Quantification of Immunoreactive Viral Influenza Proteins by

ACCELERATED ARTICLE pubs.acs.org/ac

Quantification of Immunoreactive Viral Influenza Proteins by Immunoaffinity Capture and Isotope-Dilution Liquid Chromatography
Tandem Mass Spectrometry Carrie L. Pierce,† Tracie L. Williams,† Hercules Moura,† James L. Pirkle,† Nancy J. Cox,‡ James Stevens,‡ Ruben O. Donis,‡ and John R. Barr*,† †

Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, Georgia 30341, United States ‡ Influenza Division, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Georgia 30333, United States

bS Supporting Information ABSTRACT: An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibodybound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography
tandem mass spectrometry. IC-IDMS relies on antibody
antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/μL) of the total H3 HA (66.69 fmol/μL) from the commercial TIV and 93.6% (57.23 fmol/μL) of the total H3 HA (61.14 fmol/μL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.

I

nfluenza viruses are highly transmissible respiratory pathogens that result in substantial morbidity and mortality.1 Vaccination is the most effective means to reduce influenza infection and is an integral part of public health strategies for the control of influenza. Inactivated seasonal influenza vaccines for human use contain two influenza A virus subtypes, H1N1 and H3N2, along with a representative influenza type B virus. These trivalent influenza vaccines (TIV) provide protection primarily by eliciting the production of protective antibodies to hemagglutinin (HA), the primary viral antigenic component of influenza vaccines. The TIV must meet regulatory standards, and formulation of the final product requires a minimum amount of 15 μg of HA from each subtype (H1, H3, and B) in each 0.5 mL dose. The HA content in TIV is quantified by single radial immunodiffusion (SRID).2
5 The SRID assay of Wood et al.3 was first reported in 1977 and is based on the formation of a lattice between the HA antigen and homologous polyclonal antibodies (pAbs) in a supporting agarose gel matrix.4 In this assay, virusspecific calibrated anti-HA polyclonal antibodies from hyperimmunized sheep are incorporated into the agar gel, and the vaccine HA antigen is dispensed into wells in the agar. Upon incubation, This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society

the HA diffuses radially, resulting in precipitation rings of antibody
antigen complexes. The diameter of rings formed by vaccine preparations using the SRID assay is proportional to HA content and is used to determine its concentration from the regression line obtained from HA in the calibrated influenza antigen standards provided by national regulatory agencies. These strain-specific HA antigen standards are purified and inactivated whole influenza virions. Their HA contents are quantified by one of the commonly employed laboratory methods to determine total protein concentration (e.g., Kjeldhal, Bradford, Lowry, bicinchoninic acid (BCA)). Standards are subsequently analyzed by SDS-PAGE followed by a densitometric scan of the Coomassie-stained gel image to establish the fraction of total viral protein that is HA. The fraction of the protein that is HA and the total protein assay are used to calculate the estimated HA content of the vaccine sample being analyzed. Received: March 14, 2011 Accepted: May 18, 2011 Published: May 18, 2011 4729

dx.doi.org/10.1021/ac2006526 | Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry The SRID assay relies on at least two assumptions. First, the SRID reference antigens consist of formalin or β-propiolactoneinactivated purified whole virus reagents. As described above, HA content in these standard reagents is determined by estimating total protein in the purified whole virus followed by SDS-PAGE to estimate how much of the total protein is HA. No immunologic reaction is involved in determining how much HA is in the antigen standards. These standards are then used in the SRID assay, which assigns a concentration of HA based on immunologic precipitation. To make the required standard curve, the SRID assay must also assume that 100% of the HA in the antigen standard is immunoreactive. Second, assay antisera are produced by hyperimmunization of sheep with bromelain-cleaved HA. Reference antigens are simply inactivated whole virus, while vaccine test samples are either split virion or subviron virus preparations that primarily contain purified surface antigens (HA and NA). Antigenic differences that may exist among these various viral preparations might produce different results with SRID. The SRID assay, therefore, must assume that these antigenic differences are negligible. Influenza viruses circulating in the population undergo antigenic drift, and new viruses are developed to update the composition of the seasonal influenza vaccine. New strategies to improve the speed of bulk vaccine production and reduce the time to formulate TIVs are needed to support vaccine manufacturing and release of the final product in a timely fashion to protect public health. To address the timeliness and reliability of HA quantification in TIVs, we recently described a quantitative isotope dilution (ID) liquid chromatography
tandem mass spectrometry (LC
MS/MS) method for quantification of total HA.6 The ID-LC
MS/MS method improves the accuracy, precision, and specificity of HA measurements and may decrease the time needed to develop vaccines for newly emerging influenza viruses. Tryptic peptides conserved among HA subtypes were identified, selected as stoichiometric representatives of the HA proteins from which they were cleaved, and quantified against a spiked internal standard to yield a measure of HA concentration. This method has also proven to be successful for the simultaneous quantification of all three influenza HA subtypes in complex matrixes, including commercially produced TIV. However, the ID-LC
MS/MS method quantified the total HA regardless of whether or not it is in an antigenically correct conformation. The immunogenicity of the HA is predicated on the presence of the HA surface epitopes that are recognized by neutralizing antibodies.7 Structural alterations of the HA can affect surface epitopes and reduce antibody binding. Therefore, we implemented an immunoaffinity selection step using antibodies to the intact HA to identify and separate the HA containing intact surface epitopes from altered HA. The HA captured by the immunoaffinity step was then quantified by IDLC
MS/MS. This method, combining immunoaffinity and isotope dilution mass spectrometry, can determine the amount of HA in TIV that binds to specific antibodies and the amount that remains unbound. Isotope dilution mass spectrometry (IDMS) for accurate quantification of proteins in complex mixtures was demonstrated in our laboratory in 1996,8 and several successful approaches using magnetic beads as affinity devices for target species such as proteins and peptides have been published.9
13 ID-LC
MS/MS is an established quantification method14,15 and a selective and sensitive method for the characterization of captured species from magnetic affinity platforms.

