Simultaneous quantification of major capsid protein of human papillomavirus 16 and human papillomavirus 18 in multivalent human papillomavirus vaccines by liquid chromatography-tandem mass spectrometry.

Human papillomavirus (HPV) vaccination is the most effective mean to prevent HPV infection and cervical carcinoma. Licensed HPV prophylactic vaccines are formulated to contain a defined amount of different major capsid protein (L1), the critical antigen to elicit protection. No method is currently available to simultaneously quantify individual L1s in multivalent vaccines, presenting a daunting challenge for the quality control of HPV vaccines. Here, HPV16 and HPV18 L1 can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using tryptic digestion without pre-digestion reduction and alkylation in multiple reaction monitoring (MRM) mode. Two signature peptides were selected to be the markers of the two L1s and can be well separated within 5.1 min. Their linear calibration curves were both obtained in the range of 20-500 nmol/L (R > 0.990). To HPV16 L1, intra/inter assay precisions and accuracies of the assay were below 11% and between 83.96-113.57%. While for HPV18 L1, they were below 12% and between 81.40-103.49%. In addition, the limits of quantitation (LOQ) were as low as 2.8 nmol/L for HPV16 L1 and 1.7 nmol/L for HPV18 L1, respectively, representing about 68 and 112 times more sensitive than those obtained with Smith Bicinchoninic Acid (BCA) assay. This LC-MS/MS method can be applied to the quantification of both bulk products and the final multivalent vaccines. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy and throughput; it could become the method of choice for absolute quantification of proteins in multivalent vaccines.