Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine.

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Site-specific PEGylation at histidine tags.

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.

Development of an ELISA-Based Potency Assay for Inactivated Influenza Vaccines Using Cross-Reactive Nanobodies.

Inactivated vaccines are the main influenza vaccines used today; these are usually presented as split (detergent-disrupted) or subunit vaccines, while whole-virus-inactivated influenza vaccines are rare. The single radial immune diffusion (SRD) assay has been used as the gold standard potency assay for inactivated influenza vaccines for decades; however, more recently, various alternative potency assays have been proposed. A new potency test should be able to measure the amount of functional antigen in the vaccine, which in the case of influenza vaccines is the haemagglutinin (HA) protein. Potency tests should also be able to detect the loss of potency caused by changes to the structural and functional integrity of HA. To detect such changes, most alternative potency tests proposed to date use antibodies that react with native HA. Due to the frequent changes in influenza vaccine composition, antibodies may need to be updated in line with changes in vaccine viruses. We have developed two ELISA-based potency assays for group 1 influenza A viruses using cross-reactive nanobodies. The nanobodies detect influenza viruses of subtype H1N1 spanning more than three decades, as well as H5N1 viruses, in ELISA. We found that the new ELISA potency assays are sensitive to the nature of the reference antigen (standard) used to quantify vaccine antigens; using standards matched in their presentation to the vaccine type improved correspondence between the ELISA and SRD assays.

The influence of the closure format on the storage stability and moisture content of freeze-dried influenza antigen.

Low moisture content is seen as crucial to achieving long term stability of freeze dried biologics and reference materials. Highly hygroscopic freeze-dried material are susceptible to moisture ingress over time which can lead to degradation and loss of biological potency. This study compared vials with unprocessed stoppers, vials with vacuum-oven dried stoppers and glass ampoules in order to determine the superior long term storage format in terms of moisture ingress and potency. B/Phuket influenza antigen was chosen as the model biological standard and the lyophilized antigen was stored at -20, 25 and 45 °C over a 1 year period. Ampoules had no significant moisture change across all storage temperatures as would be anticipated. Moisture content results at -20 °C showed no significant differences between ampoules, vials with vacuum-oven dried stoppers and vials with unprocessed stoppers over 12 months. Vials with vacuum-oven dried stoppers performed similarly to ampoules at -20 °C and 20 °C, but had a small increase in moisture content after 6 months at 45 °C. Vials with unprocessed stoppers preformed the worst and exhibited the largest moisture ingress after just 3 months at both 20 °C and 45 °C. Single radial immunodiffusion (SRD) potency assays showed at -20 °C and 20 °C there was no significant difference between all closure formats. At 45 °C there was a drop in potency for all closure formats, but ampoules and vials with vacuum-oven dried stoppers retained higher potency than vials with unprocessed stoppers. Thus, while ampoules are still considered to be the gold standard format for long term storage stability, using vials with vacuum-oven dried stoppers provides comparable stability and moisture integrity at -20 °C and 20 °C storage.