ACCELERATED ARTICLE

With the use of the same reference polyclonal antiserum currently provided by regulatory authorities for use in the SRID method, we report the successful use of antibody coated magnetic beads for affinity selection and quantification of H3N2 influenza strains. Following antibody-selection and tryptic digest of the HA, ID-LC
MS/MS was employed for selective detection and absolute quantification of target peptide sequences conserved among human influenza A H3 viruses. IC-IDMS offers several advantages over the current SRID approach including direct, specific quantification of HA that binds to strain-matched antiserum. In addition, the selectivity offered by the mass spectrometer allows for detection and quantification of other proteins that are pulled out by the antibodies either in a nonspecific or cross-reactive fashion. This immunocapture IDMS method (IC-IDMS) has excellent accuracy, precision, sensitivity, and specificity and can be applied both to purified virus samples and trivalent influenza vaccine.

’ MATERIALS AND METHODS Influenza Standards and Vaccines. Polyclonal reference antiserum (sheep antihemagglutinin, A/Wisconsin/67/2005 (H3N2), CBER S-7847L3) (herein S-7847L3) and purified virus calibrated antigens (A/Wisconsin/67/2005 (H3N2), CBER ref no. 55, 58 μg/mL HA; A/Solomon Islands/3/2006 (H1N1), CBER ref no. 58, 57 μg/mL; B/Malaysia/2506/2004 (B), CBER ref no. 53, 76 μg/mL) were provided by the U.S. Food and Drug Administration’s Center for Biologics Evaluation and Research (CBER). Test S-7847L3 antiserum was reconstituted in 0.1 M PBS (PBS) and test purified virus calibrated antigens were reconstituted in 1 mL of deionized H2O. A commercial trivalent seasonal influenza vaccine (TIV), stored at 4 °C was used in all studies (2007
2008 Formula: A/Wisconsin/67/2005 (H3N2), A/ Solomon Islands/3/2006 (H1N1), B/Malaysia/2506/2004 (B). Synthesis of Native and Labeled Peptides. Custom synthetic peptides for H1, H3, and B HAs were synthesized at a 1
5 mg scale by Midwest Biotech, Inc. (Fishers, IN) and are described in Table 1. These lyophilized peptides were dissolved in 0.1% formic acid, dispensed into 200 μL aliquots in 1.5 mL vials using a Biomex NXP Laboratory Automation Workstation (Beckman Coulter, Fullerton, CA), and then lyophilized again and stored at
70 °C until use. A labeled analogue of the target peptide VNSVIEK in H1 HA was synthesized by incorporating the internal valine with 13C and 15N, producing a peptide with a 6 Da increase in mass over the unlabeled peptide. For STQAAINQINGK in H3 HA and NLNSLSELEVK in B HA, an isoleucine and leucine were 13C and 15N-labeled, respectively, producing peptides with a 7 Da increase in mass over the corresponding unlabeled peptide. Preparation of Working Stock, Calibration, and Labeled Solutions. HA protein quantification was performed by quantification of specific HA peptides unique to the HA protein of interest. In order to use these target peptides as calibration standards, accurate and precise measurement of these peptides is necessary. The peptide standards were quantified by an in-house isobaric-tagged isotope dilution mass spectrometry (IT-IDMS) method for amino acid analysis (AAA)16 that offers improved accuracy, precision, and sensitivity compared to traditional AAA approaches. The IT-IDMS method for AAA uses amino acid standards from the National Institute of Standards and Technology (NIST) so peptide content is accurate and traceable to NIST standards. All labeled and unlabeled peptide standards were 4730

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry

ACCELERATED ARTICLE

Table 1. Target Influenza Peptides and Isotopically Labeled Counterparts a target peptide

hemagglutinin

precursor

fragment ion

fragment ion

fragment ion

subtype

ion m/z

(quantitation)

(confirmation)

(confirmation)

VNSVIEK

H1

394.7 (þ2)

575.3 (y5)

689.4 (y6)

488.3 (y4)

VNSVIEK

H1

397.7 (þ2)

581.3 (y5)

695.4 (y6)

493.3 (y4)

STQAAINQINGK

H3

622.8 (þ2)

673.4 (y6)

928.5 (y9)

857.5 (y8)

STQAAINQINGK

H3

626.3 (þ2)

680.4 (y6)

935.5 (y9)

864.5 (y8)

NLNSLSELEVK

B

623.3 (þ2)

1018.5 (y9)

904.5 (y8)

704.4 (y6)

NLNSLSELEVK

B

626.8 (þ2)

1025.5 (y9)

911.5 (y8)

711.4 (y6)

target peptide

influenza

precursor

fragment ion

fragment ion

proteins

ion m/z

(detection)

(confirmation) 817.5 (y7)

YNGIITETIK

N1

576.3 (þ2)

874.5 (y8)

SGYSGIFSVEGK

N2

615.8 (þ2)

923.5 (y9)

836.4 (y8)

LNVETDTAEIR

NB

630.8 (þ2)

934.4 (y8)

1033.5 (y9)

LEDVFAGK

A/M1

439.7 (þ2)

636.3 (y6)

521.3 (y5)

EFDLDSALEWIK

B/M1

733.4 (þ2)

961.5 (y8)

846.5 (y7)

YLEEHPSAGK

A/NP

565.8 (þ2)

854.4 (y8)

725.4 (y7)

GGGTLVAEAIR

B/NP

522.3 (þ2)

771.5 (y7)

872.5 (y8)

a

Underlined amino acids correspond to 13C- and 15N-labeled amino acids. H1, hemagglutinin of H1N1 strains; H3, hemagglutinin of H3N2 strains; B, hemagglutinin of B strains, N1, neuraminidase of H1N1 strains; N2, neuraminidase of H3N2 strains; NB, neuraminidase of B strains; A/M1, influenza A matrix protein; B/M1, influenza B matrix protein; A/NP, influenza A nucleoprotein; B/NP, influenza B nucleoprotein. MS/MS = mass spectrometry/ mass spectrometry.

