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Vollständige Antikörper bestehen aus eine kristallisierbaren (Fc) Region und einer Antigen-bindenden (Fab‘) Region. Die Fab Region wurde in den letzten Jahren für therapeutische Zwecke eingesetzt. Der größte Nachteil ist die kurze Halbwertszeit in vivo. Zur Verlängerung der Halbwertszeit können chemische Modifikationen, wie die Technologie der PEGylierung, angewendet werden. Diese Arbeit konzentriert sich auf die PEGylierung von Fab‘s. Das Ziel dieser Arbeit war es eine Reversed Phase Chromatographie Methode unter Anwendung eines Charged Aerosol Detektor zur Bestimmung von Nicht-PEGylierten und PEGylierten Fab‘s zu entwickeln. Das Ziel dieser Methode ist ihre universelle Anwendbarkeit für andere Nicht-PEGylierte und PEGylierte Fab's. Die Reversed Phase Chromatographie unterliegt hydrophoben Wechselwirkungen zwischen den Molekülen und der Säule. Der Charged Aerosol Detektor wurde in dieser Arbeit aufgrund seines Vorteils, nicht UV-aktive Moleküle wie PEG zu analysieren, verwendet. Es ist eine vielversprechende Technologie die ihre Anwendung für Bereiche wie verschiedene Pharmazeutika, Proteine und noch mehr Biomoleküle findet. Die entwickelte und optimierte Methode wird für weitere Untersuchungen im Rahmen dieser Arbeit sowie für die Bestimmung von anderen Nicht-PEGylierten und PEGylierten Antikörper-Fragmenten Anwendung finden. Mit der etablierten Methode wird der Prozess der PEGylierung und De-PEGylierung von Fab und deren thermodynamischer Effekt untersucht. Als Ergebnis wurde eine Reversed Phase Chromatographie Methode unter Anwendung eines Charged Aerosol Detektor für Nicht-PEGylierte und PEGylierte Fab‘s etabliert und für die weitere Entwicklung in dieser Arbeit angewandt. Die PEGylierungs- und De-PEGylierungs- Studien wurden erfolgreich abgeschlossen. Schließlich bestätigten die thermodynamischen Studien die Literatur, in welcher angegeben ist, dass PEGylierte Moleküle stabiler als Nicht-PEGylierte Moleküle in Bezug auf ihre Thermodynamik sind. Zusammenfassend gibt diese Arbeit Informationen über Optimierung und Entwicklung einer Reversed Phase Chromatographie Methode unter Anwendung eines Charged Aerosol Detektors, über PEGylierungs und De-PEGylierungs Studien sowie über Untersuchungen zu ihrer Thermodynamik wieder. Full antibodies consist of a fragment crystallizable (Fc) region and a fragment antigen-binding (Fab’) region. The Fab region has been used for therapeutic purposes in recent years. The main drawback is their short in vivo half-life. For the extension of the half-life, chemical modifications like the technology of PEGylation may be applied. This thesis concentrates on the PEGylation of Fab’s. The objective of this thesis is to develop a reversed phase chromatography method using a Charged Aerosol Detector to determine Non-PEGylated and PEGylated Fab’s. The aim of this established method is its universal applicability for other Non-PEGylated and PEGylated Fab’s. The reversed phase chromatography is based on hydrophobicity interaction between the molecules and the column. The Charged Aerosol Detector is used in this thesis due to its advantage to analyze non-UV active molecules like PEG. It is a promising technology used in applications including different pharmaceuticals, proteins and even more biomolecules. The finally developed and optimized method was applied for further investigations during this thesis as well as for the determination of other Non-PEGylated and PEGylated Antibody-fragments. With the established method, the process of PEGylation and De-PEGylation of Fab’ and their thermodynamic effect was investigated. As a result, a reversed phase chromatography method using a Charged Aerosol Detector for Non-PEGylated and PEGylated Fab’s was established and used for further development work in this thesis. The PEGylation and De-PEGylation studies were investigated and successfully completed. Finally, the thermodynamic studies confirmed the literature stating that PEGylated molecules are more stable than Non-PEGylated molecules concerning their thermodynamics. In conclusion, this thesis provides information about optimization and development of a reversed phase chromatography method using a Charged Aerosol Detector, PEGylation and De-PEGylation studies as well as investigations concerning their thermodynamics. vorgelegt von: Jürgen Iser Masterarbeit Wien, FH Campus Wien 2013

