Selective and Quantitative Detection of Influenza Virus Proteins in

Anal. Chem. 2007, 79, 3164-3172

Selective and Quantitative Detection of Influenza Virus Proteins in Commercial Vaccines Using Two-Dimensional High-Performance Liquid Chromatography and Fluorescence Detection Virginia Garcıá-Canãs, Barry Lorbetskie, Diane Bertrand, Terry D. Cyr, and Michel Girard*

Centre for Biologics Research, Health Canada, Banting Building, Tunney’s Pasture, Ottawa, ON, Canada K1A 0L2

Influenza is a contagious disease of animals caused by the influenza virus. This pathogen is an enveloped virus that belongs to the Orthomyxoviridae family. The classification of influenza viruses as types A, B, and C is based on antigenic differences in their nucleoprotein (NP) and matrix protein (MP1). In addition, type A influenza viruses are classified according to differences in

their major surface antigens, i.e., hemagglutinin (HA) and neuraminidase (NA). In the last decades, types A and B influenza viruses have been found in human isolates and are responsible for numerous outbreaks around the world.1 Circulating influenza virus strains are under the surveillance of the World Health Organization (WHO) which, in collaboration with several national health authorities and reference laboratories, provides recommendations on the annual composition of influenza vaccines to minimize the incidence of influenza epidemics in the human population. As a result, the manufacturing processes of influenza vaccines are subjected to significant annual changes and the production is frequently hurried. In addition, the heightened fear of an influenza pandemic has raised public health concerns in recent years, and governments have devised emergency preparedness programs in order to respond to such an eventuality. In this context of uncertainty, fast and versatile techniques for antigenic surveillance and vaccine development are urgently required in order to obtain effective vaccines in a timely fashion. In addition, increasing regulatory requirements of purity and overall quality of vaccines2 means that the ability to rapidly detect and characterize specific constituents contained in these preparations is an important feature. Recent reports have demonstrated that new methodologies based on physicochemical techniques could play valuable roles in antigenic surveillance and vaccine development. Among them, new approaches, based on the combination of mass spectrometry and immunoassay, have shown promising results for the rapid characterization of the structure and antigenicity of influenza hemagglutinins.3,4 Studies on the stability of HA in vaccine bulk material have been carried out using fluorescence and circular dichroism spectroscopy.5 Negative stain electron microscopy has also been successfully used to identify differences at the molecular construct level between influenza vaccines produced by different manufacturers.6 Influenza vaccines are evaluated for their content in HA and NA, the major surface antigens. The HA content is traditionally determined by single radial immunodiffusion (SRID) and nominal

* Corresponding author. Tel: 613-952-0399. Fax: 613-941-8933. E-mail: [email protected]. (1) Horimoto, T.; Kawaoka, Y. Nat. Rev. Micobiol. 2005, 3, 591-600. (2) Dellepiane, N.; Griffiths, E.; Milstein, J. B. Bull. World Health Organization 2000, 78, 155-162.

(3) Kiselar, J. G.; Downard, K. M. Biochemistry 1999, 38, 14185-14191. (4) Morrissey, B.; Downard, K. M. Proteomics 2006, 6, 2034-2041. (5) Luykx, D. M.; Casteleijn, M. G.; Jiskoot, W.; Westdijk, J.; Jongen, P. M. Eur. J. Pharm. Sci. 2006, 23, 65-75. (6) Renfrey, S.; Watts, A. Vaccine 1994, 12, 747-752.

In this work, we report on the applicability of twodimensional high-performance liquid chromatography (2D-HPLC) for the comprehensive characterization of inactivated influenza vaccine proteins. This novel procedure features minimal sample treatment and combines the on-line coupling of size exclusion HPLC to reversedphase HPLC. A comparative analysis of commercial vaccines from three different manufacturers showed the method to be highly selective by providing characteristic reproducible chromatographic profiles for each vaccine. In addition, the method provided enhanced sensitivity for most constituents as a result of the use of native fluorescence detection in the reversed-phase HPLC step. The limits of detection (at a signal-to-noise ratio of >3) for hemagglutinin (HA) antigens were 105 and 172 ng/mL for influenza A/New Caledonia/20/99 and B/Jiangsu/10/ 2003 strains, respectively. The potential of this 2D-HPLC procedure in terms of quantitative antigen analysis was assessed by determination of the HA content of commercial vaccines. Results provided very good correlation with nominal HA values. The reproducibility (RSD) of the whole procedure was also evaluated and was found to be better than 2 and 3% for calculated antigen concentrations expressed as micrograms of HA per milliliter in commercial vaccines for samples of the same lot (n ) 5) or different lots (n ) 3), respectively. In addition, it allowed the selective detection of several influenza constituents including nucleoproteins from type A and B viruses and the highly hydrophobic matrix protein 1 from both virus strains.