reconstituted with 100 μL of 10% (v/v) formic acid and diluted with 0.1% (v/v) formic acid to yield a 5 pmol/μL stock solution. For each peptide, working stock solutions of 0.5 pmol/μL were made from the 5 pmol/μL stock solution by diluting with 0.1% formic acid. This working stock solution was used to make the calibration standards. Eight 0.5 mL stock calibration standards, ranging from 2 to 120 fmol/μL, were prepared by adding 2, 5, 10, 30, 50, 70, 90, and 120 μL of each of the unlabeled peptides, 50 μL of the labeled peptides, and 0.1% formic acid to make the final volume 0.5 mL. The 0.5 pmol/μL spiked solutions of the labeled peptides were used for the internal standards. Mean area ratios (unlabeled/labeled) were plotted against concentrations for each standard. Linear regression without weighting was applied to the data sets, and calibration curves were generated for each peptide. Regression analysis of calibration curves showed a linear relationship with R2 values of 0.997 for H3 HA, 0.993 for H1 HA, and 0.993 for B HA. Covalent IgG Immobilization on Protein G Magnetic Beads. Homogenized Dynabeads Protein G (Dynal Biotechnology, Lake Success, NY) bead suspensions (2 mL) were washed twice with 10 mL of PBS. Lyophilized CBER reference antiserum S-7847L3 reconstituted in 5 mL of PBS was added to the washed beads. This antiserum/bead slurry was incubated at room temperature (RT) overnight with gentle rotation to allow the polyclonal H3 antibodies (pAb-H3) to attach. The beads were washed three times with 10 mL of PBS followed by two washes with 10 mL of 0.2 M triethanolamine pH 8.2. Protein G beads with bound pAb-H3 were resuspended in 3 mL of freshly prepared cross-linking solution (20 nM dimethyl pimelimidate dihydrochloride in 0.2 M triethanolamine, pH 8.2) and incubated at RT for 30 min with gentle rotation. The supernatant was discarded, and the pAb-H3 beads were resuspended in 1 mL of 50 mM Tris, pH 7.5 and incubated for 15 min at RT with gentle rotation to stop the cross-linking reaction. The beads with covalently attached IgG were washed twice with a 10 mL

0.01 M PBS/0.05% Tween-20 solution followed by a final 10 mL PBS wash. Beads with covalently attached pAb-H3 were resuspended in 2 mL of PBS buffer and stored at 4 °C for up to 1 week. Influenza Sample Preparation. A/Wisconsin/67/2005 (H3N2) calibrated antigen standard was reconstituted in 1 mL of deionized H2O and used without further purification. To examine nonspecific antibody interaction of the pAb-H3 coupled beads with other HA subtypes, A/Solomon Islands/3/2006 (H1N1) and B/Malaysia/2506/2004 (B) calibrated antigen standards for SRID (provided by FDA CBER) were each reconstituted in 1 mL of deionized H2O and used without further purification. The commercial TIV sample was in solution and was also used without further purification. Per CBER instructions, test samples were pretreated with a 1% (w/v) Zwittergent 3-14 detergent (Calbiochem, EMD Biosciences, Gibbstown, NJ).17 This detergent solution is assumed to solubilize the viral HA proteins anchored in the influenza virion’s lipid envelope.18 For IC-IDMS, 1% Zwittergent-treated samples, 20 μL of TIV or 10 μL of purified virus (A/Wisconsin/67/2005 (H3N2); A/Solomon Islands/3/2006 (H1N1); B/Malaysia/ 2506/2004 (B)) was added to 20 μL of a 10% (w/v) Zwittergent 3-14 solution. Zwittergent-treated samples were then diluted with PBS to a final volume of 200 μL and incubated for 30 min at RT prior to introduction of the pAb-H3 bound Protein G beads. For untreated PBS samples, 20 μL of TIV or 10 μL of purified virus was diluted in 180 or 190 μL of PBS, respectively, and added to the pAb-H3 bound Protein G beads. Virus and Vaccine Sample Binding to Magnetic Protein G Beads. A volume of 100 μL of pAb-H3 coated Protein G bead suspensions were aliquoted, the supernatant removed from the beads, and the 200 μL influenza sample preparations introduced. All Zwittergent-treated and untreated influenza samples were incubated at 37 °C for 1 h with minimal agitation. The unbound supernatant was recovered for subsequent HA quantification, 4731

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry

ACCELERATED ARTICLE

Figure 1. Flowchart representing the IC-IDMS method. A single influenza sample is subjected to three independent antibody capture events performed in series. New pAb-H3 coated Protein G bead suspensions are used for each capture, and following each antibody capture event, the antigen
antibody bound beads are independently washed. Following the wash step, each immunocaptured HA influenza sample is independently digested. The three immunocaptured HA influenza digests are then combined in a single vial and analyzed in triplicate by LC
MS/MS. Following the third antibody capture event, the wash fractions from each binding event are combined (separately from the immunocaptured samples) with the unbound fraction, lyophilized, digested, and analyzed in triplicate by LC
MS/MS. Each reported TIV sample concentration is an average of the triplicate LC
MS/MS analyses.

