TY - JOUR A1 - Wilke, Marco A1 - Weller, Michael G. T1 - Affinitätschromatografie - Neues Trägermaterial aus Borosilikatglas JF - ChemieXtra N2 - Therapeutische Antikörper sind innerhalb weniger Jahre zur wichtigsten pharmazeutischen Produktklasse aufgestiegen. Für 2023 werden weltweite Umsätze von über 200 Milliarden USD erwartet. Auch diagnostische Antikörper sind mittlerweile unverzichtbare Produkte, auf deren Basis zahllose Immunoassays und andere Schnelltests entwickelt wurden. Neben der bereits sehr aufwendigen Herstellung von Antikörpern ist deren Aufreinigung aus komplexen Zellkulturmedien oder Blutseren und -plasmen zu einem Engpass in der Produktion und Nutzung dieser komplexen Proteine geworden. Schnelle und einfache Reinigungsmethoden für Antikörper sind daher sehr gefragt. KW - Affinitätschromatographie KW - Affinitätsextraktion KW - Antikörper KW - Glasmonolith KW - Additive Fertigung KW - IgG KW - Immunglobulin KW - HPLC KW - FPLC KW - biokompatibel KW - Borosilikatglas KW - gesintert KW - Titan-Halterung KW - Druckstabilität PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-477490 UR - https://www.chemiextra.com/ IS - 4 SP - 16 EP - 17 PB - Sigwerb CY - Zug AN - OPUS4-47749 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Celasun, S. A1 - Remmler, D. A1 - Schwaar, Timm A1 - Weller, Michael G. A1 - Du Prez, F. A1 - Börner, H. G. T1 - Digging into the sequential space of thiolactone precision polymers: A combinatorial strategy to identify functional domains JF - Angew. Chem. Int. Ed. N2 - Functional sequences of precision polymers based on thiolactone/Michael chemistry are identified from a large one-bead one-compound library. Single-bead readout by MALDI-TOF MS/MS identifies sequences that host m-THPC that is a second Generation photo-sensitizer drug. The corresponding Tla/Michael-PEG conjugates make m-THPC available in solution and drug payload as well as drug release kinetics can be fine-tuned by the precision segment. KW - Combinatorial chemistry KW - Combinatorial polymer libraries KW - Sequence-defined oligomer KW - Precision polymer sequencing KW - Pseudo peptides KW - MALDI-TOF KW - ESI MS KW - Mass spectrometry KW - Sequencing KW - PEG KW - Polyethylene glycol KW - Solubilizer KW - Drug KW - Conjugates PY - 2019 DO - https://doi.org/10.1002/anie.201810393 SN - 1521-3773 VL - 58 IS - 7 SP - 1960 EP - 1964 PB - Wiley-VCH CY - Weinheim AN - OPUS4-47323 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schwaar, Timm A1 - Lettow, Maike A1 - Remmler, Dario A1 - Börner, H. G. A1 - Weller, Michael G. T1 - Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides JF - High-Throughput N2 - Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide. KW - Peptide library KW - HTS KW - Target KW - MALDI KW - Mass spectrometry KW - Biochip KW - Lab-on-a-Chip KW - Array KW - Screening KW - Ladder sequencing KW - Binder KW - Pharmaceutical PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-478973 UR - https://www.mdpi.com/2571-5135/8/2/11 DO - https://doi.org/10.3390/ht8020011 VL - 8 IS - 2 SP - 1 EP - 15 PB - MDPI CY - Basel AN - OPUS4-47897 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Bierstedt, Andreas A1 - You, Yi A1 - van Wasen, Sebastian A1 - Bosc-Bierne, Gaby A1 - Weller, Michael G. A1 - Riedel, Jens T1 - Laser-Induced Microplasma as an Ambient Ionization Approach for the Mass-Spectrometric Analysis of Liquid Samples JF - Analytical Chemistry N2 - An airborne high repetition rate laser-induced plasma was applied as a versatile ambient ionization source for mass-spectrometric determinations of polar and nonpolar analytes in solution. The laser plasma was sustained between a home-built pneumatic nebulizer and the inlet capillary of an Orbitrap mass spectrometer. To maintain stable conditions in the droplet-rich spray environment, the plasma was directly fed by the fundamental output (λ = 1064 nm) of a current state-of-the-art diode-pumped solid-state laser. Ionization by the laser-driven plasma resulted in signals of intact analyte ions of several chemical categories. The analyte ions were found to be fully desolvated since no further increase in ion signal was observed upon heating of the inlet capillary. Due to the electroneutrality of the plasma, both positive and negative analyte ions could be formed simultaneously without altering the operational parameters of the ion source. While, typically, polar analytes with pronounced gas phase basicities worked best, nonpolar and amphoteric compounds were also detected. The latter were detected with lower ion signals and were prone to a certain degree of fragmentation induced during the ionization process. All the described attests the laser-induced microplasma by a good performance in terms of stability, robustness, sensitivity, and general applicability as a self-contained ion source for the liquid sample introduction. KW - Laser KW - Laser-induced plasma KW - Ambient ionization KW - Mass Spectrometry PY - 2019 DO - https://doi.org/10.1021/acs.analchem.9b00329 SN - 0003-2700 VL - 91 IS - 9 SP - 5922 EP - 5928 PB - American Chemical Society CY - Washington, DC, USA AN - OPUS4-47939 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schroeder, Barbara A1 - Le Xuan, Hoa A1 - Völzke, Jule L. A1 - Weller, Michael G. T1 - Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies JF - Methods and Protocols N2 - Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. KW - Antibody coating KW - Proximity-enhanced reaction KW - Immunoglobulins KW - IgG KW - Protein A KW - Protein G KW - Immunoprecipitation KW - Immunocapture KW - Regeneration KW - Biosensor KW - Immunosensor KW - Affinity chromatography KW - Immunoaffinity extraction KW - Oriented immobilization KW - Immunoassay KW - Bioconjugation PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-479786 UR - https://www.mdpi.com/2409-9279/2/2/35 DO - https://doi.org/10.3390/mps2020035 SN - 2409-9279 VL - 2 IS - 2 SP - 1 EP - 14 PB - MDPI CY - Basel, Switzerland AN - OPUS4-47978 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -