Quality Control of Therapeutic Peptides by ¹H NMR HiFSA Sequencing

Abstract

Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes or minor impurities that affect effectiveness and safety. HiFSA sequencing yields definitive identity and purity information concurrently, allowing for API quality assurance and control (QA/QC). Achieving full peptide analysis via NMR building blocks, the process lends itself to both research and commercial applications as 1D 1H NMR (HNMR) is the most sensitive and basic NMR experiment. The generated HiFSA profiles are independent of instrument or software tools and work at any magnetic field strength. Pairing with absolute or 100% qHNMR enables quantification of mixtures and/or determination of peptide conformer populations. Demonstration of the methodology uses single amino acids (AAs) and peptides of increasing size, including the octapeptide, angiotensin II, and the nonapeptide, oxytocin. The feasibility of HiFSA coupled with automated NMR and qHNMR for use in QC/QA efforts is established through case-based examples and recommended procedures.

High-Resolution Structure of ClpC1-Rufomycin and Ligand Binding Studies Provide a Framework to Design and Optimize Anti-Tuberculosis Leads

“…To assess competitive effects on bacterial growth, a luciferase-expressing strain of M. tb was utilized in a checkerboard assay at several concentrations and time points. Results yielded a fractional inhibitory concentration index of 0.8–2.0, 20 indicating that the compounds were possibly additive or have no effect when dosed in combination. 26 From the assay, the concentrations of 140 nM of ECU and 190 nM of RUFI alone produced at least a two-log drop in bioluminescence signal after a two-week incubation when compared to the untreated bacterial control (Figure 6D).…”

“…RUFI and RUFVII were isolated as described previously 20 (these compounds are of the same lot used in these studies). The anti- M. tb compound ECU and its analogues were isolated from Nonomuraea sp.…”

Classification of Flavonoid Metabolomes via Data Mining and Quantification of Hydroxyl NMR Signals

“…This has important implications for NP discovery and biomedical research: while required for unambiguous structural assignment, isolates represent a limited fraction of the plant metabolome. These abundance and chemical stability constraints conversely limit not only the coverage of chemical complexity achievable for any investigated organism but also the accessible bandwidth of the biological profiles, especially in the relatively likely case where a trace component with elevated potency turns out to be the active component . This highlights an important application of the presented HMBC and qHNMR metabolomic approach for the study of complex bioactives.…”

13C-Satellite Decoupling Strategies for Improving Accuracy in Quantitative Nuclear Magnetic Resonance

“…More importantly, our data suggest that when excessive corrections are required to account for integral interferences, qHNMR does not provide accurate results, as demonstrated by the 400 MHz qHNMR of angiotensin II. Recent approaches to data analysis, such as quantum mechanically calculated NMR signals, indicate a shift away from integral-based qNMR as a potential improvement to explore in conjunction with 13 C-decoupling methodologies . Notwithstanding the availability of high field NMR instrumentation, qH­{ 13 C}­NMR offers the added benefit of seemingly improved resolution at lower fields.…”