and the remaining beads were washed twice with 200 μL of PBS buffer each time (spent wash buffer fractions were also saved for HA quantification). Multiple binding events were necessary to maximize total binding recovery. Therefore, following the first incubation, second and third binding events, identical to the first, were performed with the collected supernatant. The unbound supernatant, following the third binding event and the unbound washes from all three binding events were pooled, evaporated to dryness, and analyzed separately (Figure 1). Preparation of Purified Virus Digests, Vaccine Digests, and LC
MS/MS Quantification. SRID calibrated antigen standards (purified virus) and TIV preparations not subjected to antibody capture were used to quantify total HA content. For these samples, 10 μL aliquots of calibrated antigen standards and 20 μL aliquots of TIV were diluted in 10 μL of a 0.1% solution of sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-1propanesulfonate (Rapigest) (Waters Corporation, Milford, MA) in 50 mM ammonium bicarbonate to solubilize proteins and improve protein digestion.19,20 The samples were heated for 5 min at 100 °C and allowed to cool to RT. After cooling, 86 pmol of sequence grade trypsin (Promega, Madison, WI) was added to each tube, and samples were incubated at 37 °C for 2 h to achieve complete digestion. Digests were allowed to cool, 10 μL of a 0.175 M HCl solution was added, and digests were incubated at RT for 30 min to reduce the pH and cleave the acid-labile surfactant. After incubation, 10 μL of each of the 0.5 pmol/μL labeled internal standard (ISTD) working stock solutions were added to each sample, and a 0.1% formic acid solution was used to dilute the final sample volume to 100 μL. The digested samples were mixed, centrifuged, and transferred to LC autosampler vials for analysis. All bound, unbound, and wash preparations were treated identically to the above preparation with the following exceptions. (1) Bound fractions, unbound lyophilized supernatants, and lyophilized wash collections were reconstituted in 10 μL (virus) and 20 μL (TIV) of deionized water prior to Rapigest

dilution. (2) Following protein digestion, fractions from binding events 1, 2, and 3 were combined, and 20 μL of each of the 0.5 pmol/μL labeled ISTD working stock solutions was added to the pooled binding events. The final sample volume was diluted to 200 μL with 0.1% formic acid to obtain a single LC
MRM-MS value for the three bound fractions. LC
MRM-MS Instrumentation Parameters. The binary solvent and sample managers on the NanoAcquity system (Waters Corporation, Milford, MA) were configured for capillary flow rates by using 0.005 in. Peeksil tubing from the injector on the sample manager to the head of the analytical column. The analytical column was a 150 mm  1 mm i.d. Symmetry300 reverse phase C18 (3.5 μm particle size, Waters Corporation, Milford, MA). The aqueous mobile phase (A) consisted of HPLC-grade water with 0.1% formic acid, while the organic phase (B) was acetonitrile (ACN) with 0.1% formic acid. A 2 μL full loop injection with three-time loop overfill was utilized for injections. The needle draw rate was set to 5 μL/min. Both preand postinjection, the needle was washed with 200 μL of mobile phase B, followed by 600 μL of a weak wash solution of 98% HPLC grade water, 2% ACN, and 0.1% formic acid. The gradient profile utilized a 30 μL/min flow rate. Initially, the mobile phase, consisting of 98% A and 2% B, was held constant for 5 min. A 1.2% change per min was then utilized over the next 15 min, where the mobile phases were 80% A and 20% B, respectively, followed by a 1.0% change per min over the next 5 min, where the mobile phases were 75% and 25%, respectively. After 27 min run time, the gradient was increased to 98% A and 2% B for the next 30 min to equilibrate the column to its initial conditions. The total run time was 57 min. The column eluent was introduced into a Thermo Scientific Vantage TSQ triple quadrupole tandem mass spectrometer with an electrospray interface (Thermo Scientific, Waltham, MA). The instrument was operated in positive ion mode with multiple reaction monitoring m/z quantifying ion pair transitions of m/z 4732

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry

ACCELERATED ARTICLE

Table 2. Amount of Hemagglutinin Captured Using pAb-H3 in Purified Virus and Commercial Trivalent Influenza Vaccine (fmol/μL Tested)a IC-IDMS digest

total digest

percent

(mean ( std deviation)

(mean ( std deviation)

bound (%)

H3N2
STQAAINQINGK; A/Wisconsin/67/2005 (0.1 M PBS) H3N2
STQAAINQINGK: A/Wisconsin/67/2005 (1% Zwittergent 3-14)

57.46 ( 2.59 57.23 ( 0.41

62.41 ( 2.37 61.14 ( 2.34

92.07 93.60

H3N2
STQAAINQINGK; trivalent influenza vaccine (0.1 M PBS)

51.62 ( 2.24

64.61 ( 1.18

79.89

H3N2
STQAAINQINGK; trivalent influenza vaccine (1% Zwittergent 3-14)

55.26 ( 0.95

66.69 ( 2.13

82.86 22.69

identifier

H1N1
VNSVIEK; A/Solomon Islands/3/2006 (0.1 M PBS)

15.93 ( 0.97

70.22. ( 1.12

H1N1
VNSVIEK; A/Solomon Islands/3/2006 (1% Zwittergent 3-14)

3.21 ( 0.06

66.78 ( 1.35

4.81

H1N1
VNSVIEK; trivalent influenza vaccine (0.1 M PBS)

24.73 ( 1.28

96.78 ( 2.63

25.55

H1N1
VNSVIEK; trivalent influenza vaccine (1% Zwittergent 3-14)

4.90 ( 0.29

98.15 ( 1.36

4.99

B
NLNSLSELEVK; B/Malaysia/2506/2004 (0.1 M PBS) B
NLNSLSELEVK; B/Malaysia/2506/2004 (1% Zwittergent 3-14)

17.39 ( 0.96 nondetect

73.85 ( 3.80 78.41 ( 2.10

23.55 not reported

B
NLNSLSELEVK; trivalent influenza vaccine (0.1 M PBS)

32.85 ( 1.67

106.72 ( 2.45

30.78

B
NLNSLSELEVK; trivalent influenza vaccine (1% Zwittergent 3-14)