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10.1021/ac0621120 CCC: $37.00 Published 2007 Am. Chem. Soc. Published on Web 03/16/2007

values of 15 µg of HA per strain per dose (0.5 mL) are used for immunization. In the SRID assay, a reference HA antigen reagent is used for calibration and specific antibodies are required in order to produce immunoprecipitation of the specific antigen in a semisolid medium. Quantitative results are obtained by measuring the radius of the stained immunoprecipitation zone, which correlates with the amount of HA. The main advantage of this technique is its high specificity and low level of technology. Nevertheless, it is labor-intensive, time-consuming, requires the production of antibodies, and is subject to interferences.7 These limitations have encouraged the development of innovative procedures for the characterization of constituents in influenza preparations. For instance, different modes of high-resolution chromatographic techniques have been employed. The potential of size exclusion chromatography (SEC) was demonstrated for the purification of influenza viral components8 and the profiling of vaccine constituents.9 Similarly, their separation by ionexchange chromatography was assessed; however, due to low recoveries, this chromatographic mode was found to be unacceptable.8 Recently, an immunochromatographic method was reported for the quantitative determination of NA in split and attenuated vaccines.10 Likewise, a few reports on the use of reversed-phase chromatography for the analysis of influenza constituents have been published. Early work based on the use of conventional porous sorbents resulted in low recoveries.11 More recent and promising results have been reported using sorbents with large pore size for the detection of HA12 and nonporous sorbents for the selective detection of several influenza constituents, including HA, NP, and MP1 in influenza bulk material and commercial vaccines.13 In the latter case, an elaborate sample processing step is required to obtain detectable levels of many constituents in diluted samples such as influenza trivalent vaccines. Although these reversed-phase (RP)-HPLC methods have been shown to provide a good alternative to conventional immunologic methods for the detection of several influenza constituents in bulk preparations, their potential application to the characterization of commercial influenza vaccines obtained by different manufacturing processes has not been reported yet. The annual influenza vaccines contain proteins from three strains (trivalent vaccines), two of which are of type A and one of type B. The vaccines are prepared from the independently grown and purified A and B influenza strains. Vaccines also contain a number of excipients that are added to enhance stability of the constituents toward chemical and enzymatic degradation and prevent aggregation. This implies that no less than 30 components are present in vaccines. In addition, both HA and NA are glycosylated proteins and exist as glycoform populations, adding to the complexity of the mixture. (7) Willkommen, H.; Platen, S.; Staber, H. Acta Virol. 1983, 27, 407-411. (8) Calam, D. H.; Davidson, J. J. Chromatogr. 1984, 296, 285-292. (9) Girard M., Garcia-Can ãs V., Lorbetskie B., Cyr T.D., Bertrand D., Hefford M.A.; Smith S. J. Chromatogr., A, Submitted (10) Tanimoto, T.; Nakatsu, R.; Fuke, I.; Ishikawa, T.; Ishibashi, M.; Yamanishi, K.; Takahashi, M.; Tamura, S. Vaccine 2005, 23, 4598-4609. (11) Phelan, M. A.; Cohen, K. A. J. Chromatogr. 1983, 266, 55-66. (12) Kaptein, J. C.; Saidi, M. D.; Dijkstra, R.; Kars, C.; Tjon, J.; Weverling, G. J.; de Vocht, M. L.; Kompier, R.; van Montfort, B. A.; Guichoux, Y. J.; Goudsmit, J.; Lagerwert, F. M. Vaccine 2006, 14, 3137-3144. (13) Garcia-Can ãs, V.; Lorbetskie, B.; Girard, M. J. Chromatogr., A 2006, 1123, 225-232.

The recent application of multidimensional analytical techniques to provide enhanced specificity for the analysis of complex mixtures produced by the pharmaceutical industry has been striking.14 Among these sophisticated techniques, multidimensional liquid chromatography has emerged as a separation technique that provides increased peak capacity and concomitant resolution.15 Of particular note is the extensive application of multidimensional liquid chromatography within the proteomic research field during the past decade;16-19 however, its application to characterization of biopharmaceuticals is rarely reported. In this work, the capabilities of two-dimensional highperformance liquid chromatography (2D-HPLC) for the characterization of inactivated influenza vaccine constituents have been explored. A novel procedure requiring minimal sample pretreatment and combining the on-line coupling of SEC to RP-HPLC with native fluorescence detection has been developed. The selectivity of the method has been examined through a comparative analysis of commercial vaccines from three different manufacturers. In addition, the potential of this 2D-HPLC procedure in terms of the quantitative analysis of antigens is assessed by determination of the HA content of commercial vaccines. EXPERIMENTAL SECTION Chemicals. All chemicals were of analytical reagent grade and used as received. Sodium chloride, sodium phosphate, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), trifluoroacetic acid (TFA), and iodoacetamide were from Sigma (St. Louis, MO) and trypsin, mass spectrometry grade, was obtained from Promega (Madison, WI). Acetonitrile (ACN) and 2-propanol were purchased from Merck KGaA (Darmstadt, Germany). Distilled water was deionized on a Nanopure Diamond system (Barnstead International, Dubuque, IA). Samples. Trypsin inhibitor (MW 20 kDa), trypsinogen (MW 25 kDa), and carbonic anhydrase (MW 29 kDa) were obtained from Sigma. The lyophilized proteins were dissolved in 0.5% SDS and 12 mM DTT and incubated in a water bath at 100 °C for 3 min prior to analysis. Trivalent influenza vaccines A, B, and C, produced by three different manufacturers, were used: three lots of each of vaccines A and B produced in season 2005-2006; two lots of vaccine C from 2005 to 2006, one lot each of vaccine A from 2000 to 2001, 2001-2002, and 2002-2003, and vaccine B from 2001 to 2002 and 2002-2003. The virus strains used to producetrivalentvaccineswereA/NewCaledonia/20/99,A/Panama/ 2007/99, and B/Yamanashi/166/98 in season 2000-2001; A/New Caledonia/20/99, A/Panama/2007/99, and B/Victoria/504/2000 in season 2001-2002; A/New Caledonia/20/99, A/Panama/2007/ 99, and B/Hong Kong/330/2001 in season 2002-2003; and A/New Caledonia/20/99, A/New York/55/2004, and B/Jiangsu/ 10/2003 in season 2005. Prior to 2D chromatographic analysis, trivalent vaccines samples were treated as follows: 10 µL of a (14) Hoke, S. H.; Morand, K. L.; Greis, K. D.; Baker, T. R.; Harbol, K. L.; Dobson, R. L.; Int. J. Mass Spectrom. 2001, 212, 135-196. (15) Giddings, J. C. High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10, 319-323. (16) Wagner, K.; Racaityte, K.; Unger, K. K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. Chromatogr., A 2000, 893, 293-305. (17) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247. (18) Vollmer, M.; Horth, P.; Nagele, E. Anal. Chem. 2004, 76, 5180-5185. (19) Shin, Y. K.; Lee, H. J.; Lee, J. S.; Paik, Y. K. Proteomics 2006, 6, 11431150.

