ABSTRACT
BACKGROUND: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immunogenic response in organism. METHODS: This article includes proposed detection and characterization of aggregated, as well as other modified forms of monoclonal antibodies (mAb), by using selected chromatographic and spectrometric methods. Additionally, selected mAb's aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells' (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immunogenic response of peripheral blood mononuclear cells (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMCs to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. RESULT AND CONCLUSION: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays, could be used as platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products which contain monoclonal antibodies as active pharmaceutical ingredients.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Cells, Cultured , Chromatography, Gel , Cytokines/biosynthesis , Dendritic Cells/immunology , Drug Discovery , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulin G/immunology , Leukocytes, Mononuclear/immunology , Molecular WeightABSTRACT
Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.
