Capillary gel electrophoresis of therapeutic oligonucleotides--analysis of single- and double-stranded forms

Recently, several therapeutic double-stranded (ds) oligonucleotides (ODNs) are in pharmaceutical development. During quality control, these therapeutic molecules have to be characterized with respect to their identity, their content and their impurity profile. It follows that the ds molecule as well as its process- and product-related impurities have to be quantified. The single strands are considered as process as well as product-related impurities in the ds drug substance. Applying well known, conventional, single-base resolution CE-CGE systems developed for the quality control of single-stranded antisense ODNs in the early 1990s, it turned out that the ds ODNs under investigation are migrating in broad, splitted peaks between the peaks reaction zones are observed. It follows that the quantification of the single strands in the drug substance as well as quantification of other product-related impurities, e.g. n-1; n-2 (loss of one and two bases (n), respectively) etc., are not possible without adaptation of the test system. The paper shows how the test system was adjusted in order to determine single-stranded strands as well as ds strands next to each other quantitatively in the ds drug substance under investigation.