During the stability evaluation of β-artemether containing finished drug products, a consistent and disproportional increase in the UV-peak areas of β-artemether degradation products, when compared to the peak area decline of β-artemether itself, was observed. This suggested that the response factors of the formed β-artemether degradants were significantly higher than β-artemether. Dry heat stressing of β-artemether powder, as a single compound, using different temperatures (125–150 °C), times (10–90 min) and environmental conditions (neutral, KMnO 4 and zinc), resulted in the formation of 17 degradants. The vast majority of degradants seen during the long-term and accelerated ICH stability study of the drug product, were also observed here. The obtained stress results allowed the calculation of the overall average relative response factor (RRF) of β-artemether degradants, i.e. 21.2, whereas the individual RRF values of the 9 most prominent selected degradants ranged from 4.9 to 42.4. Finally, Ames tests were performed on β-artemether as well as a representative stressed sample mixture, experimentally assessing their mutagenic properties. Both were found to be negative, suggesting no mutagenicity problems of the degradants at high concentrations. Our general approach and specific results solve the developmental quality issue of mass balance during stability studies and the related genotoxicity concerns of the key antimalarial drug β-artemether and its degradants.

Comparison of hydrophilic interaction chromatography (HILIC) columns coupled with an evaporative light scattering detector (ELSD) or charged aerosol detector (CAD) was done for the detection of gabapentin in pharmaceutical formulations. The chromatographic separations were achieved on four HILIC columns: ZIC HILIC, ZIC pHILIC, Luna HILIC, and Atlantis HILIC. Experimental factors such as mobile phase composition, acetonitrile content, and mobile phase pH were evaluated. Validation of method was done in terms of linearity, sensitivity, accuracy, and precision. The performance of ELSD detection method is comparable to that of CAD. The intra-day and inter-day variations were below 1.7% and 3.2% for CAD and 2.8%, and 3.4% for ELSD, respectively. In addition, detection sensitivities of ELSD, CAD, and UV detectors were also compared for HILIC and reversed phase (RP) modes and the highest sensitivities were obtained in the HILIC mode when connected with CAD and ELSD. The developed HILIC aerosol based detection methods were successfully applied to the analysis of gabapentin in commercial tablets and capsules.

The present work studies the effectiveness of the use of triacylglycerols (TAGs) for the quantification of olive oil in blends with vegetable oils. The determinations were obtained using high-performance liquid chromatography (HPLC) coupled to a Charged Aerosol Detector (CAD), in combination with Partial Least Squares (PLS) regression and using interval PLS (iPLS) for variable selection. Results revealed that PLS models can predict olive oil concentrations with reasonable errors. Variable selection through iPLS did not improve predictions significantly, but revealed the chemical information important in the chromatogram to quantify olive oil in vegetable oil blends.

This work presents a method for an efficient differentiation of olive oil and several types of vegetable oils using chemometric tools. Triacylglycerides (TAGs) profiles of 126 samples of different categories and varieties of olive oils, and types of edible oils, including corn, sunflower, peanut, soybean, rapeseed, canola, seed, sesame, grape seed, and some mixed oils, have been analyzed. High-performance liquid chromatography coupled to a charged aerosol detector was used to characterize TAGs. The complete chromatograms were evaluated by PCA, PLS-DA, and MCR in combination with suitable preprocessing. The chromatographic data show two clusters; one for olive oil samples and another for the non-olive oils. Commercial oil blends are located between the groups, depending on the concentration of olive oil in the sample. As a result, a good classification among olive oils and non-olive oils and a chemical justification of such classification was achieved.

