High-sensitivity quantification of antisense oligonucleotides for pharmacokinetic characterization

Area of Science:

• Pharmacology
• Biochemistry
• Analytical Chemistry

Background:

• Antisense oligonucleotides (ASOs) represent a rapidly advancing therapeutic class.
• Accurate pharmacokinetic profiling and quantification of ASOs are essential for their successful drug development.
• Current quantification methods like LC-MS and hybridization immunoassays may not meet the required sensitivity levels.

Purpose of the Study:

• To develop a highly sensitive method for quantifying ASOs.
• To compare the performance of a novel branched DNA assay against established hybridization immunoassays for ASO quantification.

Main Methods:

• Development of a branched DNA (bDNA) assay specifically designed for ASO quantification.
• Comparative analysis of the bDNA assay with dual-probe hybridization electrochemiluminescence and single-probe hybridization ELISA.

Main Results:

• The developed bDNA assay demonstrated significantly enhanced sensitivity, achieving a lower limit of quantification (LLOQ) of 31.25 pg/ml in plasma.
• This represents a 6.4-fold and 16-fold improvement in sensitivity compared to dual-probe hybridization electrochemiluminescence and single-probe hybridization ELISA, respectively.
• The bDNA assay exhibited adequate precision, accuracy, selectivity, and specificity, with minimal matrix interference and reduced hemolysis interference.

Conclusions:

• Branched DNA technology provides a significantly more sensitive approach for ASO quantification.
• This method offers advantages over existing techniques, particularly in reducing interference from hemolysis, making it suitable for pharmacokinetic studies.