Biosensor based quantification analyses for process development: Influenza vaccine production and

plasma fractionation Åsa Frostell-Karlsson, Johan Kärnhall, Tomas Björkman, Martin Hall and Camilla Estmer Nilsson. GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden

First presented at the Plasma Product Biotechnology Meeting, May 9–13, 2011, in Cyprus.

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Introduction •  Reliable analytical tools are important for economical

process development, production and batch release of vaccines and human plasma proteins.

•  A biosensor assay, using BiacoreTM T200 (1), was compared with single radial immunodiffusion assay (SRID) (2) regarding variability, sensitivity and speed of analysis for concentration determination of human influenza virus in process development.

•  Biacore analysis was also used for quantification of total IgG and albumin and the distribution of the IgG subclasses from all steps of chromatographic plasma fractionation. Results were compared with Biuret for total protein concentration and electrophoresis for purity analysis.

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Influenza vaccine quantification Assay setup

Figure 1. Inhibition assay principle (A). HA (red circles) is immobilized on the dextran matrix. Virus is mixed with a fixed concentration of serum and injected over the surface. Free antibodies (not bound to virus) bind to the surface HA, giving a response. Low concentration of virus in the sample gives high antibody binding, and vice versa. (B) 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.

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Influenza vaccine quantification Results: In-process samples

Figure 2. Quantification of process samples;. 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.

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Influenza vaccine quantification Results: Trivalent vaccines

Figure 3 (Continued on next slide). Analyses of three commercial vaccines. The vaccines were quantified using SRID (red) and Biacore™ (blue). The Biacore 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. The bold line represents the specified concentration from the manufacturer of 30 μg/ml of each strain in the vaccine. 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.

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Influenza vaccine quantification Results: Trivalent vaccines

Figure 3 (Continued from previous slide). Analyses of three commercial vaccines. The vaccines were quantified using SRID (red) and Biacore™ (blue). The Biacore 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. The bold line represents the specified concentration from the manufacturer of 30 μg/ml of each strain in the vaccine. 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.

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Plasma fractionation analysis Quantification of human IgG, albumin and IgG subclass distribution

Figure 4. Assay principle. (A) Anti-IgG, or for albumin quantification anti-albumin, (green) is immobilized on the dextran matrix. Plasma sample is injected over the surface and IgG (alt. albumin) binds to the immobilized antibody, resulting in a response. (B) Overlay plot showing sensorgrams of injected IgG (or albumin) standard. Response levels are measured between report points (marked as X) before and after injection. After each injection the surface is regenerated in preparation for a new injection.

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Plasma fractionation analysis Quantification of human IgG, albumin and IgG subclass distribution

Figure 6. IgG standard curve performance over time. Analyses of more than 1000 process samples were performed on the same surface over a time period of over a week, i.e. a master curve may be used giving In-process result in < 10 min. All standards (IgG, albumin, IgG subclasses) were calibrated against an international standard (3).

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Plasma fractionation analysis Quantification of human IgG, albumin and IgG subclass distribution

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Plasma fractionation analysis Quantification of human IgG, albumin and IgG subclass distribution

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Conclusions Biacore™ flu assay, compared with SRID:

• Higher sensitivity (detection range 0.5-10 μg/ml)

• Higher precision

• Shorter analysis and hands on time

Biacore plasma protein assays, compared with traditional methods:

• Specific protein quantification rather than total protein, more reliable data

• Very robust: ~1000 plasma process samples analyzed on the same surface

• Shorter analysis and hands on time. In-process result in <10 minutes

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References 1. Estmer Nilsson, C., Abbas, S., Bennemo, M., Larsson, A., Hämäläinen, M. and

Frostell-Karlsson, Å. A novel assay for influenza virus quantification using surface plasmon resonance. 2010, Vaccine 28, 759-766.

2. Wood, J M., Schild, GC., Newman, RW. and Seagroatt, VA. 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. 1977. 5, 237-247.

3. Blirup-Jensen, S., Johnson, A. M., and Larsen, M. Protein standardization V: value transfer. A practical protocol for the assignment of serum protein values from a Reference Material to a Target Material, Clin. Chem. Lab. Med. 2008, 46, 1470-1479.

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Acknowledgments

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