The increasing occurrence of toxic cyanobacterial blooms has led to a requirement for robust monitoring strategies and whilst several validated procedures have been developed these can be limited by the lack of high quality calibration standards. High quality standards must have confirmation of identity, purity and concentration by multiple methods. One aspect, purity, is rarely addressed but is essential. This is the first evaluation of the charged aerosol detector (CAD) to determine the benefits of incorporating a universal detector for more accurate purity determination of these peptides. Microcystins were detected at 5–10 ng on the column using the CAD, providing comparable quantification limits to those obtained using traditional UV detection. Purity determination of test compounds that had been partially purified, had showed that highest purity was at 238 nm > UV TIC > ESI TIC > CAD indicating that increased impurities could be detected using the CAD thus providing a more accurate indication of compound quality. Compounds purified by preparative HPLC were shown to have relative purities between 97% and 99%, however, when evaluated by CAD this dropped to 90–94% supporting the multi-detector strategy as essential for production of high quality compounds.

Recent literature has described the exciting development of a new universal detection technology for high-performance liquid chromatography (HPLC), as well as some exploratory work on its application to quantitative measurement of solutes at millimolar concentrations. The new methodology, known as charged aerosol detection (CAD), has been recognized as a viable alternative to evaporative light-scattering detection and refractive index detection that, like CAD, respond to molecular structures independently of their absorbance, or lack thereof, in the ultraviolet region of the electromagnetic spectrum. In this article, the authors exemplify their use of CAD in-line with HPLC and mass spectrometry (MS) to provide both stand-alone and complementary information that aids decision making for sample storage and processing practices in the compound management setting. Illustrations include monitoring contaminants leached from different plate materials into the solvent dimethyl sulphoxide (DMSO) and profiling the concentrations of solutions destined for liquid storage and dispensing to assays, with the aim of improving processes.

In recent decades the use of liposomal preparations as drug delivery systems has become very attractive in pharmaceutical development. Therefore, thorough characterization and quantification of the lipids which form liposomes is wished from both investigators and regulatory authorities when the application in humans is being considered. In this study a new HPLC method for the detection of lipids in liposomal formulations was established using corona charged aerosol detection (CAD) which has the advantage to be independent of the chemical properties of the analytes. The superiority of this method over UV detection was demonstrated. Compared to UV detection no absorption effects of the organic solvent in the mobile phase interfering with the lipid signals were observed with CAD. CAD showed good linearity ( R 2 > 0.990) for all liposomal compounds. The acceptance criteria for precision including repeatability were met. The average recovery for each of the excipients of the liposomal formulation was in the range of 90.0–110%.

The reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of dabigatran etexilate (DAB) in capsules using an ultraviolet detector (UV) and a charged aerosol detector (CAD) connected in series. The RP-LC method was carried out on a GL Sciences Inc. Inertsil ODS-2 column (250 mm × 4.6 mm I.D.). The mobile phase consisted of acetonitrile and ammonium acetate buffer (pH 5.5; 10 mM) (65 : 35, v/v) (flow rate 1.0 mL min−1). CAD detection was performed using a nitrogen pressure of 35 psi and a range of 100 pA and the UV detection at 225 nm. The chromatographic separation was obtained with a retention time of 6.7 min. Validation parameters such as specificity, linearity, precision, accuracy and robustness were evaluated. The method was linear over the concentration range of 1–70 μg mL−1 in both detectors. The specificity and stability-indicating capability of the method were proven through the degradation studies, which also show that there was no interference from the excipients. The method was precise and accurate, with the Plackett–Burman experimental design used to evaluate robustness, delivering results within the acceptable range. The method was successfully applied for DAB analysis and the results of the CAD and UV detectors were compared, showing a non-significant difference (P > 0.05).

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.