Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step

Space-borne Bose-Einstein condensation for precision interferometry

Space offers virtually unlimited free-fall in gravity. Bose-Einstein condensation (BEC) enables ineffable low kinetic energies corresponding to pico- or even femtokelvins. The combination of both features makes atom interferometers with unprecedented sensitivity for inertial forces possible and opens a new era for quantum gas experiments. On January 23, 2017, we created Bose-Einstein condensates in space on the sounding rocket mission MAIUS-1 and conducted 110 experiments central to matter-wave interferometry. In particular, we have explored laser cooling and trapping in the presence of large accelerations as experienced during launch, and have studied the evolution, manipulation and interferometry employing Bragg scattering of BECs during the six-minute space flight. In this letter, we focus on the phase transition and the collective dynamics of BECs, whose impact is magnified by the extended free-fall time. Our experiments demonstrate a high reproducibility of the manipulation of BECs on the atom chip reflecting the exquisite control features and the robustness of our experiment. These properties are crucial to novel protocols for creating quantum matter with designed collective excitations at the lowest kinetic energy scales close to femtokelvins.Comment: 6 pages, 4 figure

Adatoms underneath Single Porphyrin Molecules on Au(111)

The adsorption of porphyrin derivatives on a Au(111) surface was studied by scanning tunneling microscopy and spectroscopy at low temperatures in combination with density functional theory calculations. Different molecular appearances were found and could be assigned to the presence of single gold adatoms bonded by a coordination bond underneath the molecular monolayer, causing a characteristic change of the electronic structure of the molecules. Moreover, this interpretation could be confirmed by manipulation experiments of individual molecules on and off a single gold atom. This study provides a detailed understanding of the role of metal adatoms in surface–molecule bonding and anchoring and of the appearance of single molecules, and it should prove relevant for the imaging of related molecule–metal systems

Photoswitchable Catalysts: Correlating Structure and Conformational Dynamics with Reactivity by a Combined Experimental and Computational Approach

Photocontrol of a piperidine's Bronsted basicity was achieved by incorporation of a bulky azobenzene group and could be translated into pronounced reactivity differences between ON- and OFF-states in general base catalysis. This enabled successful photomodulation of the catalyst's activity in the nitroaldol reaction (Henry reaction). A modular synthetic route to the photoswitchable catalysts was developed and allowed for preparation and characterization of three azobenzene-derived bases as well as one stilbene-derived base. Solid-state structures obtained by X-ray crystal structure analysis confirmed efficient blocking of the active site in the E isomer representing the OFF-states, whereas a freely accessible active site was revealed for a representative Z isomer in the crystal. To correlate structure with reactivity of the catalysts, conformational dynamics were thoroughly studied in solution by NMR spectroscopy, taking advantage of residual dipolar couplings (RDCs), in combination with comprehensive DFT computational investigations of conformations and proton affinities.</p

Photoisomerization Ability of Molecular Switches Adsorbed on Au(111): Comparison between Azobenzene and Stilbene Derivatives

High resolution electron energy loss spectroscopy and two-photon photoemission was employed to derive the adsorption geometry, electronic structure, and the photoisomerization ability of the molecular switch tetra-tert-butyl-stilbene (TBS) on Au(111). The results are compared with the azobenzene analogue, tetra-tert-butyl-azobenzene (TBA), adsorbed on Au(111). TBS was found to adsorb on Au(111) in a planar (trans) configuration similar to TBA. The energetic positions of several TBS-induced electronic states were determined, and in comparison to TBA, the higher occupied molecular states (e.g., the highest occupied molecular orbital, HOMO) are located at similar energetic positions. While surface-bound TBA can be switched with light between its trans and cis configurations, in TBS this switching ability is lost. In TBA on Au(111), the trans → cis isomerization is driven by a substrate-mediated charge transfer process, whereby photogenerated hot holes in the Au d band lead to transient positive ion formation (transfer of the holes to the TBA HOMO level). Even though the energetic positions of the HOMOs in TBA and TBS are almost identical and thus a charge transfer should be feasible, this reaction pathway is obviously not efficient to induce the trans → cis isomerization in TBS on Au(111). Quantum chemical calculations of the potential energy surfaces for the free molecules support this conclusion. They show that cation formation facilitates the isomerization for TBA much more pronounced than for TBS due to the larger gradients at the Franck−Condon point and the much smaller barriers on the potential energy surface in the case of the TBA