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solution containing 8% SDS and 192 mM DTT was added to 150 µL of the vaccine samples, and then the samples were incubated in a water bath at 100 °C for 3 min. Trivalent vaccines used in 1D-HPLC separations were treated as follows: 10 µL of a solution containing 192 mM DTT was added to 150-µL aliquots of the vaccine, and then the samples were incubated in a water bath at 100 °C for 3 min. Lyophilized hemagglutinin antigen reagents from A/New Caledonia/20/99 (H1N1) (IVR-116 reassortment) with a content of 54 µg of HA/vial and B/Jiangsu/10/2003 with a content of 121 µg of HA/vial were obtained from the Office of Vaccines Research and Review, Center for Biologics Evaluation and Research (Food and Drug Administration). Chromatographic Separations. (a) Instrumentation. The fully automated 2D-HPLC instrument consisted of a Waters Alliance 2695 chromatograph equipped with an autosampler, a sample cooling device, and an electronically controlled 2-position, 10-port valve (Waters). The valve enabled the loading of the primary column (size exclusion column) effluent onto the secondary column (reversed-phase column) through a 200-µL sample loop. In one of the two positions, the effluent from the primary column filled the loop. When the valve was turned to the second position, the effluent from the primary column flowed to waste, while the effluent inside the loop was directed to the secondary column. The detection of the analytes from the first dimension was performed with a Waters 2487 dual λ absorbance detector with 10-mm-path length flow cell. The second dimension consisted of a 1525µ Binary HPLC pump system equipped with a column heater and two detectors connected in series: the first was a Waters 2996 photodiode array detector with a 10-mm-path length flow cell, and the second was a Waters 2475 multichannel fluorescence detector with 8-µL flow cell volume working at λex 290 nm and λem 335 nm. In each analysis, the valve was programmed to turn twice from one position to the other. The first time, the turn allowed the transfer of the fraction from the primary to the secondary column through the 200-µL loop. The second time, the valve was switched after the secondary column had been re-equilibrated, restoring the system to the initial conditions for the next run. Data acquisition and integration were performed with Empower Pro Software from Waters. A matrix file was constructed in MS Excel using chromatographic data obtained from 2D analysis. The matrix was read by Matlab software (The Mathworks, Inc.) and presented as a 2D contour plot. 2D plots represent the separation taking place in the first dimension (SEC) along the x-axis, with a time scale from 8.0 to 11.5 min, while separations occurring in the second dimension (RP-HPLC) are shown along the y-axis, with a time scale of 35 min. For one-dimension experiments (1D-HPLC), reversed-phase HPLC separations were performed in a single-column instrument configuration using a Waters Alliance 2695 chromatograph equipped with a column heater and connected to a photodiode array and a fluorescence detectors connected in series. (b) Separation Conditions. The samples were stored in vials at 22 °C in the temperature-controlled tray of the autosampler. The chromatographic columns used in this investigation were TSKgel G4000SWXL 300 × 7.8 mm, 8-µm particles, 450-Å pore size (TosoHaas, Montgomeryville, PA) for the first dimension and MICRA HPLC NPS-ODSI, 4.6 mm × 33 mm, 1.5-µm particles 3166