5.58 ( 0.38

104.21 ( 2.98

5.35

a

Quantification of H3, H1, and B HA captured by pAb-H3 using liquid chromatography
tandem mass spectrometry (LC
MS/MS) in the presence and absence of 1% Zwittergent for A/Wisconsin/67/2005 (CBER ref no. 55), A/Solomon Islands/3/2006 (CBER ref no. 58), and B/Malaysia/2506/ 2004 (CBER ref no. 53) purified virus and commercial trivalent influenza vaccine. For purified virus immunocapture HA measurements, 10 μL of purified virus (n = 3) were subjected to antibody capture, tryptic digestion, and LC-MS/MS analysis. Triplicate results for each immunocapture preparation were averaged, and the mean (fmol/μL tested and percent recovery) and the method’s standard deviation for n = 3 sample preparations is reported. For total HA measurements, 10 μL samples of purified virus (n = 3) were tryptic digested and analyzed by LC
MS/MS in triplicate. Triplicate results for each total preparation were averaged, and the mean (fmol/μL tested) and the method’s standard deviation for n = 3 sample preparations is reported. For TIV immunocapture HA measurements, 20 μL of seasonal TIV (n = 3) were subjected to antibody capture, tryptic digestion, and LC-MS/ MS analysis. Triplicate results for each immunocapture preparation were averaged, and the mean (fmol/μL tested and percent recovery) and the method’s standard deviation for n = 3 sample preparations were reported. For total HA measurements, 20 μL samples of seasonal TIV (n = 3) were tryptic digested and analyzed by LC
MS/MS in triplicate. Triplicate results for each total preparation were averaged and the mean (fmol/μL tested) and the method’s standard deviation for n = 3 sample preparations were reported.

622.8 f m/z 673.4, m/z 394.7 f m/z 575.3, and m/z 623.3 f m/z 1018.5 for the H3, H1, and B HA native peptides and m/z 626.3 f m/z 680.4, m/z 397.7 f m/z 581.3, and m/z 623.4 f m/z 1025.5 for the corresponding labeled peptides. Two additional ion transition pairs utilizing the same conditions were monitored for HA peptide confirmation and are provided in Table 1. Two peptide transitional pairs were monitored for each non-HA influenza protein: one for detection purposes and one for confirmation. Ion transition pairs for detection and confirmation of neuraminidase of H1N1 strains (N1), neuraminidase of H3N2 strains (N2), neuraminidase of B strains (NB), influenza A matrix protein (A/M1), influenza B matrix protein (B/M1), influenza A nucleoprotein (A/NP), and influenza B nucleoprotein (B/NP) are given in Table 1. Instrument parameters were as follows: spray voltage 4000 V, sheath gas 4, auxiliary gas 2, capillary tube temperature 300 °C, and collision gas of 1.5 mTorr. Collision energies and tube lens were optimized for each peptide. Data processing and instrument control were performed with the Thermo Scientific Xcalibur software.

’ RESULTS AND DISCUSSION Our aim for this work was to use the same antibodies used in SRID to bind to and precipitate HA with neutralization epitopes and then to utilize ID-MS/MS to measure the HA as an accurate and specific means for HA quantification in purified virus and commercial trivalent influenza vaccine (TIV) samples. Identical SRID Zwittergent 3-14 detergent, CBER reference antigens, CBER S-7847L3 antiserum, and reagents were used to mimic SRID antibody
antigen binding in order to obtain IC-IDMS results comparable to the formation of the SRID precipitation ring.

Precipitation of large heteromeric complexes between HA and other proteins might compromise the accuracy and precision of the assay. To promote complex disruption, we examined the effects of mild detergents on HA capture and wash procedures in the assay. The binding of native HA protein in a neutral aqueous environment (PBS) was used as a reference. In the absence of detergents, viral HA subunits can aggregate and HA has been shown to adopt a characteristic rosette structure.21
23 Addition of excess detergent has been shown to prevent aggregate rosette formation in solubilized HA influenza virus.21,24 The current SRID assay treats all reference and unknown vaccine, antigen reagents, and test virus antigens with a 1% Zwittergent 3-14 solution for 30 min at RT before samples are added to the wells in the single-radial immunoplates;25,26 thus, to mimic these conditions we also treated our purified virus and TIV samples with 1% Zwittergent 3-14 for 30 min at RT and performed the 1 h incubation in a 1% Zwittergent solution. For both the TIV and A/Wisconsin/67/2005 virus 1% Zwittergent-treated samples, no significant differences were observed in the amount of H3 HA specifically bound to the pAb-H3 when compared to the PBS reference (Table 2). Nonspecific binding of H1 and B HAs by the pAb-H3 was observed in both the PBS and 1% Zwittergenttreated TIV samples; however, this amount was significantly reduced in the 1% Zwittergent-treated samples (Table 2). For completeness, we tested the effects of Zwittergent 3-14-treated TIV samples above and below the critical micelle concentration (CMC) of 0.011% (w/v) (Figure S-1 in the Supporting Information). Additionally, n-octyl p-D-glucopyranoside (OG) has been reported to prevent HA aggregation,22 and prior to Zwittergent 3-14, the SRID assay protocol recommended pretreating samples with OG. To observe the impact of OG 4733

