Method Validation of High Performance Anion Exchange Chromatography with Pulse Amperometric Detection (HPAEC-PAD) for Meningococcal Serotype C Antigen Determination

Abstract

Background: Quantitative analysis of meningococcal vaccine serotype C antigen is an important test for quality control of vaccine. Vaccine must be confirmed that contains the specific antigen corresponding to the registration dossier. So, the quality, efficacy and safety of vaccine are importance for public confidence in vaccines. HPAEC-PAD is known for its high accuracy and precision, establishing it as the gold standard method recommended by WHO for the analysis of carbohydrates, oligosaccharides, and polysaccharides. However, this method has not yet been used to check meningococcal vaccine quality in Thailand.

Objective: Optimization and validation of Meningococcal serotype C antigen content were investigated for use as standard methods in the laboratory.

Methods: The optimum conditions and validate the method were studied in various parameters, including linearity and range, precision, accuracy, specificity, limit of detection, limit of quantification and robustness.

Results: The standard curve was linear in the concentration range of 0.47 and 15.00 µg/ml, and the correlation coefficient was 0.9996. The precision of total antigen and free antigen on six times in the same day showed a percentage of standard deviation of 0.55 and 1.90, respectively, whereas the precision of total antigen and free antigen on six different days showed a percentage of standard deviation of 3.71 and 6.83, respectively. The accuracy of measuring the standard substances used in the meningococcal vaccine was investigated, and the results showed that the percentage of recovery of total antigen was between 81.01 and 116.05. The percentage of recovery of free antigen was between 80.78 and 114.25. The limit of detection and quantification were shown as 0.15 and 0.46 µg/ml, respectively. The specificity showed there was no interference from other substances and could separate the target antigen from others. In addition, this method was durable when analysts were changed. Therefore, the results proved that all parameters of the validation method passed the acceptance criteria.

Conclusions: The study revealed that the Meningococcal serotype C antigen content by HPAEC-PAD passed validation and all met the requirements. Therefore, this method is suitable for use as a standard method for determining Meningococcal serotype C antigen content.