(Eprogen, Darien, IL) for the second dimension. Specific conditions for each column were as follows. For size exclusion column (first dimension), isocratic elution at 1 mL/min with phosphate buffer (0.1 M sodium phosphate, 0.2 M NaCl, pH 7) containing different amounts of SDS. When the valve turns to the second position, the following sequence was used for reversed-phase chromatographic separations at a flow rate of 0.5 mL/min: (1) salt-washing step by flushing with 95%:5% (A/B) until gradient elution starts at minute 20. Eluent A was 0.1% aqueous TFA and eluent B was 0.1% TFA in 25% ACN and 75% 2-propanol; (2) gradient elution for solvent B was as follows, 5-20% in 15 min, 20-26% in 3 min, 26-31% in 5 min, 31-40% in 4.5 min and 4095% in 25 min; (3) strongly adsorbed components were eluted off the column by flushing with 5%:95% (A/B) for 8 min at the end of the gradient, and (4) re-equilibration of the column was achieved by flushing with 95%:5% (A/B) for 5 min. Mobile phases were filtered through 0.2-µm nylon filter membranes (Phenomenex, Torrance, CA). For 1D-HPLC experiments, the gradient elution started at 1 min after the injection and was the same as that used for the reversed-phase chromatographic separations in the 2D-HPLC experiments. (c) Mass Spectrometry Analysis. The proteins were identified by mass spectrometric analysis of fractions corresponding to peaks eluting from the secondary column collected from 4 × 100 µL injections of vaccine samples. Pooled fractions from successive runs were first reduced with 100 µL of 100 mM DTT for 80 min and then alkylated with 210 µL of 500 mM iodoacetamide for 90 min at room temperature and followed by membrane filtration (Amicon, 10 k MWCO; Millipore Corp., MA) and buffer exchange to 150 µL of 50 mM ammonium bicarbonate. The filtration retentates were digested with 2 µL of 0.1 µg/µL trypsin at 37 °C. After overnight digestion, the solution was reacidified by adding 1 µL of 10% TFA and 10 µL of 2% formic acid. The solution was evaporated to ∼50 µL by vacuum centrifugation, and subsequently, 20 µL was transferred to a HPLC vial and then analyzed by mass spectrometry. The mass spectrometer used was a Waters MicroMass Global (QTOF) system coupled to a Waters capillary HPLC. The tryptic peptides were separated on a Waters capillary HPLC column (Atlantis C18, 3-µm-diameter packing, 75 µm × 150 mm NanoEase column at controlled room temperature (22 °C). The autosampler parameters included temperature control (15 °C), partial loop injection, 2-µL injection volume, and a sample loading flow rate of 20 µL/min for 3 min onto a trap (Atlantis dC18) bypassing the analytical column. The 10-port valve was then switched to place the trap in line with the analytical column. The column flow rate was maintained at 250 nL/min by splitting the gradient, which was delivered from the pump at a flow rate of 10 µL/min. The linear binary gradient was formed from A (0.2% formic acid, 2% acetonitrile, and 97.8% water) and B (0.2% formic acid, 10% water, and 89.8% acetonitrile). The gradient elution for B was as follows: 2% for 3 min, 2-10% in 2 min, 10-40% in 30 min, and 40-100% in 5 min. The system was maintained at 100% B for 5 min, and then returned to 2% B in 0.5 min, and finally allowed to re-equilibrate for 14.5 min. The capillary HPLC system was checked at the beginning of each day with duplicate injections of a mixture of peptides (HPLC peptide standard mixture from Sigma, which had

been diluted to 100 fmol/µL, 1.0-µL injection volume). The accuracy of the mass spectrometer was also checked each day, after calibrating with 100 fmol/µL [glu1]-fibrinopeptide B human (glufib) in 50% acetonitrile, 49.8% water, and 0.2% formic acid, by reanalyzing the glufib using the new calibration, experimental deviation from the calculated masses was determined with the maximum allowed single point deviation being 25 ppm and the maximal average deviation being 10 ppm. The mass spectrometer was operated in the positive ion electrospray mode over the mass range 450-1500 amu in the continuum mode, with a scan time of 0.9 and 0.1 s interscan time, a source temperature of 80 °C, a capillary voltage of 3.0 kV, and no nebulizer or drying gas. The system was operated in a data-directed analysis mode wherein MS/MS were automatically collected if the characteristics of the ion exceeded the selected limits. In this case, these limits related to peak intensity (>10 counts/s) and excluded the possibility of having detected the same ion within the previous 60 s. When an ion of sufficient intensity was detected in the MS scan (survey scan), a MS/MS was obtained over the mass range 100-1900 amu. The system was programmed to obtain the MS/MS spectra for up to three ions per survey scan with the collision energy varying depending on the charge state and m/z value of the ion; for example, for the +2 charge state, the collision voltage increased from 25 V at 450 amu to 55 V at 1200 amu versus 11 V at 450 amu and 26 V at 1000 amu for the +3 charge state. Argon pressure in the collision cell was kept at an optimal pressure at all times, such that resolution (mass/fwhh >9500) was maintained in the MS mode and good fragmentation was obtained in the MS/ MS mode. The proteins were identified using an in-house copy of Mascot (Matrix Science) software with the parameter setup to use the NCBInr database, to allow one missed tryptic cleavage, carbamidomethyl cysteine modification, and variable modification to include methionine oxidation and deamidation. The peptide MS and MS/MS tolerances were set to 0.5 amu for the charge state to +2 and +3. The minimal acceptance criterion was set to >95% confidence that the identified protein or a highly homologous protein was found. RESULTS AND DISCUSSION 2D-HPLC Setup. Recently, the characterization of influenza vaccines has been successfully approached by reversed-phase chromatography using nonporous silica-based columns.13 These columns provided very good selectivity for the separation of major viral constituents in influenza preparations; however, in order to achieve detectable levels of proteins in commercial vaccines, a sample concentration step based on evaporation and addition of solubilizing agents was necessary. Although the selectivity of the method allowed for the detection of several of the major proteins (such as NP, MP1, and HA) within the same analysis, the separation of other constituents was unsatisfactory. The potential of size exclusion chromatography for the purification of viral components has been demonstrated.8,20 In our hands, influenza trivalent vaccines produced by several manufacturers have been examined using this chromatographic mode over the last years. Our data suggest that chromatographic profiles (20) Welling, G. W.; Slopsema, K.; Welling-Wester, S. J. Chromatogr. 1986, 356, 307-314.

