Development of Centrifugal Partition Chromatography for the Purification of Antibody-Drug Conjugates.

Monoclonal antibody-drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K < 0.16) and its monoclonal antibody backbone, IgG (0.16 < K < 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

Pharmaceutical analysis by NMR can accommodate strict impurity thresholds: The case of choline.

The ICH guidelines recommend reporting thresholds for regular impurities in drug substances at the level of 0.05% or 0.03% (w/w) depending on the maximum daily intake. Therefore, any instrumental method of analysis applicable to the impurity analysis should be able to detect and quantify the analytes at those levels. This investigation was designed to verify the suitability of 1H NMR spectroscopy for the detection of impurities, as a first step in the process before attempting quantification. In order to minimize demand on equipment, this study employed a 400 MHz instrument for structural confirmation and signal assignments of choline (1) and O-(2-hydroxyethyl)choline (2), a known impurity. The limit of detection (LOD) of 2 in 10 mg of 1 was established as 0.01% on a 400 MHz instrument and 2% on a 60 MHz (benchtop) NMR spectrometer. Thus, impurities for which quantification is required are readily detected at 400 MHz or above. These results are in contrast to the widespread belief that 1H NMR sensitivity is insufficient for pharmaceutical impurity analysis. The choice of solvent was recognized as a critical parameter for 1H NMR LOD analysis. Furthermore, publicly available NMR raw data (HMDB) proved to be valuable for unveiling the otherwise cryptic information hidden in complex signal patterns via 1H NMR iterative Full Spin Analysis. Finally, the study uncovered the less noticed, yet characteristic, 14N-1H coupling in the -N+(CH3)3 groups, adding strong arguments for the Raw NMR Data Initiative. Collectively, the data prove that the analytical capabilities of high-field NMR easily fulfill the ICH requirements for detection of impurity in the presence of an actual substance of interest which makes it a step closer to achieving regulatory standards.

Rufomycins or Ilamycins: Naming Clarifications and Definitive Structural Assignments.

Rufomycin and ilamycin are synonymous for the same class of cyclopeptides, currently encompassing 33 structurally characterized isolates and 9 semisynthetic derivatives. Elucidation of new structures prioritized the consolidation of the names and established the structures of four diastereoisomeric rufomycins with a 2-piperidinone, named rufomycins 4-7, including full 1H/13C NMR assignments. The characteristic HSQC cross-peak for the CH-5, the hemiaminal carbon in amino acid #5, allows assignment of the stereocenters C-4 and C-5 within this ring. Semisynthetic derivatives (rufomycinSS 1, 2, and 3) were prepared from a rufomycins 4 and 6 mixture to validate the structural assignments. Based on the X-ray crystal structures of rufomycins 2 and 4, considering the NMR differences of rufomycins 7 vs 4-6 compared to rufomycinSS 1 vs 2 and 3, and taking into account that two major conformers, A and B, occur in both rufomycinSS 2 and 3, structural modeling was pursued. Collectively, this paper discusses the NMR spectroscopic differences of the stereoisomers and their possible 3D conformers and correlates these with the anti-Mycobacterium tuberculosis activity. In addition, a look at the history prioritizes names and numbering schemes for this group of antibiotics and leads to consolidated nomenclature for all currently known members, natural and semisynthetic derivatives, and serves to accommodate future discoveries.

Plain 1 H nuclear magnetic resonance analysis streamlines the quality control of antiviral favipiravir and congeneric World Health Organization essential medicines.

Favipiravir is an established antiviral that is currently being assessed as an investigational drug for the treatment of COVID-19. Favipiravir is strikingly similar to two molecules that the World Health Organization (WHO) lists as essential medicines, which also consist of a six-membered aromatic N-heterocycle bearing a carboxamide function: the anti-tuberculosis agent, pyrazinamide, and nicotinamide, also known as vitamin B3 . We demonstrate the utility of 1 H nuclear magnetic resonance (NMR) profiling, an emerging pharmacopoeial tool, for the highly specific identification, selective differentiation of congeners, and subsequent detection of drug falsification or adulteration of these medicines. The straightforward comparison of basic 1-D 1 H NMR spectra, obtained with benchtop or advanced NMR instruments alike, offers a rapid identity assay and works independently of physical reference materials. This approach accelerates and advances pharmaceutical quality control measures under situations of increased drug demand and altered economy, such as during a pandemic.

Quantum mechanical NMR full spin analysis in pharmaceutical identity testing and quality control.

