Fig. 1. Schematic representation of the vaccine sample procedure.

Fig. 2. Effect of mobile phase composition on NPS-RP-HPLC separation of influenza virus constituents. Five microliters of trivalent bulk mixtures were separated on a MICRA^® HPLC NPS-ODSI column (4.6 mm × 33 mm with 1.5 μm packing) at 60 °C with a flow-rate of 1 mL/min in linear gradient 5–95% eluent B. Eluent A was 0.1% TFA in water and eluent B was 0.1% TFA in: (A) 100% ACN; (B) 50% ACN and 50% 2-propanol; (C) 49% ACN, 49% 2-propanol and 2% HFIP and (D) 25% ACN and 75% 2-propanol.

Fig. 3. Influence of the addition of detergents to trivalent bulk mixtures on the chromatographic profile of influenza virus constituents. (A) Control (evaporated sample without detergent); (B) 0.1% Zwittergent^®3-14; (C) 1.2% OGL; (D) 0.3% Brij^®35; (E) 0.3% Triton X-100; (F) 0.1% SDS; (G) 0.1% Zwittergent^®3-14, 1.2% OGL and 0.3% Brij^®35 and (H) 0.1% Zwittergent^®3-14 and 1.2% OGL. All samples were incubated during 60 min with 6 mM DTT. 2.5 μL of treated mixtures were injected and separated under the same conditions as in Fig. 2D.

Fig. 4. Influence of the addition of organic solvents to trivalent bulk mixtures on the chromatographic profile of influenza virus constituents. (A) Control (sample diluted in water); (B) 2,2,2-trifluoroethanol; (C) trifluoroacetic acid and (D) 1,1,1,3,3,3-hexafluoro-2-propanol. 2.5 μL of treated mixtures were injected and separated under the same conditions as in Fig. 2D.

Fig. 5. Chromatographic profile of a influenza trivalent vaccine after the optimized sample treatment using Zwittergent^®3-14, OGL and 1,1,1,3,3,3-hexafluoro-2-propanol as solubilizing agents. 2.5 μL of treated mixtures were injected and separated under the same conditions as in Fig. 2D.

Fig. 6. Chromatographic analysis of three monovalent preparations from the strains (A) A/New Caledonia/20/99 (H1N1); (B) A/Wyoming/03/2003 (H3N2) and (C) B/Jiangsu/10/2003. Solid lines show the separations of 2.5 μL of the monovalent bulk material after optimized sample treatment and dotted lines represent the separation of the solubilized antigens. Separation conditions were described under Fig. 2D.

Influenza virus proteins identified in vaccines prepared as described in Section 2

Reproducibility of retention times (t_r) and corrected areas for the same day, three different days and 10 different samples. All conditions as in Fig. 5