could be used to discriminate between vaccines produced by different manufacturing processes.9 Although this technique provides important information about the composition and the state of aggregation of the influenza constituents in vaccines, insufficient resolution prevents the complete separation of several analytes. In order to circumvent the selectivity limitations mentioned above, a strategy based on the use of 2D-HPLC was envisaged. Given that both RP-HPLC using nonporous silica columns13 and size exclusion chromatography possess a demonstrated potential for the analysis of influenza vaccines, a 2D-HPLC system based on these two chromatographic modes was designed. SEC was chosen as the first dimension since, in addition to size fractionation, it allows high sample loading (i.e., high injection volumes) and consequently would provide a means of minimizing the need for sample preconcentration. As a second dimension, RP-HPLC offers an orthogonal separation mode based on hydrophobicity and has high peak capacity and selectivity. The setup involved, as an initial step, the isocratic elution of the SEC column (primary column) with a phosphate buffer, resulting in a partial size-based separation of vaccine constituents. Fractions eluting from the primary column were automatically collected with a 200-µL loop, loaded into the reversed-phase column (secondary column), and, finally, separated under gradient elution conditions. Significant improvements in sensitivity and selectivity for the detection of proteins in the RP-HPLC dimension were obtained by using native fluorescence detection in addition to UV detection. Selectivity of the 2D-HPLC Method. The applicability of the 2D-HPLC setup was first assessed with a mixture of three standard proteins of similar molecular weights (i.e., trypsin inhibitor, 20 kDa; trypsinogen, 25 kDa; and carbonic anhydrase, 29 kDa) using generally applicable elution conditions. Under these conditions, the three components were expected to be separated solely in the second dimension since the type of column used in the first dimension (SEC) does not resolve compounds with such close molecular weights. The effluent from the primary column peak containing the three proteins was sampled in the loop and transferred automatically into the secondary column. Figure 1 shows chromatograms obtained from the first (Figure 1, inset) and second dimension (Figure 1). As expected, the three proteins eluted as a single peak in the primary column (peak P, Figure 1, inset) and were well separated in the secondary column (peaks 1-3, Figure 1). Despite the availability of 1D chromatographic methods for influenza vaccines based on the chosen separation modes (that is, SEC and RP-HPLC), their direct coupling into a 2D system was not straightforward. Modifications to the SEC separation conditions were required in order to render them compatible with the RP-HPLC step. For highly hydrophobic proteins, among which are viral envelope proteins such as those found in influenza vaccines, adequate SEC methods require conditions to overcome aggregation.21 This is generally accomplished by the addition of detergents, denaturants, or organic solvents to the mobile phase to improve solubilization of hydrophobic proteins on-column, preventing aggregation as well as nonspecific interactions with (21) Welling, G. W.; Van der Zee, R.; Welling-Wester, S. In HPLC of biological macromolecules; Gooding, K. M., Regnier, F. E., Eds.; Marcel Dekker: New York, 1990; pp 373-401.


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Figure 1. 2D-HPLC analysis of a mixture of three protein standards. Chromatogram from the second dimension; peak 1, trypsinogen; peak 2, trypsin inhibitor; and peak 3, carbonic anhydrase. Inset: chromatogram from the first dimension by SEC; the fraction eluting between 10.3 and 10.5 (peak P) was transferred to the secondary column for analysis in the second dimension.

the sorbent. In our hands, SDS was found to be the most efficient detergent for the separation of influenza vaccine constituents by SEC.9 However, the presence of large amounts of SDS on the reversed-phase column has been shown to result in coelution of some compounds.13 Consequently, it became imperative to minimize the impact of SDS on the resolution of the analytes in the second dimension in order to achieve the maximum compatibility between both dimensions. Mobile phases containing different concentrations of SDS were prepared, and their effects on the analysis of commercial vaccine samples were investigated. Fractions eluting between 9.6 and 9.8 min on the SEC column were sampled in the 200-µL loop, loaded into the secondary column, and then separated under gradient elution (Figure 2). Apart from late-eluting peaks between 12.5 and 16.0 min related to excipients and small molecular weight compounds, at a concentration of 0.01% SDS, no peaks were detected in the first dimension (Figure 2A, inset), and as a result, the second dimension analysis displayed a flat baseline (Figure 2A). An increase in SDS concentration to 0.05% improved significantly the detection of constituents in the first dimension (Figure 2B, inset). The resulting separation in the second dimension also provided a satisfactory separation of the three major surface antigens from the three different strains of the virus (peaks 1, 2, and 3, Figure 2B) as reported previously13 as well as good recoveries of other compounds. At higher SDS concentrations in the SEC mobile phase, comparable levels of protein recoveries were obtained in the first dimension analysis (Figure 2C, inset, and D, inset). However, chromatograms obtained in the second dimension displayed both a progressive loss of resolution in the separation of the major antigens and a decrease in protein recovery (Figure 2C and D). These data clearly demonstrated the detrimental effect of SDS on the separation in the second dimension and, hence, the necessity to adjust the concentration of the detergent in order to achieve enough protein recovery during the first dimension without compromising the 3168 Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

Figure 2. Effect of SDS on the 2D-HPLC analysis of influenza vaccine A. (A-D) RP-HPLC analysis (second dimension) of fractions eluting from the size exclusion column under different conditions. Insets in A-D display the size exclusion chromatographic analysis (first dimension) of the sample using the following mobile-phase compositions: phosphate buffer (0.1 M sodium phosphate, 0.2 M NaCl, pH 7) containing 0.01 (A), 0.05 (B), 0.5 (C), and 1% SDS (D). Programmed valve was switched to sample the effluent between min 9.6 and 9.8 into the RP-HPLC column. Peaks 1, 2, and 3 correspond to HA1 subunits from influenza B and influenza B subtype H3N2 and H1N1, respectively.