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry detergent on antibody capture, we repeated our analyses with OG-treated TIV samples both above and below the CMC (0.73% (w/v)) of OG (Figure S-2 in the Supporting Information). These experiments show that while H3-specific interactions are seemingly unaffected by the presence of detergents prior to and during antibody capture, the impact of detergent during incubation on the H1 HA and B HA nonspecific interactions is dependent on the relative concentrations of each detergent (see the Supporting Information). Zwittergent 3-14 appeared to have no effect on the specific binding of H3 HA by pAb-H3 in the S-7847L3 antiserum while simultaneously decreasing the capture of other HA and non-HA influenza proteins from TIV and purified virus samples. Therefore, our reported ICIDMS results include incubation conditions using both PBS and 1% Zwittergent 3-14. Following antibody capture, we also examined the impact of Polysorbate 20 (Tween 20) detergent in the wash buffer and its effect on immunocaptured HA. We found that regardless of the presence or absence of Tween 20 in the wash buffer, at various concentrations both above and below the CMC (0.07% (w/v)), there were no measurable differences in selective H3 HA immunocapture and no apparent differences in pAb-H3 nonspecific interactions with H1 or B HAs (Figure S-3 in the Supporting Information). To quantify total HA (H1, H3, B) (not the immunocapture amount) in the purified virus and TIV matrixes and to evaluate precision in the ID-LC
MS/MS peptide measurements, three independent digest preparations were analyzed for the HA study peptides. Each digest was analyzed by triplicate LC
MS/MS. The mean and method standard deviation (n = 3) for the total HA in virus and TIV digest preparations are presented in Table 2. Percent relative standard deviations (% RSD; (standard deviation/mean)  100) for total HA in both purified virus and TIV preparations were less than 6%. Conditions were optimized for antibody capture and recovery of the target antigen so that nonspecific binding was minimized while recovery of the H3 HA was maximized. Binding buffer composition, incubation temperature, incubation time, Protein G bead volumes, Protein G lot numbers, S-7847L3 antiserum concentration, S-7847L3 antiserum volume, HA antigen concentration, HA antigen volume, and wash buffer composition were all evaluated and captured H3 HA recoveries were determined. To evaluate the antibody capture protocol for quantifying immunoreactive H3 HA, three antibody capture tryptic digests from purified virus and three antibody capture tryptic digests from TIV samples were prepared. Three binding events were required to maximize H3 HA recovery. At all concentrations tested, the largest percentage of H3 HA was recovered in binding event 1, followed by binding event 2, and then binding event 3. Although only a very small amount of H3 HA was recovered in the third binding event, our research goal was to perform as many binding events as needed until there was no detectable MS signal observed following antibody capture and digest. A fourth binding event did not produce a discernible H3 HA MS signal. Replicates were subjected to binding events 1, 2, and 3, enzymatically digested, and the ratio of the unlabeled target peptide to labeled target peptide was determined for quantification by IDLC
MS/MS. Bound HA recoveries were calculated by comparing the amount of recovered H3 HA from the antibody capture digest preparations (binding events 1, 2, and 3) to the amount of H3 HA quantified in the total H3 HA tryptic digest preparations. Mean total digest concentrations, mean bound digest concentrations, method standard deviations for three complete

ACCELERATED ARTICLE

Figure 2. Liquid chromatography
tandem mass spectrometry (LC
MS/MS) extracted ion chromatograms of target HA peptides for quantifying three hemagglutinin components (H1, H3, B) from TIV. Data were acquired on a Thermo Scientific TSQ Vantage. The chromatograms show affinity of the pAb-H3 to hemagglutinin of H3N2 strains and reveal nonspecific binding of the pAb-H3 to hemagglutinin of the influenza A/Solomon Islands/3/2006 H1N1 strain and the hemagglutinin of B/Malaysia/2506/2004 B strain in 1% Zwittergent 3-14 treated TIV samples. Underlined amino acids are 13C and 15N stable isotope labeled.

replicates, and percent bound recoveries are presented in Table 2. The % RSDs for the three immunocapture A/Wisconsin/67/ 2005 virus and three TIV immunocapture experiments were less than 5%. As seen in Table 2, capture in the purified virus is reproducibly high with a bound mean H3 recovery of 93.6% of the total H3, showing the effectiveness of the method. The pAbH3 captured a lower percentage of total H3 from TIV as compared to the H3 HA in inactivated virus calibrated antigen reagents. The analysis showed that 82.9% of the total H3 HA was bound by the pAb-H3 in the commercial TIV (Table 2). To verify that all the H3 HA present in the samples being analyzed could be accounted for, unbound and bead wash collections were also analyzed for the A/Wisconsin/67/2005 purified virus calibrated antigen. Total H3 HA recoveries (bound fractions þ unbound fraction þ wash collections) exceeded 98%. Thus, a very minor amount of H3 HA was lost due to sample transfer or from other experimental factors. Use of salts and buffers can be detrimental in MS and generally interferes with molecular ion formation through signal suppression and signal broadening. High concentrations of PBS present in the unbound fractions and wash collections, coupled with the large sample volumes were not well tolerated in the MS; therefore, these fractions were not analyzed for all test samples. Taken together, these results indicated that not all the HA present in the purified virus preparations can be captured by the pAb-H3, suggesting that the assumption that 100% of the HA in the reagents used to calibrate the SRID are immunoreactive may be incorrect. Further analysis of the commercial TIV was informative concerning the specificity of the pAb-H3. While the pAb-H3 recognized the H3 HA efficiently, we found that the pAb-H3 coupled beads bound other HA subtypes and even other influenza proteins. As part of the analysis of the commercial TIV, we also measured the amount of H1 HA and B HA that adhered to the pAb-H3 coupled magnetic beads. The IC-IDMS analysis employed to identify these influenza proteins is the same 4734

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry

ACCELERATED ARTICLE

Figure 3. Liquid chromatography
tandem mass spectrometry (LC
MS/MS) extracted ion chromatograms of target influenza peptides in (a) PBS and (b) 1% Zwittergent 3-14-treated seasonal TIV that indicate the pAb-H3 nonspecific immunocapture of non-HA influenza proteins. Liquid chromatography
tandem mass spectrometry (LC
MS/MS) extracted ion chromatograms of detection and confirmation ion transitions for target neuraminidase peptide LNVETDTAEIR in influenza virus B/Malaysia/2506/2004 confirming detection of pAb-H3 nonspecific immunocapture of neuraminidase in (c) PBS and (d) 1% Zwittergent-treated seasonal TIV samples. S/N, signal/noise; H3, hemagglutinin of H3N2 strains, N1, neuraminidase of H1N1 strains; N2, neuraminidase of H3N2 strains; NB, neuraminidase of B strains; A/M1, influenza A matrix protein; B/M1, influenza B matrix protein; A/NP, influenza A nucleoprotein; B/NP, influenza B nucleoprotein.

