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.

Figure 1.

The 20 common AAs with their R groups, classified by chemical characteristics and with PDB labeling schemes.

Figure 2.

General workflow of the HiFSA parameter generation.

Figure 3.

HiFSA-generated ¹H NMR profiles of common AAs at experimentally collected frequencies with the distributions of each ¹H chemical shift generated from the top-200 largest proteins of the BMRB database (accessed on July 7, 2017). Each ¹H nucleus of each AA is represented by one colored line, in the order of the NMRSTAR format. The color intensity reflects the density of the distribution.

Figure 4.

Structure of aspartame (1), glutathione (2), angiotensin II (3), and oxytocin (4).

Figure 5.

Graphical representation of the ΔJ values (Hz, oxytocin J – common AA J) of all oxytocin hydrogens versus their position within oxytocin. Gly is not included since it is a statistical outlier.

Figure 6.

Structures of DRVYIH (5) and DRIYVH (6).

Figure 7.

Proposed workflow for the generation of a general QC protocol for pharmaceutically and biologically relevant peptides using qHNMR and HiFSA profiling. Population-based QM-qHNMR analysis paired with HiFSA profiling can replace INT-qHNMR analysis.

Figure 8.

Workflow for QC testing by qHNMR and HiFSA profile.