ABSTRACT
Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.
Subject(s)
Immunoglobulin G , Magnetic Iron Oxide Nanoparticles , Materials Testing , Particle Size , Magnetic Iron Oxide Nanoparticles/chemistry , Ligands , Immunoglobulin G/chemistry , Immunoglobulin G/isolation & purification , Biocompatible Materials/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/metabolism , Surface Properties , Ferric Compounds/chemistryABSTRACT
Biopharmaceuticals and their production are on the rise. They are needed to treat and to prevent multiple diseases. Therefore, an urgent need for process intensification in downstream processing (DSP) has been identified to produce biopharmaceuticals more efficiently. The DSP currently accounts for the majority of production costs of pharmaceutically relevant proteins. This short review gathers essential research over the past 3 years that addresses novel solutions to overcome this bottleneck. The overview includes promising studies in the fields of chromatography, aqueous two-phase systems, precipitation, crystallization, magnetic separation, and filtration for the purification of pharmaceutically relevant proteins.
Subject(s)
Biological Products , Proteins , Filtration , WaterABSTRACT
The original version of this Article contained an error in the Acknowledgements, which incorrectly omitted the following: 'C.C., C.A., and J.C.H. were supported by the Fundação Calouste Gulbenkian through a grant from the Instituto Gulbenkian de Ciência and by the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and Foundation for Science and Technology (Portugal).' This has been corrected in both the PDF and HTML versions of the Article.
ABSTRACT
Some strains of the protozoan parasite Toxoplasma gondii (such as RH) are virulent in laboratory mice because they are not restricted by the Immunity-Related GTPase (IRG) resistance system in these mouse strains. In some wild-derived Eurasian mice (such as CIM) on the other hand, polymorphic IRG proteins inhibit the replication of such virulent T. gondii strains. Here we show that this resistance is due to direct binding of the IRG protein Irgb2-b1CIM to the T. gondii virulence effector ROP5 isoform B. The Irgb2-b1 interface of this interaction is highly polymorphic and under positive selection. South American T. gondii strains are virulent even in wild-derived Eurasian mice. We were able to demonstrate that this difference in virulence is due to polymorphic ROP5 isoforms that are not targeted by Irgb2-b1CIM, indicating co-adaptation of host cell resistance GTPases and T. gondii virulence effectors.
Subject(s)
GTP Phosphohydrolases/immunology , Host-Parasite Interactions/immunology , Protozoan Proteins/immunology , Toxoplasma/pathogenicity , Toxoplasmosis, Animal/immunology , Animals , Disease Resistance/genetics , Disease Resistance/immunology , Female , Fibroblasts , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HEK293 Cells , Host-Parasite Interactions/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Protein Isoforms , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Selection, Genetic/immunology , Toxoplasma/immunology , Toxoplasmosis, Animal/parasitology , Virulence/immunologyABSTRACT
Sudden infant death syndrome (SIDS) is a target for public health care in Germany. The aim of this study was to monitor data on risk-related behavior in the population of Hamburg, Germany, in order to respond to changes quickly and to estimate the effectiveness of prevention activities. Data have been gathered using the sentinel system with repeated surveys (1996, 1998, 2001, and 2006) in pediatric practices, thus allowing an estimate of the prevalence of risk factors in an urban population, both transversally and vertically. From 1996 to 2007, the SIDS rate in Hamburg fell from 0.9/1,000 live births to 0.1. The prevalence of infants sleeping prone declined from 8.1% in 1996 to 3.5% in 2006. In this small subgroup, up to 81.7% (2006) of the caretakers were well aware of the risk of sleeping prone. The prevalence of infants sleeping on their sides fell from 55.3% in 1998 to 10.6% in 2006. The sentinel setting is suitable for gathering risk-related data on SIDS. Despite the fact that, so far, no nationwide back-to-sleep campaign has been instituted in Germany, local campaigns have proved successful in reducing prone sleeping for infants. Moreover, the substantial reduction of side sleeping within a short time span going along with a reduced SIDS rate is an indicator of the effectiveness of prevention activities on a local basis.