NMR spectroscopy goes mobile: Using NMR as process analytical technology at the fume hood.

Nuclear magnetic resonance (NMR) is potentially a very powerful process analytical technology (PAT) tool as it gives an atomic resolution picture of the reaction mixture without the need for chromatography. NMR is well suited for interrogating transient intermediates, providing kinetic information via NMR active nuclei, and most importantly provides universally quantitative information for all species in solution. This contrasts with commonly used PAT instruments, such as Raman or Flow-infrared (IR), which requires a separate calibration curve for every component of the reaction mixture. To date, the large footprint of high-field (≥400 MHz) NMR spectrometers and the immobility of superconducting magnets, coupled with strict requirements for the architecture for the room it is to be installed, have been a major obstacle to using this technology right next to fume hoods where chemists perform synthetic work. Here, we describe the use of a small, lightweight 60 MHz Benchtop NMR system (Nanalysis Pro-60) located on a mobile platform, that was used to monitor both small and intermediate scale Grignard formation and coupling reactions. We also show how low field NMR can provide a deceptively simple yes/no answer (for a system that would otherwise require laborious off-line testing) in the enrichment of one component versus another in a kilogram scale distillation. Benchtop NMR was also used to derive molecule specific information from Flow-IR, a technology found in most manufacturing sites, and compare the ease at which the concentrations of the reaction mixtures can be derived by NMR versus IR.

Analysis of two commercially available bortezomib products: differences in assay of active agent and impurity profile.

The analytical properties of two commercially available bortezomib products (VELCADE(®) and Bortenat) were compared using nuclear magnetic resonance, mass spectrometry, high-performance liquid chromatography, and gas chromatography. The data showed differences between the two products. Based on these data, Bortenat samples contained more active ingredients than indicated by the label (mean, 116.5% and 117.9% of label, in 2-mg and 3.5-mg vials, respectively). In comparison, VELCADE samples contained a mean of 99.3% of active ingredient, which was consistent with the approved specification range (US, 90-110%; EU, 95-105%). Clinical data demonstrate that patients exposed to higher than recommended doses of bortezomib on the standard twice-weekly dosing schedule are likely to have an increased risk of major toxicities. Bortenat 2-mg vials contained an isovaleraldehyde impurity; the origin of this is unknown. Additionally, the ratio of boronic acid to boronic ester differed between Bortenat 2 mg (0.27:1) and 3.5 mg (0.13:1) and VELCADE (0.10:1) samples reconstituted in saline indicating that the Bortenat product is not equivalent to the VELCADE product.

Mapping the bound conformation and protein interactions of microtubule destabilizing peptides by STD-NMR spectroscopy.

Using the hemiasterlin analogs taltobulin (I, HTI-286), II, and III as model compounds, we demonstrate that relaxation-compensated STD-NMR can be used as an effective tool to efficiently provide a qualitative epitope map for microtubule destabilizing peptides. Due to the disparate relaxation behavior of the protons in these model compounds, it was essential to collect STD with very short saturation times to render an accurate picture of the binding interaction. The conformation of HTI-286 (I) in complex with the protein was determined from TRNOESY/ROESY experiments and is similar to the X-ray crystal structure conformation observed for hemiasterlin methyl ester in the absence of protein.

A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents.

Application of triple-resonance and isotope-edited-NOE methods to the study of increasingly larger macromolecules and their complexes remains a central goal of solution NMR spectroscopy. The slow reorientational motion of larger molecules leads to rapid transverse relaxation and results in losses in both resolution and sensitivity of multidimensional-multinuclear solution NMR experiments. A recently described technique employs a physical approach to increase the tumbling rate of macromolecules in an attempt to preserve access to the full range of structural restraints available to studies of smaller systems. This technique involves encapsulation of a hydrated protein in a surfactant shell which is subsequently solubilized in a low viscosity solvent. A simple, efficient and cost effective NMR cell that accommodates the moderate liquefaction pressures required in the encapsulation method is described. Application of the method to the 56 kD triose phosphate isomerase homodimer is demonstrated.