Fibre-Optic Surface Plasmon Resonance Biosensor for Monoclonal Antibody Titer Quantification

An extraordinary optical transmission fibre-optic surface plasmon resonance biosensing platform was engineered to improve its portability and sensitivity, and was applied to monitor the concentrations of monoclonal antibodies (Mabs). By refining the fabricating procedure and changing the material of the flow cell and the components of the optical fibre, the biosensor is portable and robust to external interference. After the implementation of an effective template cleaning procedure and precise control during the fabrication process, a consistent sensitivity of 509 ± 5 nm per refractive index unit (nm/RIU) was achieved. The biosensor can detect the Mab with a limit of detection (LOD) of 0.44 µg/mL. The results show that the biosensor is a potential tool for the rapid quantification of Mab titers. The biosensor can be regenerated at least 10 times with 10 mM glycine (pH = 2.5), and consistent signal changes were obtained after regeneration. Moreover, the employment of a spacer arm SM(PEG)2, used for immobilising protein A onto the gold film, was demonstrated to be unable to improve the detecting sensitivity; thus, a simple procedure without the spacer arm could be used to prepare the protein A-based biosensor. Our results demonstrate that the fibre-optic surface plasmon resonance biosensor is competent for the real-time and on-line monitoring of antibody titers in the future as a process analytical technologies (PATs) tool for bioprocess developments and the manufacture of therapeutic antibodies.

Antibody–Drug Conjugates: The Last Decade

An armed antibody (antibody–drug conjugate or ADC) is a vectorized chemotherapy, which results from the grafting of a cytotoxic agent onto a monoclonal antibody via a judiciously constructed spacer arm. ADCs have made considerable progress in 10 years. While in 2009 only gemtuzumab ozogamicin (Mylotarg®) was used clinically, in 2020, 9 Food and Drug Administration (FDA)-approved ADCs are available, and more than 80 others are in active clinical studies. This review will focus on FDA-approved and late-stage ADCs, their limitations including their toxicity and associated resistance mechanisms, as well as new emerging strategies to address these issues and attempt to widen their therapeutic window. Finally, we will discuss their combination with conventional chemotherapy or checkpoint inhibitors, and their design for applications beyond oncology, to make ADCs the magic bullet that Paul Ehrlich dreamed of.

Hapten Design and Monoclonal Antibody to Fluoroacetamide, a Small and Highly Toxic Chemical

Fluoroacetamide (FAM) is a small (77 Da) and highly toxic chemical, formerly used as a rodenticide and potentially as a poison by terrorists. Poisoning with FAM has occurred in humans, but few reliably rapid detection methods and antidotes have been reported. Therefore, producing a specific antibody to FAM is not only critical for the development of a fast diagnostic but also a potential treatment. However, achieving this goal is a great challenge, mainly due to the very low molecular weight of FAM. Here, we design two groups of FAM haptens for the first time, maximally exposing the fluorine or amino groups, with the aid of linear aliphatic or phenyl-contained spacer arms. Interestingly, whereas the hapten with fluorine at the far end of the hapten did not induce an antibody response to FAM, the hapten with an amino group at the far end and phenyl-contained spacer arm triggered a significantly specific antibody response. Finally, a monoclonal antibody (mAb) named 5D11 was successfully obtained with an IC50 value of 97 μg mL−1 and negligible cross-reactivities to the other nine functional and structural analogs.

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yields inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly-populated “excited” states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualisation of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.

Hydrolysis of sunflower seed meal lignocellulosic fraction by free and immobilized cellulases

Lignocellulosic biomass is widely abundant in nature and recognized aspotential renewable energy source. Its efficient transformation into bio-basedfuels is enabled only after adequate pretreatment, followed by enzymaticsacharification and microbial fermentation. Hereby we present application of twocellulase preparations – from Aspergillus niger and Trichoderma reesei (Celluclast®)in treating sunflower seed meal lignocellulosic fraction (SSMLF). Temperature andpH optimums of two enzymes were determined – 52 °C and pH4.8 for A. nigercellulase and 55 °C and pH4.5 for Celluclast®. At optimized conditions, milledSSMLF was hydrolyzed by both biocatalysts. With A. niger cellulase higher initialreaction rates were accomplished and yield of 70 mM glucose equivalent wasobtained with 6 % (w/v) of enzyme after 6 hours. On the other hand, applicationof Celluclast® led to lower initial reaction rates and yielded 25 mM of glucoseequivalent with 10 % (v/v) of enzyme. To ensure cost-effective application ofA. niger cellulase, the possibility of its immobilization on different supports wasinvestigated. By using porous methacrylate-based carrier with C6 spacer arm andprimary amino groups – LifetechTM ECR8409, preparation with highest activity wasproduced. This preparation was successfully applied in saccharification of SSMLFand showed unchanged catalytic efficiency comparing to free enzyme.

Immobilization of laccase from Trametes versicolor on Lifetechtm supports for applications in degradation of industrial dyes

In this study, immobilization of laccase from Trametes versicolor on eight Lifetech? supports, with different characteristics (pore size, length of the spacer arm and functional groups), was studied and optimized for intended use in bioremediation for decolorization of industrial wastewaters. Out of six tested amino-functionalized supports, the most promising carrier was proved to be porous Lifetech? ECR8309F with primary amino groups and a C2 spacer arm. Onto this support, laccase is attached by forming electrostatic interactions so that the most active preparation has shown the activity of 66876 U/g support. On the other hand, during immobilization of laccase on epoxy-functionalized Lifetech? ECR8285F, via hydrophobic interactions and covalent bonding confirmed by a desorption assay, immobilization yield of 60 % and the activity of 118929 U/g were accomplished. Furthermore, immobilized enzyme on this support showed high capacity for decolorization of dyes (Lanaset? Violet B, Lanaset? Blue 2R, bromothymol blue and bromocresol green), by combination of both adsorption and enzyme degradation. Decolorization was in the range of 88 to 96 % after 4 h, with more than 80 % achieved after only 45 min. Also, this preparation demonstrated high operational stability during seven consecutive reuses in all examined dye reaction systems.