In this work we report the application of two-dimensional gel electrophoresis (2-D electrophoresis) for the study of the quality of selected classes of biopharmaceuticals, focusing primarily on its suitability in the... more
In this work we report the application of two-dimensional gel electrophoresis (2-D electrophoresis) for the study of the quality of selected classes of biopharmaceuticals, focusing primarily on its suitability in the regulatory/pharmacopoeia environment. In order to achieve this objective, selected recombinant biological medicines, belonging to the therapeutically relevant classes of recombinant cytokines, monoclonal antibodies and their related products, fusion proteins, were thoroughly studied using one-dimensional, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D electrophoresis. Resolved proteins were subsequently subjected to in gel tryptic digestion and resulted peptides analyzed with MALDI-TOF MS analysis. Experimental data showed that this methodology can be used for the identity characterization, and the assessment of stability, structural integrity and purity of studied biologicals. Given that 2-D electrophoresis, MALDI-TOF MS and peptide mass f...
A simple, rapid and specific ion-pair HPLC method for the determination of (R,R)-glycopyrronium bromide and its related impurities is presented, and parameters affecting the chromatographic properties of these compounds are discussed.... more
A simple, rapid and specific ion-pair HPLC method for the determination of (R,R)-glycopyrronium bromide and its related impurities is presented, and parameters affecting the chromatographic properties of these compounds are discussed. Optimal analyte separation was achieved on base deactivated Nucleosil at 40 degrees C, using phosphate buffer pH 2.30 with sodium-1-decanesulfonate (0.01 M)/methanol (35/65; v/v) as eluent for isocratic elution at a flow rate 1 ml x min(-1). The analytical assay was validated according to international guidelines. The methodis suitable for in-process control and as stability indicating assay.
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Acarbose (Glucobay, Bayer AG) acts as a potent alpha-glucosidase-inhibitor, which delays the intestinal starch digestion resulting in a reduction of postprandial blood glucose and insulin levels. Acarbose is a pseudo-tetrasaccharide, with... more
Acarbose (Glucobay, Bayer AG) acts as a potent alpha-glucosidase-inhibitor, which delays the intestinal starch digestion resulting in a reduction of postprandial blood glucose and insulin levels. Acarbose is a pseudo-tetrasaccharide, with two D-glucose units linked via an alpha 1-->4 glycosidic bond to acarviosin, which is a N-glycoside composed of an unsaturated cyclitol and 4-amino-4,6-dideoxy-alpha-D-glucopyranose. Several methods for the determination of acarbose by capillary electrophoresis can be found in literature. They are based either on the derivatisation with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) or on the detection of the unsaturated cyclitol at wavelengths below 200 nm. The aim of our work was the determination of acarbose making use of a previously developed method based on reductive amination with S-phenylethylamine. The aminoalditols generated in the reaction formed differently charged borate-complexes depending on the configuration of the sugar. After su...
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In this paper we propose the use of lactic acid oligomers (OLAs) as prodrug moieties. Two synthetic approaches are presented, on the one hand a non selective oligomerisation of lactic acid and on the other hand a block synthesis to... more
In this paper we propose the use of lactic acid oligomers (OLAs) as prodrug moieties. Two synthetic approaches are presented, on the one hand a non selective oligomerisation of lactic acid and on the other hand a block synthesis to tetramers of lactic acid. Dimers of lactic acid were investigated with respect to their plasma stability and their adsorption to albumine. Ibuprofen was chosen as the first drug for OLAylation. The ester 19 of LA(1)-ibuprofen was evaluated with respect to the degradation to human plasma and the adsorption to albumine. All results indicate that lactic acid oligomers are promising prodrug moieties.
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Capillary electrophoresis is competitive to HPLC and other chromatographic methods, predominantly when charged analytes have to be separated. The time of analysis can be reduced by the use of very short capillaries applying a high... more
Capillary electrophoresis is competitive to HPLC and other chromatographic methods, predominantly when charged analytes have to be separated. The time of analysis can be reduced by the use of very short capillaries applying a high voltage. In most instruments which are commercially available the so-called 'short end' of the capillary can be used for separation, leading to very rapid separations. In this contribution we want to demonstrate this approach by using Diclofenac Sodium as an analyte.
In the course of our validation program testing blood-brain barrier (BBB) in vitro models for their usability as tools in drug discovery it was evaluated whether an established Transwell model based on porcine cell line PBMEC/C1-2 was... more
In the course of our validation program testing blood-brain barrier (BBB) in vitro models for their usability as tools in drug discovery it was evaluated whether an established Transwell model based on porcine cell line PBMEC/C1-2 was able to differentiate between the transport properties of first and second generation antihistaminic drugs. First generation antihistamines can permeate the BBB and act in the central nervous system (CNS), whereas entry to the CNS of second generation antihistamines is restricted by efflux pumps such as P-glycoprotein (P-gP) located in brain endothelial cells. P-gP functionality of PBMEC/C1-2 cells grown on Transwell filter inserts was proven by transport studies with P-gP substrate rhodamine 123 and P-gP blocker verapamil. Subsequent drug transport studies with the first generation antihistamines promethazine, diphenhydramine and pheniramine and the second generation antihistamines astemizole, ceterizine, fexofenadine and loratadine were accomplished ...
