influenza hemagglutinin (ha) quantification
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INTERPHEX - April 26, 2016
Influenza Hemagglutinin (HA) Quantification
Ben Levin Technical Product Specialist
April 26th , 2016
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www.InDevR.com
Current Gold Standard: Single Radial Immunodiffusion Assay
Reliant on seasonal reference antisera & antigens
Leaves hazardous waste
Requires in-house prep of gels or plates
Analysis & interpretation not standardized
Limited dynamic range (6-30 ug/mL)
Limited to singleplex quantification
y = 0.11x + 6.21R = 0.979
5.006.007.008.009.00
10.00
5 15 25 35Ave
rage
Dia
met
er (m
m)
Standard Concentration (g/ml)
Monovalent SRID Calibration
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VaxArray Influenza
Assess HA stability and obtain results in hours instead of days
Implement a more efficient potency assay with less reliance on seasonal reference reagents
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VaxArray Influenza Microarray & Capture Antibodies
H1
H3
B
Array Position
Epitope TypeEpitope Location
Neutralizing Reactivity
A Conformational HA1 YES A/CA/2009 (pdm-like H1)B Conformational HA2 YES Broad H1C Linear HA1 Broad H1D Conformational HA1 YES Broad H3E Conformational HA2 Broad H3F Linear HA1 Broad H3G Conformational HA1 YES Broad B/YamH Linear HA1 Broad B/YamI Conformational HA1 YES Broad B/Vic
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subtype specific monoclonal antibody
HA
fluor-labeled polyclonal antibody
glass VaxArrayslide
Vidia
VaxArray Influenza Assay Detection Methodology
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VaxArray Influenza Assay Workflow
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orStandards & Samples
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Must Use Appropriate Standard for Quantification on VaxArray
These differences had a significant impact on the calculated potency of the commercial vaccines and demonstrate the need to have well-matched reagents
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538074/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538074/
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Demonstration
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VaxArray Influenza Data Analysis
More than 1900 data points
A wealth of information
automatically analyzed
(x16/slide)
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VaxArray Influenza Imaging Package
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VaxArray Influenza Data Processing Workbook
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VaxArray Influenza Data Processing Workbook
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VaxArray Influenza Data Processing Workbook
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VaxArray Influenza Assay Summary
Commercially available multiplexed, array-based
immunoassay
Monovalent and multivalent analysis
Simple workflow with limited hands-on time
Results in under 4 hours (8-56 samples)
Automated data analysis
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VaxArray Influenza Correlation with SRID, ELISA & paBCA
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VaxArray Influenza Correlation with SRID, ELISA & paBCA
Crude Extract Column 2 EluateColumn 1 Eluate Bulk Drug Substance
19 Samples
SRID paBCAELISA VaxArray Influenza
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VaxArray Influenza Recombinant In-Process Samples and BDS
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VaxArray Influenza Cell Culture Supernatant & Allantoic Fluid
Crude Allantoic Fluid (Negative Control)
Allantoic Fluid spiked with H1 Subtype
Cell Culture Supernatant (Negative Control)
Cell Culture Supernatant with A/CA/07/2009
1:2 Dilution
1:2 Dilution
1:5 Dilution
1:5 Dilution
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Cross-Reactivity Assessment
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Correlation with SRID, ELISA, paBCA
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Correlation Analysis Summary
Excludes one statistical outlier (3,300 point) from ToC data.* Average ratio 1/(sqrt(N-1))
Kuck et al., (2014) PLOS One 9(10): e109616
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Tracking Antigen Stability in Multivalent Vaccines
Simultaneous analysis of all antigens in TIV
Rate of decay differs for each antigen
Rate of decay differs for each method
Both VaxArray and SRID indicate that H3 is most stable
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Generally Good Correlation
Antigen Manufacturer 1 Manufacturer 2Slope R2 Slope R2
H1 0.91 0.98 0.90 0.96
H3 0.48 0.76 0.89 0.96
B/Mass 0.37 0.84 -- --
Correlation Analysis:
Correlation Analysis: Overall, good correlation In general, SRID indicates
more rapid loss of potency B/Mass irreproducible
(matrix conditions?)
