This study describes the NMR-based method to determine the limit of quantitation (LOQ) and limit of detection (LOD) of cholesterol, a process-related impurity in the replication-deficient Herpes Simplex Virus (HSV) type 2 candidate vaccine HSV529. Three signature peaks from the 1D H NMR of a cholesterol reference spectrum were selected for the identification of cholesterol. The LOQ for a cholesterol working standard was found to be 1 μg/mL, and the LOD was found to be 0.1 μg/mL. The identity of cholesterol, separated from the formulation of growth supplement by thin layer chromatography (TLC), was confirmed by 1D H NMR and 2D H-C HSQC NMR. The three signature peaks of cholesterol were detected only in a six-times concentrated sample of HSV529 candidate vaccine sample and not in the single dose HSV529 vaccine sample under similar experimental conditions. Taken together, the results demonstrated that NMR is a direct method that can successfully identify and quantify cholesterol in viral vaccine samples, such as HSV529, and as well as in the growth supplement used during the upstream stages of HSV529 manufacturing.
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| http://dx.doi.org/10.1016/j.csbj.2016.10.007 | DOI Listing |
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