Improving influenza vaccine quantification; a comparative ... · Improving influenza vaccine quantification; a comparative study of SRID and SPR References 1. Wood, J.M., Schild,

Improving influenza vaccine quantification; a comparative

study of SRID and SPR

C. Estmer Nilsson, S. Abbas, M. Bennemo, A. Larsson, M. Hämäläinen and Å. Frostell-Karlsson GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

First presented at: Vaccine Technology II Meeting, Portugal, 2008

2 / Improving influenza vaccine quantification; a

comparative study of SRID and SPR

Background

Reliable analytical tools are valuable for economical process development, production and batch release of vaccines. The current method recommended by the European Pharmacopoeia and WHO for influenza virus titer determination, single radial immuno-diffusion assay (SRID), is straightforward and easy but leaves plenty of room for improvement when it comes to assay variability and speed of analysis.



Background

In addition to having low sensitivity and precision, the method is both time-consuming and quite labor-intensive. In this study, a Biacore™ assay based on surface plasmon resonance-technology (SPR) was developed to achieve improved performance for concentration determination of the three human influenza virus types (A/H1N1, A/H3N2 and B) from cell derived vaccine process development.



Background

Robustness, precision and detection range were investigated and compared to SRID analysis. In addition, the lack of cross reactivity of different influenza virus subtypes allowed the possibility of introducing a single, combined assay for batch release of influenza vaccine. Results showed the SPR-based assay to have higher sensitivity (detection range 0.5 μg/ml to 10 μg/ml), higher precision and significantly shorter analysis and hands on time compared with SRID.



Assay Setup

Fig 1. Inhibition assay principle (A, B)5. HA (red circles) is first immobilized on the dextran matrix. Virus is then mixed with a fixed concentration of serum and injected over the surface. Free antibodies (not bound to virus at equilibrium) bind to the surface HA, giving a response. Low concentration of virus in the sample (A) gives high antibody binding, while high virus concentration (B) results in low binding level. (C) Overlay plot showing sensorgrams of injected serum mixed with a concentration series of virus standard. Report points (marked as X) are taken before and after injection and response levels are measured between those (arrow). After each injection the surface is regenerated in preparation for a new injection.



Recovery

Fig 2. Serum response levels from a quantification of B/Jiangsu/10/2005. Reference antigen was diluted and mixed with corresponding serum to concentrations between 0.5 and 16 μg/ml. Standard, samples and a control sample (6.0 μg/ml) were analyzed repeatedly on a surface immobilized with recombinant HA B. Calculated concentrations are presented in the table at the right.



In-process samples

Fig 3. Quantification of process samples; results from biosensor and SRID. TBV = trivalent bulk vaccine, MBV = monovalent bulk vaccine, Harvest = supernatant from infected MDCK cell culture, UFD = ultra- and diafiltration, Start = UFD filtrated virus diluted 10x , Eluate = fractions from chromatography , NFF= normal flow filtration, DF= diafiltration, na= not analyzed, LOD= limit of detection. *B/Brisbane MBV and TBV samples were provided by Solvay Pharmaceuticals.



Specificity

Fig 4. Influenza subtype specificity. Binding levels of different strains of sera were analyzed on three surfaces, A/H3N2 (A), A/H1N1 (B) and B (C), in the same experiment. The samples contained either serum, or serum mixed with 2 μg/ml of respective reference antigen.



Vaccine assay; Biacore T200 vs SRID

Fig 5. Analyses of three commercial vaccines. The vaccines were quantified using SRID (red bar) and biosensor (blue bar). The biosensor analysis was performed on one sensor chip, using three surfaces simultaneously immobilized with B, A/H1N1 and A/H3N2 HA proteins respectively, running the trivalent vaccines over all three surfaces in a single experiment. Coefficient of variation (CV %) of the replicates are presented over each column (n=2). The blue line represents the specified concentration from the manufacturer of 30 μg/ml of each strain in the vaccine (15 μg per dose). na = not analyzed in replicates. Vaccines are from season 2006-2007; B/Malaysia/2506/2004, A/H1N1/New Caledonia/20/99 and A/H3N2/Wisconsin/67/2005. *TBV = trivalent bulk vaccine.



Conclusions

Results showed the assay to have higher sensitivity (detection range 0.5 μg/ml to 10 μg/ml), higher precision and significantly lower analysis and hands on time compared with SRID.



References 1.  Wood, J.M., Schild, G.C., Newman, R.W. and Seagroatt V.A. Application of an

improved single-radial-immunodiffusion technique for the assay of haemagglutinin antigen content of whole virus and subunit influenza vaccines. J. Biol Stand. 5(3), 237-247 (1977).

2. European Pharmacopoeia 5th Edition 5.06. Influenza vaccine (whole virion, inactivated, prepared in cell cultures).

3. Guidance for Industry. FDA. 2006. Characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious diseases.

4. Estmer Nilsson, C., Abbas, S., Bennemo, M., Larsson, A., Hämäläinen, M. and Å. Frostell-Karlsson. A novel assay for influenza virus quantification using surface plasmon resonance. Vaccine 28, 759-766 (2010).

5. Karlsson, R., Ståhlberg, R. Surface plasmon resonance detection and multispot sensing for direct monitoring of interactions involving low-molecular-weight analytes and for determinination of low affinities. Anal Biochem. 228 (2), 274-280 (1995).



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Improving influenza vaccine quantification; a comparative ... · Improving influenza vaccine quantification; a comparative study of SRID and SPR References 1. Wood, J.M., Schild,