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Calibration of complex analytical systems is always a difficult task. Nevertheless, a suitable approach has to be designed before the systems can be introduced into routine analysis. In literature, many methods have been described for the... more
Calibration of complex analytical systems is always a difficult task. Nevertheless, a suitable approach has to be designed before the systems can be introduced into routine analysis. In literature, many methods have been described for the purpose of calibrating such systems, but only a few of them deal with capillary elctrophoresis. Here, we want to demonstrate a general approach to how the calibration of this type of analytical instrument becomes feasible.
Intravitreally applied triamcinolone acetonide (TA) is used to treat a variety of macular diseases. Commercially available products of TA are mainly intended for intramuscular application and contain benzyl alcohol (BA) as a... more
Intravitreally applied triamcinolone acetonide (TA) is used to treat a variety of macular diseases. Commercially available products of TA are mainly intended for intramuscular application and contain benzyl alcohol (BA) as a bacteriostatic preservative. Since this agent damages ocular tissues, different methods such as filtration techniques and centrifugation are usually used to eliminate BA from commercial products (40 mg/mL TA, 9.9 mg/mL BA). In this study, we evaluated these methods in regard to their ability to eliminate benzyl alcohol and to guarantee standard doses of triamcinolone acetonide. A new formulation without BA (TA 40 mg/mL) was developped according to the following criteria: autoclavability, stability, and suitability for intravitreal use. For QA/QC evaluation a new rapid and simple HPLC procedure (C18 RP column, mobile phase consisting of methanol-water, 48:52, v/v) to quantify the respective compounds was developed and validated according to ICH guidelines. The HP...
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Research Interests: Bioinformatics, Genetics, Life Sciences, Stability, Quality Control, and 14 moreProtein Stability, Biomedical Research, Temperature, Monoclonal Antibodies, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Glycosylation, Molecular sciences, Molecular weight, Protein isoforms, Two-Dimensional Gel Electrophoresis, Recombinant Proteins, SDS PAGE, Hydrogen-Ion Concentration, and Isoelectric point
Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not... more
Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not in primary astrocytes. In spite of this promising activity, a broad therapeutic application of vitamin D metabolites and analogs is still restricted because of their poor bioavailability and their hypercalcemic actions. Compared to 1alpha,25(OH)2D3, its natural 3alpha-epimer exhibits far higher metabolic stability and a reduced calcemic effect. Focusing on a possible therapeutic advantage of the 3alpha-conformation, we have examined the apoptotic potential of a representative set of vitamin D analogs, each of them in the 3alpha- and 3beta-conformation, and of natural vitamin D metabolites in the rat C6 glioma cell line. Exposure of these cells to the synthetic analogs resulted in all cases in a pronounced reduction of cell density (tested by incorporation of neutral red) and induction of apoptosis, monitored by staining nuclei with Hoechst 33258 dye and by following DNA fragmentation by capillary electrophoresis. The 3alpha-epimers showed equivalent or even higher activity on C6 cells than their respective 3beta forms. For their potent effects on growth and apoptosis of tumor cells and their high metabolic stability combined with a low calcemic potential, we speculate that these 3a-epimers could provide advantages for a prospective treatment of glioma.
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... 10 J. Sweeney , Eur. J. Org. Chem. 2009 , 4911 –4919 . ... 17 ML Berger , C. Schodl , CR Noe , Eur. J. Med. Chem. 1998 , 33 , 3 –14 . ...
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In the course of the validation of blood-brain barrier in vitro models the aim of this work was to characterize two promising continuous cell lines with regard to their tightness properties. PBMEC/C1-2 and ECV304 cells were cultured in... more
In the course of the validation of blood-brain barrier in vitro models the aim of this work was to characterize two promising continuous cell lines with regard to their tightness properties. PBMEC/C1-2 and ECV304 cells were cultured in several media with different compositions on Transwell inserts. Inducibility and functionality of tightness were investigated by transendothelial electrical resistance (TEER) and by transport studies with transcellular marker diazepam, glycine antagonist Bu101 and paracellular marker APTS-dextran. Inducibility, expression and localization of tight junctional proteins were assessed by western blotting and immunofluorescence microscopy. Presence of factors derived from glioma cell line C6 resulted in increased TEER in both cell lines. Comparison to APTS-dextran data across Caco-2 layers emphasized that correlations between permeability of the paracellular marker and TEER are dependent on each investigated cell line and the corresponding growth medium. Presence and inducibility of tight junctional proteins ZO-1 and Occludin were proven for ECV304 layers. Cell line ECV304 seemed to be suitable for TEER dependent transport studies, whereas PBMEC/C1-2 showed higher potential for P-gP studies.