Christian G. Bauer

Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.

Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification

A bienzyme electrode based on the amplification of a signal has been developed which allows the determination of picoto nanomolar concentrations of p-aminophenol. The active element of the sensor comprised of coimmobilised laccase and glucose dehydrogenase enzymes coupled with an oxygen electrode. Laccase catalyzes p-aminophenol oxidation by oxygen to give p-iminoquinone. The latter is reduced by excess of glucose in the presence of glucose dehydrogenase and results in recycling of the substrate. The detection is realized by measuring the decrease in oxygen concentration. The detection limit for p-aminophenol is 100 pM. The feasibility of the determination of a number of other substrates (polyphenols, polyamines, ferrocene derivatives) in the nanomolar range has been demonstrated. A significant background signal has been found for p-aminophenylphosphate. This background is probably caused by the ability of laccase to catalyze the oxidative dephosphorylation. In the presence of phosphate ions this background is practically completely eliminated. 50 pM of alkaline phosphatase could be determined after a 2 min incubation in p-aminophenylphosphate solution by determination of the p-aminophenol formed as the result of hydrolysis. The whole analysis time does not exceed 5 min. The new technique is suitable for application in alkaline phosphatase based enzyme immunoassays.

Application of a sensitive catechol detector for determination of tyrosinase inhibitors

Abstract A substrate regenerating bienzyme sensor was used to measure a variety of organic and inorganic inhibitors with high sensitivity. The bienzyme system consists of the two cooperating enzymes, cytosolic quinoprotein glucose dehydrogenase and mushroom tyrosinase. The cooperation takes place on the substrate/product level. Under kinetic control, compounds that affect one of the enzymes can be detected with high sensitivity. For the construction of the sensor, the enzymes were immobilized in polyvinyl alcohol, coupled to a Clark-type oxygen electrode and the oxygen consumption was monitored during catechol conversion. In this way, carboxylic acids, kojic acids, inorganic ions and thiourea derivatives were studied. Theoretical considerations reveal the relationship between amplification gain and inhibitor concentration.

COUPLING OF IMMUNOASSAYS WITH ENZYMATIC RECYCLING ELECTRODES

Enzymatic substrate regeneration is a tool to enhance the sensitivity of enzyme electrodes both for substrate analysis and immunoassays. The combination of immunoreactions and electrode based substrate recycling connects specific recognition of an analyte with highly sensitive detection. Most important for this field of application is the sensitivity, which permits to detect a label at very low concentration. Enzymatic substrate regenerating systems are reviewed which are based on sensors using acceptor dependent dehydrogenases in combination with phenol oxidases and the lactate oxidase/lactate dehydrogenase couple and applied as sensitive label detectors in electrochemical immunoassays. Label compounds are either redox-active dyes or enzymes. The label enzymes measured so far are alkaline phosphatase and ß-galactosidase.

Biosensor technology for the detection of illegal drugs II: antibody development and detection techniques

In a joint project of Deutsche Aerospace, Boehringer Mannheim and the University of Potsdam portable devices for the detection of illegal drugs, based on biosensor technology, are being developed. The concept enrichment of the drug from the gas phase and detection by immunological means. This publication covers the development of specific antibodies and various detection procedures. Antibodies with a high affinity for cocaine have been developed with the aid of specially synthesized immunogens. A competitive detection procedure with biosensors based on optical grating couplers and applying particulate labels has been established, showing a lower detection limit of 10-10 mol/l for cocaine. Additionally, a combination of a displacement-immunoreactor and an enzymatically amplified electrode was investigated, which at present still suffers from insufficient sensitivity of the immunoreactor. An alternative, fleece-matrix based test procedure, where enrichment and detection steps are integrated in a single unit, is promising in terms of simplicity and sensitivity. A simple swab-test for the detection of cocaine at surfaces has been developed, which has a lower detection limit of about 10 ng and which can be performed within one minute.