Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array

The purity of a drug substance can influence its toxicity and potency, so i mpurities must be specifically determined. In the case of synthetic oligode oxyribonucleotide drugs, however, product complexity makes complete impurit y speciation difficult. The goal of the present work was to develop a new a nalytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleoti des. A complete series of oligodeoxyribonucleotide probes were designed, ea ch complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with (35). Under stringently optimized conditions, only the perfectly seque nce-matched oligodeoxyribonucleotide hybridized to each probe, while all ot her deletion sequences were removed by washing with buffer. The S-35 signal intensity of the bound oligodeoxyribonucleotide was proportional to the co ncentration of each (n-1)mer deletion sequence in the analyte solution. Thi s method has been applied to a number of synthetic phosphorothioate oligode oxyribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n-1)mer deletion sequences present.