Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC

Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods

Characterization and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry

Large RNA including mRNA (mRNA) has emerged as an important new class of therapeutics. Recently, this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study, we have developed an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilized on magnetic particles were performed in conjunction with high-resolution liquid chromatography–mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach, a >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS-CoV-2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation, can be completed within 90 min. The ability to rapidly identify, characterize, and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation, and impurity analysis

Efficient synthesis of DNA duplexes containing reduced acetaldehyde interstrand cross-links

DNA interstrand cross-links (ICLs) prevent DNA replication and transcription and can lead to potentially lethal events, such as cancer or bone marrow failure. ICLs are typically repaired by proteins within the Fanconi Anemia (FA) pathway, although the details of the pathway are not fully established. Methods to generate DNA containing ICLs are key to furthering the understanding of DNA cross-link repair. A major route to ICL formation in vivo involves reaction of DNA with acetaldehyde, derived from ethanol metabolism. This reaction forms a three-carbon bridged ICL involving the amino groups of adjacent guanines in opposite strands of a duplex resulting in amino and imino functionalities. A stable reduced form of the ICL has applications in understanding the recognition and repair of these types of adducts. Previous routes to creating DNA duplexes containing these adducts have involved lengthy post-DNA synthesis chemistry followed by reduction of the imine. Here, an efficient and high-yielding approach to the reduced ICL using a novel N2-((R)-4-trifluoroacetamidobutan-2-yl)-2′-deoxyguanosine phosphoramidite is described. Following standard automated DNA synthesis and deprotection, the ICL is formed overnight in over 90% yield upon incubation at room temperature with a complementary oligodeoxyribonucleotide containing 2-fluoro-2′-deoxyinosine. The cross-linked duplex displayed a melting transition 25 °C higher than control sequences. Importantly, we show using the Xenopus egg extract system that an ICL synthesized by this method is repaired by the FA pathway. The simplicity and efficiency of this methodology for preparing reduced acetaldehyde ICLs will facilitate access to these DNA architectures for future studies on cross-link repair

Reduction of tlr2 gene expression in allergic and nonallergic rhinitis

Immunomodulators, including toll-like receptors (TLRs) and defensins, produced in response to pathogenic stimuli, can direct the developing immune system toward a TH1 nonallergic phenotype. Increased human f-defensin (HBD) 4 expression is associated with infection. Objective: To determine whether reduced mucosal levels of TLRs and defensins contribute to the inflammation seen in chronic allergic and nonallergic rhinitis. Methods: Real-time polymerase chain reaction was used to determine gene expression levels of HBDs 1 through 4 and TLRs 2 and 4. Immunohistochemical analysis was used to study the localization and distribution of protein for a-defensins 1 through 3, HBD2, neutrophil elastase, and TLR2 in sections of nasal turbinate tissue from adults with persistent allergic and idiopathic rhinitis, healthy nasal mucosa, and tonsil tissue. Results: Allergic mucosa showed a significant (P =3D .02) reduction in TLR2 messenger RNA expression compared with control mucosa and generally reduced expression for TLR4 and HBDs. Although not significant, the nonallergic group also showed reduced expression for TLRs and HBDs. With the exception of HBD4, increased target gene levels were seen in tonsil tissue. Protein expression of HBD2 and TLR2 was localized in lining and submucosal glandular epithelium but insignificant differences were seen for HBDs, TLRs, neutrophils, and a-defensin between the rhinitic and control patient groups. Conclusions: Subjects with allergic and nonallergic rhinitis show reduced TLR and HBD gene expression. The significant reduction in TLR2 gene expression in allergic adults supports the concept that increased TLR2 protects against the development of allergy. The low levels of HBD4 detected in both rhinitis groups suggest lack of an underlying infection pathophysiological featur

EXTRINSIC ACTIVATION OF HUMAN COAGULATION FACTOR-IX AND FACTOR-X ON THE ENDOTHELIAL SURFACE

In previous kinetic studies, the catalytic efficiency of the activation of human coagulation factors IX and X by factor VIIa in the presence of purified tissue factor apoprotein was found to be essentially equal. These activation reactions were now studied on the surface of human umbilical vein endothelial cells. The cells were stimulated with endotoxin to express tissue factor. This tissue factor activity was saturable with factor VIIa and could be inhibited by rabbit antibodies against human tissue factor apoprotein. Only stimulated cells supported factor VIIa activity. No difference in the reactivity of factor VII and VIIa was observed in the presence of factor X, due to rapid feedback activation of factor VII by factor Xa. However, the activation of factor IX by factor VII shows a 10 min lag-phase, which reflects that the activation of factor VII by factor IXa is a less efficient process. The kinetic parameters for the factor VIIa dependent activation of factor IX and factor X on the endothelial surface were: Km 0.09-mu-M, Vmax 0.13 pmol/min, and Km 0.071-mu-M, Vmax 0.41 pmol/min, respectively. The same ratio between the Vmax for factor X and factor IX activation was observed as in a cell free system. However, the Km of factor IX was 4-fold higher on the endothelial surface than in the cell free system. Together, these kinetic parameters will favour factor X activation 5-fold over factor IX activation at physiological concentrations of these proteins. The activation of factor X by factor VIIa on the endothelial surface was characterized by a short lag-phase, which was absent in factor IX activation. Further, both the activation of factor X and factor IX were down regulated by factor Xa