(T1430-02-08) LC-MS Workflow for Rapid and Precise Impurity Profiling of Oligonucleotides and GLP-1a

Purpose: Therapeutic oligonucleotides and glucagon-like-peptide-1 analogs (GLP-1a) have garnered significant attention in the biopharma industry. Their ability to be precisely tailored for treating cancer and rare diseases, as well as regulating metabolic behavior to address type 2 diabetes and obesity, while minimizing adverse side effects, underscores their immense value. However, the surge in demand for these innovative treatments has introduced analytical challenges, particularly in impurity profiling and stability monitoring, which are crucial for ensuring safety and efficacy. Monitoring product and process-related impurities and critical quality attributes (CQAs) is essential before releasing these drug products. However, this task presents significant challenges in quality control (QC) environments, as traditional UV-based methods often lack the necessary sensitivity and specificity for effective impurity monitoring.

Methods: The separation and characterization were done on a LC-MS workflow incorporating a compact single-quadruple mass detector. Oligonucleotides analysis: A 25-mer full-length product (FLP) oligonucleotide, with trace amounts of failed sequences, were analyzed through an ion-pairing RPLC method using an ethylene bridged hybrid C18 column. The ion-pairing reagents were dibutylamine (DBA)/triethylamine (TEA) and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), pH 8.5. The UV detector was set to 260 nm for monitoring the relevant samples and impurities. The mass detector was set on a negative mode with a scan range from 350 to 1500 m/z. Relative selected ion recordings (SIR) of associated impurity ions and FLP were monitored. GLP-1a analysis: Research grade exenatide was purchased from two vendors. The sample was analyzed on a Charged Surface Hybrid C18 column with a RPLC method. 0.1% formic acid in water/acetonitrile was configured as mobile phases. A PDA detector was used for absorption data acquisition with λ =190-400 nm. The mass detector was set on a positive mode with a scan range from 250 to 1500 m/z.

Results: The separation and identification of shortmer oligonucleotides impurities from the parent full length product was accomplished by precise control over the ion-paring concentration and gradient delivery. The UV spectrum reveals a broad impurity peak (14.56% of the total area). With the increased specificity of the mass detector in SIR mode, individual impurities are distinguished for more accurate assessment (Figure 1). Using GLP-1 analogs as a case study, exenatide samples purchased from two different vendors were tested for comparability using this LC-UV/MS workflow (Figure 2). UV detection indicated high purity for both samples, while MS spectra data indicates an impurity present in vendor 2 with the presence of an additional ion series.

Conclusion: The impurities that were co-eluted or overlooked using LC-UV alone highlight the value of orthogonal mass detection in confirming sample identity and efficiently screening for impurities in manufacturing settings. These characterization and development efforts enable laboratories to quickly create QC-friendly methods for monitoring critical attributes or impurities in drug products.


Figure 1. UV and selected ion recording (SIR) spectra of full length phosphorate oligonucleotides.


Figure 2. UV and MS-Base Peak Ion (BPI) chromatogram and spectra data for the GLP-1 analog Exanatide from two vendors.