Wanda Santana (Presenter)
Centers for Disease Control and Prevention
Bio: Wanda I. Santana, M.S. is a research chemist at the Biological Mass Spectrometry Laboratory at the Centers for Disease Control and Prevention. Ms. Santana received her Master of Science in Chemistry at Georgia State University and joined CDC in 2005. She has developed and applied methods that can detect and quantify protein toxins, such as botulinum neurotoxin, in human clinical samples. Most recently, she has optimized, validated and applied isotope dilution mass spectrometry (IDMS) methods to quantify hemagglutinin, neuraminidase, nucleoprotein and matrix protein in potency testing reagents and vaccines to improve the quality of influenza vaccines and transfer methods to FDA and appropriate international laboratories.
Authorship: Wanda I. Santana, Jonathan L. Bundy, Yulanda Williamson, Tracie L. Williams and John R. Barr
Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Clinical Chemistry Branch, 4770 Buford Hwy, N.E., Atlanta, GA 30341
Size exclusion chromatography (SEC) has been employed to separate HA species under the same conditions employed in the SRID assay. In order to determine the amount of the active form of HA in the vaccine, we have used a protocol where vaccine samples are first separated by SEC followed by LC-MS/MS quantitation of the protein components. The same SEC fractions may also be subject to SRID analysis to determine the fractions with SRID activity. A measure of potency can be calculated by summing the concentrations of all fractions that are SRID active. We have also demonstrated that the SRID active fractions can reproducibly be isolated via size exclusion chromatography and that the assay is also stability indicating, based on changes observed in the SEC profile of vaccines subjected to pH based stress.
The regulated protein in influenza vaccine is hemagglutinin (HA). Currently, vaccine potency is determined by measuring activity with the single radial immunodiffusion assay (SRID), which has been correlated with the production of neutralizing antibodies to HA in vivo. This assay requires the production of both reference antigens and polyclonal antibodies raised against HA, which takes several months and can be problematic. In case of an influenza pandemic, where time is of the essence, it is desirable to develop an alternative assay that can operate independently or an adjunct of SRID. Size exclusion chromatography (SEC) has been employed to separate HA species under the same conditions employed in the SRID assay. It has been proposed based on these studies that the form of HA exhibiting the greatest activity by SRID is the trimer, followed by higher aggregates of HA, with monomers having little or no activity In order to determine the amount of the active form of HA in the vaccine, we have used a protocol where vaccine samples are first separated by SEC followed by LC-MS/MS quantitation of the protein components. The same SEC fractions may also be subject to SRID analysis to determine the fractions with SRID activity. A measure of potency can be calculated by summing the concentrations of all fractions that are SRID active. We have also demonstrated that the SRID active fractions can reproducibly be isolated via size exclusion chromatography and that the assay is also stability indicating, based on changes observed in the SEC profile of vaccines subjected to pH based stress.
Vaccine preparation: Bulk vaccine preparations from A/California/07/2009 pandemic influenza strains were used in this work. Before SEC or direct SRID analysis, samples were treated with Zwittergent 3-14 to a concentration of 1% (w/v) for 30 min. Some samples were subjected to simulated stress by lowering to pH ~4.0 by the use of 500 mM sodium acetate pH 4.0 buffer, followed by naturalization with 1M Tris, pH 8.0. Size Exclusion Chromatography: SEC was performed on a Waters H Class UPLC system using an Agilent BioSEC-3 column 4.6 x 30 mm. The mobile phase was PBS (Sigma-Aldrich with) 0.1% (w/v) Zwittergent 3-14 at a flow rate of 100 ìl·min-1. Chromatographic runs were 1 hr. in length, with 36 1-min fractions collected. Fractions eluting from 16-21 min and 22-27 min, known to contain HA, were collected using an Agilent fraction collector triggered by contact closure from the H-Class system and pooled for subsequent SRID or MS analyses. Single radial immunodiffusion: SRID was performed on some fractions collected by SEC using a protocol developed by the DVP/CBER/ FDA. The appropriate reference antigens and antisera provided either by FDA or the UK NIBSC (A/California/07/2009- derived) were used as a standard. SRID response was measured electronically by the use of a GT Vison Immulab data system coupled to a high resolution flatbed scanner. Isotope dilution mass spectrometry: IDMS was used for detection of HA on a Thermo Fisher Scientific TSQ Vantage as previously reported. Three tryptic peptides arising from HA were monitored via SRM to quantitate HA content of the SEC fractions. The target peptides observed were EQLSSVSSFER, m/z 634.8; EQLSSVSSF(U-10)ER, m/z 639.8; TLDYHDSNVK, m/z 596.3; TLDYHDSNV(U-6)K, m/z 599.3; VNSVIEK, m/z 394.7; VNSV(U-6)IEK, m/z 397.7.
SEC can be successfully employed to fractionate various size classes of HA in influenza vaccine bulks upon treatment under SRID-like conditions. Egg based, cell based and recombinant vaccines each contain varied proportions of HA aggregates and “trimeric” HA as measured by SEC. Depending upon the vaccine type, the percentage of total HA in the sample present in the “trimeric” form varies. Examining a panel of egg based vaccines from various manufacturers reveals a diversity of compositions. When all regions of the SEC chromatogram are assessed for HA content, the bulk of total HA is recovered. The fractions from three regions (I, II and III) were collected and assayed for HA content by IDMS. Total HA content of the unfractionated bulk was also measured by IDMS and the recoveries were also calculated. The total HA by IDMS was 328.20 ug/ml; Region I 94.73 ug/ml; Region II 215.67 ug/ml and Region III below LOD. SRID analyses reveal the different HA forms are responsible for the response observed in SRID, with the “trimeric” fraction having a similar response to the total vaccine, the aggregate fraction (larger species by SEC) being significantly less. In the case shown here, no monomeric HA was detected. Upon pH based stress, measurable changes were observed in the chromatograms of vaccine bulks as well as the SRID analyses, demonstrating that SEC is potentially stability indicating.
References & Acknowledgements:
Bill McCormick of CBER/FDA is thanked for helpful conversations.
The work reported in this poster was funded by an Interagency Agreement between the
Centers for Disease Control and the Biomedical Advanced Research and Development
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