as described herein for H3 HA, except the monitored peptides are those specific for H1 and B HAs. The H1 HA and B HA peptides of interest and their labeled counterparts are provided in Table 1. Figure 2 shows the IC-IDMS analysis of the 1% Zwittergenttreated commercial TIV HA proteins that were captured by the pAb-H3 that were attached to the magnetic beads. Besides the expected H3 HA, additional peaks for H1 and B HAs were detected indicating that these hemagglutinins were also captured by the pAb-H3-coupled beads. Mean total digest concentrations, mean bound digest concentrations, method standard deviations for the three complete replicates, and percent bound recoveries are presented in Table 2. In the absence of 1% Zwittergent, of the total H1 HA in the commercial TIV, 25.6% was captured by pAbH3 and of the total B HA in the commercial TIV, 30.8% was captured by pAb-H3. In the presence of the 1% Zwittergent, of the total H1 HA in the commercial TIV, 5.0% was captured by pAb-H3 and of the total B HA in the commercial TIV, 5.4% was captured by pAb-H3. Thus, in the presence of the 1% Zwittergent pretreatment and immunocapture, the H1 HA and B HA cross reactivity or nonspecific interactions were diminished but not eliminated. Whether the nonspecific interactions observed are due to nonspecific protein
protein binding or are a result of true pAb-H3 cross-reactivity with the H1 and B subtypes is unclear. We further investigated nonspecific interactions by extending the analysis of the commercial TIV to non-HA proteins in the TIV matrix, specifically, neuraminidase of H1N1 strains (N1), neuraminidase of H3N2 strains (N2); neuraminidase of B strains (NB); influenza A matrix protein (A/M1), influenza B matrix protein (B/M1), influenza A nucleoprotein (A/NP), and

influenza B nucleoprotein (B/NP). Again, the analytical approach was the same as described for H3 HA except the monitored peptides are different. The detection and confirmation target peptides for LC
MS/MS analysis of these proteins are provided in Table 1. We were able to verify NB, N1, N2, A/ M1, B/M1, A/NP, and B/NP binding, demonstrating additional pAb-H3 nonspecific interactions (Figure 3a
d) in both the PBS and Zwittergent-treated TIV samples. As part of the analysis of the purified virus calibrated antigens, we also measured the amount of H1 HA and B HA in A/Solomon Islands/3/2006 and B/Malaysia/2506/2004 that was captured by the pAb-H3-coupled magnetic beads. Mean total digest concentrations, mean bound digest concentrations, method standard deviations for the three complete replicates, and percent bound recoveries are presented in Table 2. In the absence of 1% Zwittergent, of the total H1 HA in the A/Solomon Islands/3/2006 purified virus, 22.7% was captured by pAb-H3 and of the total B HA in the B/Malaysia/2506/2004 purified virus, 23.6% was captured by pAbH3. In the presence of the 1% Zwittergent, of the total H1 HA in the A/Solomon Islands/3/2006 purified virus, 4.8% was captured by pAb-H3. In the presence of 1% Zwittergent, nonspecific interactions of the pAb-H3 with B HA in the B/Malaysia/2506/2004 purified virus were not observed. Additionally, with the use of the same analytical approach, A/Wisconsin/67/2005 for SRID was further analyzed for the presence of non-HA proteins coupled to the antibody bound beads. We monitored for type A N1, N2, M1, NP and type B NB, NP M1 target peptides. We were able to confirm N2, A/M1, and A/NP nonspecific binding in both the PBS and Zwittergenttreated purified A/Wisconsin/67/2005 virus preparations and 4735

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry also found that in the presence of the 1% Zwittergent pretreatment and immunocapture, nonspecific interactions were diminished but not eliminated. The mean HA molar concentration values obtained were utilized to determine the amount (in micrograms) of each HA subtype in the TIV with a dose of 500 μL. Taking into account the dilution factor of 5 (20 μL of TIV in a 100 μL preparation), the average molecular mass of HA as 76 kDa, and the volume of a dose (500 μL), the total HA amounts of H1, H3, and B were calculated to be 18.65 ( 0.26, 12.67 ( 0.40, and 19.80 ( 0.56 μg per 0.5 mL dose, respectively. Similarly, the mean HA molar concentration values obtained were utilized to determine the amount (in micrograms) of each HA subtype in the purified virus calibrated antigens (A/Solomon Islands/3/2006 no. 58; A/Wisconsin/67/2005 no. 55; B/Malaysia/2506/2004 no. 53). As before, but taking into account the dilution factor of 10 (10 μL of virus in a 100-μL preparation), the total HA amounts of H1, H3, and B were calculated to be 50.75 ( 1.03, 46.47 ( 1.78, and 59.59 ( 1.60 μg/mL, respectively. These studies demonstrate the use of IC-IDMS for the simultaneous quantification of influenza proteins in calibrated antigen standards for SRID and TIV samples. The HA bound by the antibody-coated beads is postulated to represent molecules with the same characteristics as those involved in the formation of a measurable precipitation ring in the SRID assay. The specificity of the mass spectrometer allows for each component to be individually and selectively quantified.

’ CONCLUSIONS We report a new method (IC-IDMS) based on immunoaffinity selection of HA using polyclonal antibody-coated magnetic beads, followed by analysis of bound proteins by ID-LC
MS/MS. IC-IDMS was successfully used for quantifying H3 HA content in purified virus preparations and commercial TIV samples and also allowed simultaneous quantification of other bound influenza HA and detection of non-HA influenza proteins. Accurate quantification of HA containing antibody neutralization epitopes is critically important for influenza vaccination programs. A dose of HA below the recommended 15 μg may result in insufficient immunogenicity and reduced protection, while excessive amounts of HA could increase vaccine production costs and cause vaccine supply shortages and potential adverse vaccine-associated reactions.27 SRID, using homologous HA-specific antisera and calibrated antigen standard reagents, is the method currently used for the quantification of HA in influenza vaccines. The IC-IDMS method reported here has several advantages over the current SRID method, including accuracy traceable to NIST amino acid standards, better precision, better sensitivity, and better selectivity offered by using mass spectrometry as the detector. Further evaluation would be justified to determine its comparative performance characteristics. The IC-IDMS method can quantify H3 HA in TIV and other samples. Thus, in principle the IC-IDMS method could provide the capability to measure the concentration of the three HA types/subtypes in TIV over time and under varying storage conditions to assess their stability. The IC-IDMS antibody magnetic-bead-based platform described is amenable to automation and high throughput while offering high quality quantitative hemagglutinin analyses.

