Quantification of residual DTT by high-performance anion-exchange chromatography coupled with pulsed amperometric detection

VLP (virus-like particle) have proven to be safer vaccine candidates compared to live-attenuated or inactivated viral vaccines. As part of the manufacturing process of VLP-based vaccines, dithiothreitol (DTT) and other reducing agents are commonly used in the disassembly of VLPs, followed by a subsequent reassembly process for the removal of the added reducing agents. This disassembly and reassembly processes improve VLP integrity, stability and immunoreactivity. In the manufacture of VLPs, it is essential that DTT removal is ensured since it is a highly toxic substance. Residual DTT content has to be monitored throughout the manufacturing process flow of the final pharmaceutical product. The available method for DTT estimation involves chemical derivatization which is complex and may require 100 % derivatization of low levels of DTT. In this study, we report a simple, novel and sensitive method for DTT quantification based on the combination of HPAEC-PAD and an electrochemical detector. The developed method has a linear range from 1 to 10 ng/mL with a limit of quantification of 100 pg. It is cost-effective and more sensitive than current available fluorescent and HPLC-MS-based methods for detecting residual DTT in viral and VLP-based vaccines. This method can be implemented to monitor residual DTT levels in any vaccine or product where DTT is used as a process reagent.