Quantitation of residual valproic acid in flu vaccine drug substance
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Section snippets
Materials and methods
All chemicals and reagents were purchased from VWR Scientific (Waltham, MA), unless otherwise stated. Vaccine materials used for development were produced at the Vaccine Production Program Laboratory (Gaithersburg, MD). The HA stabilized-stem ferritin fusion protein was expressed transiently using pDNA polyethylimine chemical transfection of a HEK293E cell line. The purification comprised of an initial ultrafiltration and diafiltration step to condition the harvest for chromatography, which
Introduction of an internal standard (NNA)
In order to mitigate the impact due to unavoidable loss of VPA in the course of extraction, transfer, and derivatization, nonanoic acid (C9H18O2) (NNA, pKa = 4.95) was used as the internal standard because of its similarity to VPA (C8H16O2) in chemical structure (Supplementary Fig. S1). NNA has one additional methylene group (CH2) compared to VPA, its derivative (NNA-AcPh) is more hydrophobic than that of VPA (VPA-AcPh), leading to a baseline separation between the two in the HPLC.
Derivatization improvement and the profile of HPLC analysis of VPA
The existing
Conclusion
An efficient method for the quantitative measurement of VPA was developed and evaluated for the determination of residual VPA in the drug substance of influenza vaccine. The method is capable of quantitating VPA with an LOD of 2.3 µg/mL, LOQ of 6.8 µg/mL. The efficiency of liquid phase extraction has been greatly improved by the use of saturated NaCl solution to salt out the analyte from the vaccine drug substance. The application of NNA as the internal standard has mitigated the impact of
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgment
The authors would like to thank Nathan Barefoot, Yile Li, Andrew Shaddeau, Jessica Bahorich for the scientific discussions, Kristin Leach for providing purification step samples, Lana Hogan for reviewing and editing the manuscript. Authors are grateful to the organizational and scientific leadership provided by Kevin Carlton and Jason Gall.
Funding
This work was supported by the Intramural Research Program of the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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