Issues related to pharmaceutical quality are arising at an alarming rate. Pharmaceutical quality concerns both the Active Pharmaceutical Ingredients (APIs) and the Finished Drug Product/ Formulation. Recently, there has been a significant increase in the number of reports of harmful impurities in marketed drug formulations. Impurities range from solvents, reactants, adulterants, and catalysts to synthetic byproducts. Quality concerns in commercial preparations may also arise due to shelf life stability. Furthermore, a number of falsified and substandard drug cases have been reported. Most of the techniques which are currently in place can, at best, detect the impurities, but cannot identify them unless they are already known and can be compared to a standard. On the other hand, 1H NMR spectroscopy detects all the hydrogen containing species, typically provides information to elucidate structures partially or even completely, and through its absolute quantitative capabilities even can detect the presence hydrogen-free species indirectly. The structural properties that produce 1H NMR signals as characteristic representations of a given molecule are the chemical shifts (δ in ppm) and coupling constants (J in Hz). Along with the line widths (ω1/2 in Hz), these parameters are bound to both the molecule and the NMR experimental conditions by quantum mechanical (QM) principles. This means that the 1H NMR spectra of APIs can be precisely calculated and compared to the experimental data. This review explains how 1H NMR spectroscopy coupled with Full Spin Analysis can contribute towards the quality control of pharmaceuticals by improving structural dereplication and achieving simultaneous quantification of both APIs and their contaminants.

Synthesis and antimicrobial evaluation of piperic acid amides and their lower homologues.

Seven piperic acid amides along with their lower homologs (12) were synthesized using HATU-DIPEA coupling reagent. All the synthesized derivatives were evaluated for their antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and vancomycin-resistant P. aeruginosa. They were found to be more active on P. aeruginosa than on S. aureus. However, they did not exhibit potent activity on Vancomycin resistant P. aeruginosa. Among the tested compounds, methylenedioxycinnamic acid amide of anthranilic acid (MDCA-AA, 2a) was found to be most active against S. aureus with MIC of 3.125 µg/ml. The PAS and INH amides of piperic acid were screened against Mycobacterium tuberculosis H37Ra strain. They were found to be most active among all the tested compounds but were found to be less active than the standard drug, isoniazid.

Separation of Unprecedented Degradants of Domperidone by Ultra-Performance Convergence Chromatography and Their Structure Elucidation.

Domperidone, a gastroprokinetic agent, is a common drug to treat emesis. It was subjected to acid, base-mediated hydrolysis, peroxide-mediated oxidation, photolysis and thermal degradation according to ICH guidelines to observe stability of the selected drug under the stress conditions. Although the drug is resistant to base hydrolysis, photolysis and thermal stressors, two degradants (DP-ISO1 and DP-ISO2) were formed in acid mediated hydrolysis. Oxidation with hydrogen peroxide also resulted in one product (DP-OX). All three degradants were isolated from the crude reaction mixture by preparative high-performance liquid chromatography and supercritical fluid chromatography. Structures of isolated compounds were unambiguously characterized as 5-chloro-1-(1-(3-(6-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)propyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (DP-ISO1), 5-chloro-1-(3-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (DP-ISO2), 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-1-(3-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)propyl)piperidine 1-oxide (DP-OX) by analysis of mass spectrometry, 1D and 2D nuclear magnetic resonance spectra. To the best of our knowledge, DP-ISO1 and DP-ISO2 are new and DP-OX was previously reported as domperidone impurity.

Structure elucidation of novel degradation products of thiocolchicoside by NMR spectroscopy.

Thiocolchicoside is a natural product analogue often used for its spasmolytic action. To know more about its stability under various stress conditions, the drug was stirred in acid, base and peroxide solutions. In acid hydrolysis, two products were obtained and in both, the glucose got cleaved. In one of them the acetyl group also got cleaved. A set of two diastereomers were formed during the peroxide mediated hydrolysis. The base mediated hydrolysis resulted in formation of three novel degradants. They have six membered rings in their structures instead of a seven membered cycloheptatrienone. Structures of known and novel degradation products have been elucidated by extensive analysis of HRMS, 1D and 2D NMR spectroscopic techniques.

Flavone C-glycosides from Trichuriella monsoniae (L.f.) Bennet.

In the first phytochemical investigation of Trichuriella monsoniae, three known flavonoidal C-glycosides, isoswertisin 1, 2″-O-ß-d-galactosyl isoswertisin 2 and 2″-O-ß-d-xylosyl isoswertisin 3 were isolated from the methanolic extract of the whole plant. Their structures were elucidated by extensive NMR spectroscopic studies including 2D NMR and HRMS, and the structure of 2 was supported by single crystal X-ray data studies. Further, NMR assignments for 3 are being reported for the first time.

New malabaricane triterpenes from the oleoresin of Ailanthus malabarica.

Ten malabaricane type triterpenes were isolated from the oleoresin of Ailanthus malabarica, out of which six (1-6) were new. For three of the known compounds (7-9), NMR assignments are being reported for the first time. Compound 10, a known one, is a new report from this source. The structures were established by extensive 1D and 2D NMR spectroscopy. The oleoresin and some of the isolates did not possess antimicrobial activity and did not lyse RBCs.