G
• HPSEC, high performance size exclusion chromatography
• RI, refractive index
• UV, ultraviolet
• tR, retention time
• Mw, weight-average molecular weight
• Mn, number-average molecular weight
• Mr, molecular weight
• kDa, kilo dalton
• ADH, adipic acid dihydrazide
• RIVM, The National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu)
• NVI, Netherlands Vaccine Institute
• Intravacc, Institute for Translational Vaccinology
• NIH, National Institutes of Health
• WHO, World Health Organization
• EP, European Pharmacopeia
• NIBSC, National Institute for Biological Standards and Control (UK)

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are indebted to all Intravacc (originating from RIVM/ NVI) personal who contributed to the Hib technology transfer project especially Annemarie Bowman-Kelder, Robert van der Put and Rudy Hertroys for generating useful data and to Gideon Kersten for reviewing the manuscript.

Funding

The Hib technology transfer project was funded by the various partners (BF, BE, SII, and Glovax/ SIBP), through bilateral license agreements. In the beginning a seed capital from Intravacc was used to study the feasibility of the project and generate proof-of-concept data.