Analytical challenge

Abstract

The structure and functionality of oligonucleotides make them one of the strongest candidates for the future of pharmaceuticals with the potential to treat the most severe of illnesses. Manufacture using stepwise solid phase synthesis results in impurities accumulating at each step, as well as impurities arising from reagents and impure starting materials. With only a handful of FDA approved oligonucleotide pharmaceuticals on the market, the ability to identify and accurately quantify these impurities is vital in reaching a future enriched in oligonucleotide treatments. Our solution Scientists within the Oligonucleotide Services team at Intertek have extensive experience in running industry standard methods in a GMP certified laboratory using ion-pair chromatography for reporting identification, assay and impurities. We have a comprehensive understanding of the importance of using best practices, combined with experience of performing the complex data processing required for quantification of coeluting peaks. This understanding and previous experience is invaluable for us to transfer to new projects to allow generation of high-quality data for new molecules. We perform assay and impurity testing using dedicated Agilent 1260 Infinity systems coupled to an Agilent 6130 single quadrupole mass spectrometer using negative ion electrospray. Identification, assay and impurity analysis are demonstrated in this case study, which presents data from an unmodified DNA 20-mer. Reference standard data processing and System Suitability Testing (SST) The first step of data processing was to apply a blank subtraction to the UV chromatograms, using the blank UV chromatogram with a baseline closest to that of the reference standard UV chromatogram. Reference standard UV chromatograms were then integrated to determine system suitability by assessing retention time, peak area relative standard deviation and linearity. To assess linearity a standard UV calibration curve (Figure 1) was obtained by plotting the UV area against the amount of UV-pure standard on the column. The UV calibration line enables the determination of concentration of UV pure material in the sample. MS data were assessed to confirm the m/z value observed for the 4-charge state of the full-length product (FLP) was within 0.2 m/z of the calculated value. A second calibration curve was then plotted from MS data using the peak area of the FLP extracted ion chromatogram (EIC). The EIC peak area was plotted against injection volume (Figure 2). The MS calibration line is used to quantify co-eluting impurities under the main peak of the UV chromatogram. The UV and MS calibration lines gave R 2 values of ≥0.99, which passed the system suitability criteria.