The synthesis and characterization of a Hepatitis B virus vaccine (HBsIC-ISS) candidate composed of Hepatitis B surface antigen (HBsAg) bionanoparticles conjugated to multiple copies of immunostimulatory sequence oligodeoxynucleotides is presented. An efficient tangential flow filtration (TFF) method has been developed to purify the conjugated bionanoparticles from the excess conjugation reagents. The TFF technique presented can serve as a rapid and convenient alternative to current methods like ultracentrifugation for the separation of excess small molecule/polymeric conjugation reagents from chemically modified viruses and other viruslike particles.
We have synthesized and characterized
a novel phosphorothioate
CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate
adjuvant, termed DV230-Ficoll. This adjuvant was constructed from
an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and
the CpG-ODN DV230. Herein, we describe the evaluation of the purity
and reactivity of linkers of different lengths for CpG-ODN-Ficoll
conjugation, optimization of linker coupling, and conjugation of thiol-functionalized
CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical
characterization of independently produced lots of DV230-Ficoll reveal
a bioconjugate with a particle size of approximately 50 nm and covalent
attachment of more than 100 molecules of CpG per Ficoll. Solutions
of purified DV230-Ficoll were stable for at least 12 months at frozen
and refrigerated temperatures and stability was further enhanced in
lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll
conjugate demonstrated improved in vitro potency for induction of
IFN-α from human peripheral blood mononuclear cells and induced
higher titer neutralizing antibody responses against coadministered
anthrax recombinant protective antigen in mice. The processes described
here establish a reproducible and robust process for the synthesis
of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant
suitable for manufacture and use in vaccines.
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