Development and evaluation of ion exchange-type chiral stationary phases for liquid chromatography and subcritical fluid chromatography

Hochleistungsflüssigchromatographie (HPLC) unter Verwendung von chiralen stationären Phasen (CSPs) ist in der Enantiomerenanalytik und in den enantioselektiven Trenntechniken von größter Bedeutung. So wird diese Technik im universitären Bereich und vor allem in der (pharmazeutischen) Industrie massiv eingesetzt, um chirale Verbindungen in enantiomerenreiner Form zu gewinnen. In der vorliegenden Arbeit wird die Herstellung von CSPs auf Ionenaustauscherbasis beschrieben, welche mittels HPLC und Superkritischer (Subkritischer) Fluid Chromatographie (SFC) auf ihre chiralen Trenneigenschaften hin evaluiert wurden. Im Allgemeinen können die in dieser Dissertation durchgeführten Forschungen auf zwei Bereiche aufgeteilt werden: Der erste Teil beschreibt eine umfassende Evaluierung von Chinin- und Chinidin- basierten Anionenaustauschern (QN-AX – und QD-AX CSP), welche mittels SFC betrieben wurden. Die chiralen Anionenaustauscher erwiesen sich als vollkommen SFC tauglich, da die Trenneigenschaften im Hinblick auf chirale Säuren vergleichbar waren mit den Ergebnissen aus bereits bekannten HPLC Messungen. Weiters konnte festgestellt werden, dass auch in der SFC ein ionenaustausch-dominierter Retentionsmechanismus vorliegt und dass die chirale Erkennung enthalpisch kontrolliert ist. Des Weiteren konnte chromatographisch die in situ Bildung von Methylkohlensäure (und deren dissoziierte Form, nämlich Methylcarbonat) in super(sub)kritischen CO2 – Methanol Mischungen festgestellt werden. Dieses chemische Phänomen ermöglicht Anionenaustauschchromatographie ohne Zugabe von (Puffer)Salzen, da die in situ gebildete Methylkohlensäure als Gegenion fungiert und somit Elution der sauren Analytmoleküle ermöglicht. Diese Erkenntnisse eröffnen neue Möglichkeiten und Wege für präparative Trennungen von chiralen Säuren, da unerwünschte Salzadditiva in der mobilen Phase vermieden werden können. Ferner wurden die QN-AX und QD-AX CSPs zur Enantiomerentrennung von neuartigen chiralen Sulfonsäuren (bzw. deren Sulfonatsalzen) mittels HPLC und SFC verwendet. Es konnte eine unkomplizierte Methode erstellt werden, welche die Basislinientrennung aller applizierten Sulfonsäureanalyte ermöglichte. In einer weiteren Applikationsstudie wurden beide vorher genannten Säulen zur indirekten Bestimmung der Absolutkonfiguration einer Reihe von chiralen sauren Wirkstoffen verwendet. Der zweite Teil der vorliegenden Dissertation beschreibt die Synthese von neuartigen Cinchona-Alkaloid-basierten zwitterionischen chiralen Selektoren (ZWIX-SOs). Durch nachfolgende Immobilisierung der SOs auf Kieselgel wurden zwitterionische chirale stationäre Phasen (ZWIX-CSPs) erhalten. Diese ZWIX-CSPs ermöglichten alle drei Arten von Ionenaustauschmodi, nämlich den schwachen Anionenaustauschmodus zur Trennung von chiralen Säuren, den starken Kationenaustauschmodus zur Trennung von chiralen Basen (Aminen) und, am wichtigsten, den Zwitterionenaustauschmodus zur Enantiomerentrennung von amphoteren Verbindungen wie freien Aminosäuren und kurzkettigen Peptiden. Bei der Synthese dieser ZWIX-SOs wurden Chinin oder Chinidin als Edukte verwendet, welche dann mit strukturell unterschiedlichen, sulfonsäuregruppen-enthaltenden Resten derivatisiert wurden (jeweils über eine Carbamatbindung an der C9-Position). Auf diese Art und Weise konnte ein kleines Set an Chinin- oder Chinidin-basierten ZWIX-CSPs hergestellt werden, welche nachfolgend für Studien der Struktur – Enantioselektivitäts-Beziehungen mittels HPLC verwendet wurden. ZWIX-CSPs mit chiralen Substituenten in Nachbarschaftsstellung zur Sulfonsäuregruppe waren ihren Analoga mit achiralen Seitenketten auf der Kationenaustauscherseite bezüglich Trennleistungen überlegen. Sie zeigten die größte Anwendungsbreite für Aminosäure-Enantiomerentrennungen, wohingegen Dipeptid-Analyte auf allen ZWIX-CSPs ähnlich gut getrennt werden konnten. Eine weitere vorteilhafte Eigenschaft bei Verwendung dieser ZWIX-CSPs war die Möglichkeit zur Umkehr der Elutionsreihenfolge der Aminosäure-Enantiomere, welche durch ein Wechseln von der Chinin-basierten zur pseudoenantiomeren Chinidin-basierten ZWIX-CSP realisiert werden konnte.High Performance Liquid Chromatography (HPLC) using chiral stationary phases (CSPs) is of central importance in both industry and academia for either analysis of enantiomeric purity of chiral molecules or (preparative) separation of enantiomers to obtain enantiomerically pure compounds. The present dissertation describes the preparation of low molecular weight ion exchange-type CSPs and their evaluation using HPLC and Supercritical (or Subcritical) Fluid Chromatography (SFC). The research carried out in the thesis can be divided into two projects. The first part of the thesis presents a comprehensive evaluation of quinine- and quinidine-based chiral anion exchanger stationary phases (QN-AX- or QD-AX CSPs, respectively) for SFC. Both CSPs proved to be fully applicable in SFC which was manifested in similar enantioseparation properties for chiral acids as compared to HPLC. The retention mechanism was found to be ion exchange dominated and thermodynamic analysis revealed an enthalpically controlled chiral recognition mechanism, thus resembling HPLC behaviour. Additionally, we could chromatographically confirm the in situ formation of methylcarbonic acid (and its dissociated species methylcarbonate, respectively) in supercritical CO2-based methanolic fluids. This phenomenon enables salt free chiral anion exchange chromatography by merely applying super(sub)critical CO2-methanolic mobile phases, because the in situ formed methylcarbonate functions as a counterion and thus enables elution of the acidic analytes. This finding opens up new possibilities for preparative separations of chiral acids, as troublesome mobile phase additives, such as salts or acids, can be avoided. Furthermore, QN-AX and QD-AX CSPs were applied for enantioseparation of novel chiral sulfonic acids with HPLC and SFC. We could establish a straightforward method for baseline resolving all of the investigated chiral sulfonic acids (or their sulfonate salts, respectively). In another application study, the absolute configuration of structurally related chiral acidic drug compounds was indirectly assigned by means of chiral HPLC using QN-AX and QD-AX CSPs. The second part of the thesis deals with the synthesis of cinchona alkaloid-based zwitterionic chiral selectors (ZWIX-SOs) and their immobilization onto silica gel to yield the corresponding zwitterionic chiral stationary phases (ZWIX-CSPs). The ZWIX-CSPs provided three modes of ion exchange, namely weak anion exchange mode for separation of chiral acids, strong cation exchange mode for resolving chiral bases (amines) and, most importantly, zwitterion exchange mode for the separation of amphoteric solutes, such as underivatized amino acids and small peptides. The synthesis followed a semi-synthetic approach using quinine or quinidine as starting materials, which were then derivatized with structurally different sulfonate group containing moieties (via a carbamate bond at the C9 position of the cinchona alkaloid). A small library of either quinine- or quinidine-based ZWIX-CSPs was prepared to carry out structure - enantioselectivity relationship studies for zwitterionic analytes by means of chromatography. In short, ZWIX-CSPs with chiral moieties in vicinity to their strong cation exchanger sites exhibited the broadest scope of application for the enantioseparation of amino acids, whereas dipeptides could be well resolved on all ZWIX-CSP independently from the chiral or achiral nature of the strong cation exchange subunit. A special benefit of ZWIX-CSPs is their ability to invert elution orders of amino acid enantiomers, which is easily accomplished by switching from a quinine-based to a pseudoenantiomeric quinidine-based ZWIX-CSP

