Bioanalytical methods are needed to analyze protein conjugated antisense oligonucleotides (POCs) to accurately quantify the active antisense oligonucleotide (ASO) payloads, assess its pharmacokinetics and biodistribution in plasma and tissues, and ensure patient safety by evaluating potential immunogenicity and toxicity. Because the conjugate, the free ASO, and the linked ASO fragment can all be present, specialized techniques are required to differentiate and quantify these components, which is essential for supporting the development of these complex biotherapeutics. The unique properties of POCs present significant analytical challenges that necessitate specialized methods.
We set out to develop a methodology for quantifying total ASO in POCs that could be universally applied across similar POCs. The
study compared mass spectrometry and fluorescence detection platforms to determine optimal sensitivity, selectivity,
and adaptability. Additionally, it aimed to establish a single extraction method suitable for both plasma and tissue
analysis.
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