Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Biotechnology

Background:

• Numerous liquid chromatography-mass spectrometry (LC-MS) methods exist for quantifying antisense oligonucleotides (ASOs).
• Limited data are available on the reproducibility and robustness of a single ASO bioanalytical method across different laboratories.
• Standardization of bioanalytical methods is crucial for reliable ASO drug development and clinical application.

Purpose of the Study:

• To evaluate the reproducibility and robustness of a single ion-pairing LC-MS (IP-LC-MS) bioanalytical method for ASOs across multiple independent laboratories.
• To establish a multicenter-validated method for ASO quantification.
• To identify potential challenges in the application of ASO bioanalytical methods.

Main Methods:

• A single IP-LC-MS method was used to measure mipomersen, a model ASO.
• Seven independent laboratories participated in the study.
• Standard bioanalytical method validation parameters (calibration curve, accuracy, precision, selectivity) were assessed.

Main Results:

• All laboratories met the validation criteria for calibration curve, accuracy, precision, and selectivity according to conventional guidelines.
• The IP-LC-MS method demonstrated sensitivity, robustness, and reproducibility across the participating sites.
• Significant carryover (>20%) was detected in three of the seven laboratories.

Conclusions:

• The study successfully demonstrated a multicenter-validated IP-LC-MS bioanalytical method for ASOs.
• The method is suitable for sensitive, robust, and reproducible quantification of ASOs.
• Carryover is a critical parameter that needs careful monitoring and mitigation in future applications of this method.