Lateral frontal cortex volume reduction in Tourette syndrome revealed by VBM

<p>Abstract</p> <p>Background</p> <p>Structural changes have been found predominantly in the frontal cortex and in the striatum in children and adolescents with Gilles de la Tourette syndrome (GTS). The influence of comorbid symptomatology is unclear. Here we sought to address the question of gray matter abnormalities in GTS patients <it>with </it>co-morbid obsessive-compulsive disorder (OCD) and/or attention deficit hyperactivity disorder (ADHD) using voxel-based morphometry (VBM) in twenty-nine adult actually unmedicated GTS patients and twenty-five healthy control subjects.</p> <p>Results</p> <p>In GTS we detected a cluster of decreased gray matter volume in the left inferior frontal gyrus (IFG), but no regions demonstrating volume increases. By comparing subgroups of GTS with comorbid ADHD to the subgroup with comorbid OCD, we found a left-sided amygdalar volume increase.</p> <p>Conclusions</p> <p>From our results it is suggested that the left IFG may constitute a common underlying structural correlate of GTS with co-morbid OCD/ADHD. A volume reduction in this brain region that has been previously identified as a key region in OCD and was associated with the active inhibition of attentional processes may reflect the failure to control behavior. Amygdala volume increase is discussed on the background of a linkage of this structure with ADHD symptomatology. Correlations with clinical data revealed gray matter volume changes in specific brain areas that have been described in these conditions each.</p

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species

Extending the limits of size exclusion chromatography: Simultaneousseparation of free payloads and related species from antibody drugconjugates and their aggregates

tSize exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partiallyexcluded molecules from the pores of the stationary phase, based on their difference in hydrodynamicvolume. In this work, a baseline resolution was obtained between the monomeric antibody drug conjugate(ADC) and high molecular weight species (HMWS). Besides HMWS, small free payloads, linkers and linker-payloads of ADCs, which would not be discriminated solely based on their size (MW < 1.5 kDa), were alsoseparated on the same SEC column by applying sequentially an acetonitrile gradient after the elutionof the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amountunder native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For thispurpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 m particles and 250 Å pore size,was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was alsodeveloped to further extend the possibility offered by this experimental setup. The SECxRPLC multipleheart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 L samplingloops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to besuitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SECmethod was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC methodfacilitated separating some additional impurities. The streamlined methodology will further support thedevelopment and characterization of ADC product

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.</p