resolution in the second dimension. From these considerations, a concentration of 0.05% SDS in the SEC mobile phase was found to be optimal and was selected for use in subsequent analyses. Modifications of the RP-HPLC conditions were also required in order to improve separations of the analytes. Among these modifications, a washing step was performed to prevent salt contained in the transferred fraction from precipitating on the reversed-phase column. The flow rate was also decreased from 1 to 0.5 mL/min to avoid difficulties with back pressure. Finally, the gradient was adjusted for a better resolution of the influenza vaccine constituents. Selectivity improvements of the 2D configuration were highlighted by comparison with the 1D RP-HPLC method. For 1D analyses, trivalent vaccine samples were reduced with DTT in the absence of SDS because of the deleterious effect of SDS on the separation of influenza constituents for this type of column.13 Subsequently, 50 µL of sample was injected onto the reversedphase column and separated under the same gradient elution described for the 2D-HPLC (see Experimental Section). For 2D analyses, separations were carried out by injecting 100 µL of treated sample onto the size exclusion column and collecting the eluent at selected times for subsequent chromatographic analysis in the reversed-phase column. The improved selectivity of the 2D system is illustrated in Figures 3 and 4. For example, peaks 12, 13, and 14 are only partially resolved in the 1D RP-HPLC system

Figure 3. Comparison of 1D-reversed-phase chromatographic analysis of vaccine A (season 2005-2006) between 1D- and 2DHPLC methods. The 50 µL of sample was injected directly onto the RP-HPLC column and separated under gradient elution (A); 100 µL of sample was injected onto the size exclusion column and the eluted fractions (B) and (C) were sampled into the RP-HPLC column and separated under gradient elution. The inset displays the size exclusion chromatogram (first dimension) and the sampled fractions. Fraction B, from minute 11.2 to 11.4; fraction C, from minute 10.2 to 10.4. Other separation conditions as in Figure 2B. Peaks: 12, MP1 from influenza A; 13, chain B influenza virus M1 protein; 14, MP1 from influenza B.

(Figure 3A). The same peaks were completely resolved in the 2D-HPLC system as a result of the sample fractionation in the size exclusion column (Figure 3B and C). In this instance, peak 13 was found to be a component of fraction B of the SEC analysis (Figure 3 inset). Similarly, the separation of one of the major surface antigens (peak 1, Figure 4A) was hampered by the interfering signal from a vaccine excipient (peak 15, Figure 4A) in the 1D configuration. Moreover, this coelution problem imposed restrictions to the quantitative analysis of the antigens. Using the 2D-HPLC method, the interfering excipient was separated from the compound of interest (Figure 4B and C). These results demonstrate that the 2D-HPLC method provided enhanced selectivity for the separation of compounds of interest in the samples. Characterization of Influenza Vaccines and Identification of Constituents. In general, the preparation of inactivated influenza vaccines from viral suspensions consists of several steps. Among them are virus inactivation and disruption, steps that involve the use of detergents or organic solvents approved by the national control authority. The viral components are subsequently concentrated and purified. Depending on the stringency of the purification step, inactivated vaccines can be classified as split virus vaccines or subunit vaccines. Vaccine producers have adopted

Figure 4. Comparison on reversed-phase chromatographic analysis of vaccine B (season 2005-2006) between 1D- and 2D-HPLC methods. The 50 µL of sample was injected directly onto the RPHPLC column and separated under gradient elution (A); 100 µL of sample was injected onto the size exclusion column and the eluted fractions (B) and (C) were sampled into the RP-HPLC column and separated under gradient elution. The inset displays the size exclusion chromatogram (first dimension) and the sampled fractions. Fraction B, from minute 11.2 to 11.4; fraction C, from minute 9.8 to 10.0. Other separation conditions as in Figure 2B. Peaks 1, 2, and 3 correspond to HA1 subunits from influenza B and influenza A subtype H3N2 and H1N1, respectively. Peak 15, vaccine excipient.

different procedures to carry out the preparation of commercial vaccines, and as a result, the composition of influenza vaccines has been reported to differ among manufacturers.6,24 Differences in composition among three commercial vaccines produced by different manufacturers in the season 2005-2006 were assessed using the 2D-HPLC methodology. Panels A-C in Figure 5 show contour plots generated from 2D-HPLC analyses of vaccines A, B, and C, respectively. While plots showed many of the same components, marked differences were apparent. For instance, clear differences were observed in the low molecular weight SEC fraction eluting between 11.3 and 11.5 min, where spots 10 and 15 were detected solely in the analysis of vaccine B (Figure 5B). In addition, vaccine C (Figure 5C) could be distinguished from vaccine A (Figure 5A) by the presence of spot 6 in the former. The analysis of fractions containing higher molecular weight compounds also showed clear differences in composition. For example, spots 5 and 11 were only detected in the analysis of vaccine B (Figure 5B), and their presence along with spots 10 and 15 provided a distinct profile for this vaccine. In addition, (22) Arzt, S.; Baudin, F.; Barge, A.; Timmins, P.; Burmeister, W. P.; Ruigrok, R. W. Virology 2001, 279, 439-446. (23) Wood, J. M.; Schild, G. C.; Newman, R. W.; Seagroatt, V. J. Biol. Stand. 1977, 5, 237-247. (24) Ku ¨ rsteiner, O.; Moser, C.; Lazar, H.; Durrer, P. Vaccine In press.


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Figure 5. 2D plots obtained from the 2D-HPLC analysis of influenza vaccines produced in season 2005-2006: (A and D) vaccine A; (B) vaccine B; (C and F) vaccine C; and (E) vaccine A produced in season 2000-2001. Samples in A, B, C, and E were treated with DTT and SDS reagents as indicated in Experimental Section. Samples D and F were injected directly without previous sample treatment. For spot numbers and asterisks, refer to the text. Table 1. Identified Influenza Virus Proteins spot no.