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VaxArray Influenza Antigenic DriftMultiplexed
A/H1
B/Yam-like
B/Vic-like
A/H3
A/NewCal/20/1999
A/Duck/Shantou/1283/2001 (H3N8)
B/Malaysia/2506/2004
B/Jilin/20/2003
Historic
A/CA/07/2009
A/Vic/361/2011
B/Bris/60/2008
B/Mass/2/2012
RecentRecombinant HA
Viral HA
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Publication Information and Application & Technical Notes
Stability Indicating Array Layout and Specificity
Protein Quantification in Crude Samples
Virion Splitting Studies
Monovalent and Multivalent Vaccines Accuracy and Precision
Correlation with SRID
Manuscript published in PLOS ONE October 20, 2014, titled "Titer on Chip: New Analytical Tool for Influenza Vaccine Potency Determination is available at http://dx.plos.org/10.1371/journal.pone.0109616.
Application Notes: Technical Notes:
Calibration Standards
Best Practice for Accurate Concentratio
Wash Method Comparison
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http://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-1-Stability-Indicating_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-2_Multiplex_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-3_Virion-Splitting_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2014/10/Flu-ToC-App-Note-4-Crude-Samples-R10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-1_Specificity_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-2_Accuracy-and-Precision_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-3_SRID-Correlation_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-1-Stability-Indicating_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-1_Specificity_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2014/10/Flu-ToC-App-Note-4-Crude-Samples-R10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-3_Virion-Splitting_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-App-Note-2_Multiplex_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-2_Accuracy-and-Precision_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-3_SRID-Correlation_Rev10A.pdfhttp://dx.plos.org/10.1371/journal.pone.0109616http://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-3_SRID-Correlation_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-3_SRID-Correlation_Rev10A.pdfhttp://indevr.com/newsite/wp-content/uploads/2013/12/Flu-ToC-Tech-Note-3_SRID-Correlation_Rev10A.pdf
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Advancements in Progress
- High throughput configuration- Possible 96-well plate configuration- Barcode scanning integration
- 21CFR Part 11 capable software- Refined user interface- Accommodate regulatory guidelines and customer feedback
- Additional Assays- Potentially pandemic influenza strains- Neuraminidase assay
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Acknowledgements & Contact Information
Thank You!
Protein Sciences Corporation
BEI and Influenza Reagent Resource
Reagents group at CBER/FDA
Funding from NIH/NIAID (IVMI)
InDevR Contact Information:
Email: [email protected]
Phone: +1-303-402-9100
INTERPHEX Booth #:
3762
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mailto:[email protected]
Slide Number 1Current Gold Standard: Single Radial Immunodiffusion AssayVaxArray InfluenzaVaxArray Influenza Microarray & Capture AntibodiesSlide Number 5VaxArray Influenza Assay WorkflowMust Use Appropriate Standard for Quantification on VaxArrayDemonstrationVaxArray Influenza Data AnalysisVaxArray Influenza Imaging PackageVaxArray Influenza Data Processing WorkbookVaxArray Influenza Data Processing WorkbookVaxArray Influenza Data Processing WorkbookVaxArray Influenza Assay SummaryVaxArray Influenza Correlation with SRID, ELISA & paBCAVaxArray Influenza Correlation with SRID, ELISA & paBCAVaxArray Influenza Recombinant In-Process Samples and BDSVaxArray Influenza Cell Culture Supernatant & Allantoic FluidCross-Reactivity AssessmentCorrelation with SRID, ELISA, paBCA Correlation Analysis SummaryTracking Antigen Stability in Multivalent VaccinesGenerally Good CorrelationVaxArray Influenza Antigenic DriftPublication Information and Application & Technical NotesAdvancements in ProgressAcknowledgements & Contact Information