RESUMEN
Oligonucleotide characterization is a rapidly advancing field in the biopharmaceutical industry. Understanding critical quality attributes, such as intact mass and impurities, requires a toolbox of analytical techniques, which commonly includes liquid chromatography-mass spectrometry (LC-MS). Oligonucleotide LC-MS analysis frequently requires sample run times upward of 15 min to achieve separation of multiple oligonucleotide species. Additionally, LC methods frequently employ mobile phase additives such as triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol that are not always desired for use in MS instrumentation. Here, microfluidic capillary electrophoresis mass spectrometry (CE-MS) via ZipChip technology was employed to enable rapid intact mass analysis of oligonucleotide single strands. Baseline separation of equal length oligonucleotides was achieved in less than 4 min. Additionally, the potential of the ZipChip platform for separation of oligonucleotide full-length products (FLPs) and their impurities was evaluated.
Asunto(s)
Microfluídica , Oligonucleótidos , Oligonucleótidos/química , Espectrometría de Masas/métodos , Cromatografía Liquida , Electroforesis Capilar/métodosRESUMEN
The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme-substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the alpha and zeta isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the epsilon isoform of diacylglycerol kinase exhibiting the greatest selectivity.