MW (kDa)

Accession Number

name

no. of peptides

Mowse score

1 2 3 4 5 7 12 13 14

38 37 37 57 57 62 28 18 28

ABB02186 AAG47816 BAC82888 CAA91086 CAA91086 AAD29167 AAM75161 1EA3_B AAA67100

hemagglutinin [influenza B virus] hemagglutinin [influenza A virus, subtype H3N2] hemagglutinin [influenza A virus, subtype H1N1] nucleoprotein [influenza A virus] nucleoprotein [influenza A virus] nucleoprotein [influenza B virus] matrix protein M1 [influenza A virus] chain B, influenza virus M1 protein matrix protein [influenza B virus]

9 5 12 33 17 19 38 5 44

221 96 250 673 305 453 610 97 591

compounds 12 and 9 were characteristic of vaccines A and C (Figure 5A and C), respectively. A number of spots showing constant or differential patterns among the three vaccine profiles were selected for identification by off-line mass spectrometry. The corresponding peaks were collected and processed for peptide mass mapping and mass spectrometry. The identities of the protein spots along with the data from the peptide mass mapping are summarized in Table 1. The major spots 1, 2, and 3 were detected in the three vaccines and corresponded to HA1 subunit of strain B, HA1 subunit of 3170


strain A [H3N2], and HA1 subunit of strain A [H1N1], respectively. These results are also consistent with SEC data where the three proteins were found in a fraction eluting at a retention time corresponding to that of a globular protein of MWapp ∼45 kDa. Significantly, the presence of NP showed differential patterns between vaccines. In both vaccines A and B, NP from influenza A and B were selectively identified in spots 4 and 7, respectively. However, these components were not present in vaccine C. The absence of NP in vaccine C is consistent with the fact that vaccine C is a subunit vaccine consisting essentially of purified surface

Table 2. Reproducibility of Retention Times (RT) and Peak Areas for Same-Day and Between-Day Replicate Injectionsa first dimension (SEC)b

RT (min) RSD (%) peak area (flu units) RSD (%) a

second dimension (RP-HPLC)c

same sample (n ) 5), same day

same sample (n ) 3), between-day (n ) 3)

same sample (n ) 5), same day

same sample (n ) 3), between-day (n ) 3)

9.77 0.01 5 9502 06 0.04

9.77 0.04 5 986 850 0.69

40.32 0.07 89 268 490 0.70

40.32 0.11 90 019 813 1.50

All conditions as in Figure 2B and 2B inset. b Analysis of peak marked with asterisk (/) in Figure 2B, inset. c Analysis of peak 1 in Figure 2B.

Table 3. Hemagglutinin Content of B/J and A/NC Strains in Commercial Influenza Vaccines Produced in the Different Seasons by Different Manufacturers B/J season

manufacturer (n)a

µg of HA/ mL (SD)b

2005-2006

A, same lot (5) C, same lot (3) A, different lots (3) B, different lots (3) A, same lot (2) B, same lot (2) A, same lot (2) B, same lot (2) A, same lot (2)

29.88 (0.48) 30.41 (0.50) 30.26 (0.85) 30.96 (0.91) ndd nd nd nd nd

2002-2003 2001-2002 2000-01

A/NC % RSD

µg of HA/ mL (SD) b

% RSD

% deviationc

1.62 1.67 2.81 2.94

29.71 (0.55) 30.55 (0.54) 30.41 (0.66) 30.08 (0.52) 28.81 25.49 24.64 19.79 24.89

1.88 1.80 2.19 1.74

-0.97 1.83 1.37 0.27 -7.30 -14.53 -17.37 -34.03 -17.03

a Numbers in parentheses indicate the total number of injections. b Values are average calculated from the total number of injections, n. c Calculated for A/NC values. d B/Jiangsu strain was not part of vaccine manufacturing during these years.

antigens where other virus components have been removed. MP1 from influenza A and B were identified in spots 12 and 14, respectively, and eluted as expected from the size exclusion column as proteins of MWapp ∼30 kDa. In addition, chain B of MP1 was identified in spot 13, which eluted from the size exclusion column as a globular protein of ∼18 kDa. The presence of this small protein in vaccines may be explained as a result of proteolytic cleavage of monomeric MP1 during vaccine manufacturing or sample preparation/analysis. Studies on the structure of MP1 have reported its susceptibility to proteolysis in the proximity of amino acid residue 164.22 Cleavage at that site would result in two fragments, a globular N-terminal fragment of 18 kDa (chain B) and a C-terminal fragment of 10 kDa, which has been shown to feature an elongated shape. Under the conditions assayed in the present study, the C-terminal fragment of MP1 could not be identified in the 2D plots which, in part, may be explained by its reported strong tendency to aggregate.22 In order to obtain information about differences in composition between vaccines produced by the same manufacturer in different seasons, analysis of a sample of influenza vaccine A produced in 2000-2001 was carried out using the described methodology and is shown in Figure 5E. When compared to the vaccine produced in 2005-2006 (Figure 5A), a number of similarities and differences could be observed. For instance, the major spots 1, 2, 3, 4, and 12 were found in both plots while spot 6 was only present in the plot of the older vaccine (Figure 5E). The absence of spot 7 was noticeable in the older vaccines as well as a change in the pattern

of the most hydrophobic compounds (marked with asterisks in both plots). Differences observed may be related to differences in the strains used. However, results show that interyear vaccines can be differentiated. The 2D-HPLC analyses of untreated samples of influenza vaccine A and C (season 2005-2006) were also carried out. In these cases the sample treatment step consisting of the addition of DTT to reduce disulfide bonds was omitted. The 2D plots of the untreated vaccines A and C (Figure 5D and F, respectively) showed profiles significantly different from those obtained from the corresponding treated samples. The 2D plots featured mostly low-intensity signals and the absence of the major spots observed in the corresponding treated samples (Figure 5A and C). These results support the necessity of including a disulfide cleavage step into the present analytical methodology for the characterization of the major proteins in influenza vaccines. Reproducibility of the 2D Method and Quantitation of HA Antigens in Commercial Influenza Vaccines. The reproducibility of the method was evaluated for same-day and betweenday replicate injections of vaccine samples. Values of RSD were calculated for retention times and peak areas in both dimensions and are summarized in Table 2. Results are expressed for the peak sampled in the SEC dimension at RT ) 9.77 min (MWapp ∼45 kDa) and its related peak in the RP-HPLC dimension corresponding to HA1 from influenza B/Jiangsu10/2003 at RT ) 40.32 min. Overall, highly reproducible data were obtained in both dimensions. As expected, slightly higher RSD values were found Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

