Amgen: Method Development and Evaluation of the Techniques for Oligonucleotide Concentration Determination

Oligonucleotide-based therapeutics are emerging as promising modality against a variety of diseases. The determination of oligonucleotide concentration is a critical quality attribute as it directly relates to the patient’s safety and efficacy. A typical dose may require a dosing concentration of > 150 mg/mL. Traditionally, concentration determinations have been assessed through a fixed path length instrument. Due to limitations of the spectrophotometer with these high absorbance products, sample concentrations in this range must involve a timeconsuming dilution step. However, alternate technologies exist for high concentration samples which require no sample dilution, such as the NanoDrop™ and SoloVPE instruments using Slope Technology™.

A study was performed to 1) compare the range of oligonucleotide concentrations using the SoloVPE and NanoDrop™ instruments including the evaluation of multiple wavelengths and 2) evaluate the precision and accuracy of both instruments to a standard. The study encompassed an oligonucleotide concentration range up to ~200 mg/mL. Extinction coefficients of the target oligonucleotide at multiple wavelengths were determined. With the optimal wavelength determined, high concentration determinations were possible. Accuracy was assessed via a direct comparison between the instruments. Statistical analysis evaluated the equivalency of the two instruments