ACCELERATED ARTICLE

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We acknowledge Dr. Adrian Woolfitt and Maria Solano for AAA analyses. We also acknowledge the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration for providing valuable hemagglutinin samples and antiserum used in this study. Reference in this article to any specific commercial products, process service, manufacturer, or company does not constitute an endorsement or a recommendation by the U.S. Government or the Centers for Disease Control and Prevention. The findings and conclusions reported in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. ’ REFERENCES (1) Bright, R. A.; Carter, D. M.; Daniluk, S.; Toapanta, F. R.; Ahmad, A.; Gavrilov, V.; Massare, M.; Pushko, P.; Mytle, N.; Rowe, T.; Smith, G.; Ross, T. M. Vaccine 2007, 25, 3871–3878. (2) Wood, J. M.; Schild, G. C.; Newman, R. W.; Seagroatt, V. J. Biol. Stand. 1977, 5, 237–247. (3) Wood, J. M.; Schild, G. C.; Newman, R. W.; Seagroatt, V. Dev. Biol. Stand. 1977, 39, 193–200. (4) Mostow, S. R.; Schild, G. C.; Dowdle, W. R.; Wood, R. J. J. Clin. Microbiol. 1975, 2, 531–540. (5) Schild, G. C.; Pereira, M. S.; Chakraverty, P. Bull. World Health Org. 1975, 52, 43–50. (6) Williams, T. L.; Luna, L.; Guo, Z.; Cox, N. J.; Pirkle, J. L.; Donis, R. O.; Barr, J. R. Vaccine 2008, 26, 2510–2520. (7) Feng, J.; Gulati, U.; Zhang, X.; Keitel, W. A.; Thompson, D. M.; James, J. A.; Thompson, L. F.; Air, G. M. Vaccine 2009, 27, 6358–6362. (8) Barr, J. R.; Maggio, V. L.; Patterson, D. G., Jr.; Cooper, G. R.; Henderson, L. O.; Turner, W. E.; Smith, S. J.; Hannon, W. H.; Needham, L. L.; Sampson, E. J. Clin. Chem. 1996, 42, 1676–1682. (9) Boyer, A. E.; Quinn, C. P.; Hoffmaster, A. R.; Kozel, T. R.; Saile, E.; Marston, C. K.; Percival, A.; Plikaytis, B. D.; Woolfitt, A. R.; Gallegos, M.; Sabourin, P.; McWilliams, L. G.; Pirkle, J. L.; Barr, J. R. Infect. Immun. 2009, 77, 3432–3441. (10) Kalb, S. R.; Moura, H.; Boyer, A. E.; McWilliams, L. G.; Pirkle, J. L.; Barr, J. R. Anal. Biochem. 2006, 351, 84–92. (11) Kalb, S. R.; Lou, J.; Garcia-Rodriguez, C.; Geren, I. N.; Smith, T. J.; Moura, H.; Marks, J. D.; Smith, L. A.; Pirkle, J. L.; Barr, J. R. PLoS One 2009, 4, e5355. (12) Kalb, S. R.; Barr, J. R. Anal. Chem. 2009, 81, 2037–2042. (13) Boyer, A. E.; Quinn, C. P.; Woolfitt, A. R.; Pirkle, J. L.; McWilliams, L. G.; Stamey, K. L.; Bagarozzi, D. A.; Hart, J. C., Jr.; Barr, J. R. Anal. Chem. 2007, 79, 8463–8470. (14) Mayya, V.; D, K. H. Expert Rev. Proteomics 2006, 3, 597–610. (15) Bronstrup, M. Expert Rev. Proteomics 2004, 1, 503–512. (16) Woolfitt, A. R.; Solano, M. I.; Williams, T. L.; Pirkle, J. L.; Barr, J. R. Anal. Chem. 2009, 81, 3979–3985. (17) U.S. Department of Health and Human Services; Food and Drug Administration; Center for Biologics Evaluation and Research; Influenza Virus Vaccine Potency Reagents for Single-Radial-Immunodiffusion; A/Wisconsin/67/2005 X-161b; CBER Ref. #55. Rockville, MD, 2006. 4736

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737

Analytical Chemistry

ACCELERATED ARTICLE

(18) Bruhl, P.; Kerschbaum, A.; Kistner, O.; Barrett, N.; Dorner, F.; Gerencer, M. Vaccine 2000, 19, 1149–1158. (19) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Anal. Chem. 2003, 75, 6023–6028. (20) Suder, P.; Bierczynska, A.; Konig, S.; Silberring, J. Rapid Commun. Mass Spectrom. 2004, 18, 822–824. (21) Laver, W. G.; Valentine, R. C. Virology 1969, 38, 105–119. (22) Remeta, D. P.; Krumbiegel, M.; Minetti, C. A.; Puri, A.; Ginsburg, A.; Blumenthal, R. Biochemistry 2002, 41, 2044–2054. (23) Ruigrok, R. W.; Martin, S. R.; Wharton, S. A.; Skehel, J. J.; Bayley, P. M.; Wiley, D. C. Virology 1986, 155, 484–497. (24) Johannsen, R.; Moser, H.; Hinz, J.; Friesen, H. J.; Gruschkau, H. J. Biol. Stand. 1983, 11, 341–352. (25) Williams, M. S. Vet. Microbiol. 1993, 37, 253–262. (26) World Health Organization; National Institute for Biological Standards and Control (NIBSC); Influenza Reagent Antigen Reagent for Single-Radial-Diffusion Assay of A/Wisconsin/67/2005 (H3N2) (NYMCX161-B). NIBSC code: 06/120, Instructions for use (Version 3.0, Dated 09/07/2007). Hertfordshite, UK. 2007, 1
2. (27) Wood, J. M. Philos. Trans. R. Soc. London, B: Biol. Sci. 2001, 356, 1953–1960.

4737

dx.doi.org/10.1021/ac2006526 |Anal. Chem. 2011, 83, 4729–4737