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for retention times and peak areas of 0.11 and 1.50%, respectively, for between-day injections. Similar RSD values were obtained for other peaks. The HA content of commercial vaccines is typically measured against a reference HA antigen reagent prepared and calibrated by control laboratories (CBER, FDA). Standard solutions of reference HA antigen reagents were prepared at five different concentrations within the range of 5.3-231 µg/mL of the nominal concentration by dissolving known amounts of lyophilized reference antigens A/New Caledonia/20/99 (H1N1) (IVR-116 reassortment) and B/Jiangsu/10/2003 in a solution containing 0.5% SDS and 12 mM DTT. After incubation at 100 °C for 3 min, standards solutions were injected onto the size exclusion column and fractions containing HA (eluting between 9.6 and 9.8 min) were sampled in the 200-µL loop and loaded into the secondary column. The fluorescence intensity of the HA1 subunits of both strains (peaks 1 and 3, in Figure 2B) increased linearly as a function of the HA concentration over the range of concentrations assayed (data not shown). The calibration curves obtained by plotting peak areas (y-axis) corresponding to HA1 subunits of A/New Caledonia/20/99 (H1N1) (IVR-116 reassortment) and B/Jiangsu/10/2 against the injected antigen amounts (x-axis) within the range 0.28-11.56 and 0.29-11.06 µg, respectively, provided the equations: y ) 61992939x + 6790832 (R2 ) 0.9995) and y) 28110058x + 6909071 (R2 ) 0.9993), respectively. Limits of detection (S/N g 3) for fluorescence detection at λex 290 nm and λem 335 nm were calculated to be 105 and 172 ng/mL for A/NC and B/J, respectively. Limits of quantification, calculated for an S/N ratio of 10, were equal to 0.35 and 0.83 µg/mL for A/NC and B/J, respectively, showing a sensitivity comparable with that usually reported for detection of HA by single radial immunodiffusion, i.e., ∼2 µg/mL.23 Some considerations with respect to robustness of the presented methodology could be drawn from the optimization experiments. For instance, the SDS concentration in the mobile phase used for SEC and the addition of DTT to the sample are critical factors to consider in order to obtain reliable results as shown in Figures 2 and 5. The interday precision study showed that mobile phases were stable for at least one week. The influence of the mobile-phase composition on the selectivity of the second dimension has been reported previously.13 The HA content of commercial vaccines was determined by a process identical to that used for the standard solutions. The concentration of HA was calculated by interpolation of the measured peak area in the appropriate standard curve prepared from the reference reagent. Results are shown in Table 3. The HA content in current commercial vaccines is set at 15 µg per strain/dose for a typical 0.5-mL dose. Calculated values were close to the expected amounts for both strains, with RSD values lower than 2% for samples from the same lot (n ) 5). Moreover, RSD

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values lower than 3% were obtained for different lots (n ) 3) of both vaccines. These data provided strong evidence that the procedure was adequate for quantitative purposes in terms of HA content. The A/New Caledonia/20/99 (A/NC) influenza strain has been used for the preparation of vaccines since the season 2000-2001. Vaccine samples from several of the past years containing the A/NC strain were analyzed using the developed separation method, and the HA1 content was determined (Table 3). As indicated above, the calculated HA1 content for trivalent vaccines produced in 2005-2006 were very close to the nominal value of 30 µg of HA per strain/mL. As may be expected for vaccines produced in previous years, the calculated HA content of A/NC was lower than the nominal with percent deviation values progressively larger for the older vaccines (% deviation ) (calculated HA - nominal HA) × 100/nominal HA). For instance, the estimated HA content of A/NC in samples from 2000 to 2001 was lower than the nominal value by 34.0%. The decrease in detectable HA was likely due to protein degradation as a result of sample aging. However, further studies would be necessary in order to understand or confirm this apparent lower concentration of HA in expired vaccines. CONCLUSIONS It has been demonstrated that the use of the developed 2DHPLC method combined with fluorescence detection improves significantly the selectivity for the separation of influenza virus constituents in vaccines. The method is highly reproducible and efficient and can be used with confidence for analyzing influenza vaccines to detect some major virus antigens, without prior sample concentration. An additional advantage of the method is that fractions of interest can be targeted for subsequent analysis. The 2D-HPLC method also provided accurate determination of HA content in commercial influenza vaccines, corroborated by the good agreement between experimental and expected values. Several proteins including HA1, NP, and MP from the two different influenza types, influenza A and B, could be selectively separated and conclusively identified in individual vaccines. This procedure may also be applicable to distinguish between vaccines produced under different manufacturing processes. ACKNOWLEDGMENT V.G.-C. thanks Health Canada for a Visiting Fellowship in a Canadian Government Laboratory.

Received for review November 9, 2006. Accepted January 27, 2006. AC0621120

Selective and Quantitative Detection of Influenza Virus Proteins in

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