Nanoplasmonic Avidity-Based Detection and Quantification of IgG Aggregates.

Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A-mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein A-functionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 µg/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 µg/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.

Process integrated biosensors for real-time monitoring of antibodies for automated affinity purification.

Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 µg mL-1. In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min-1. This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.

Self-Assembly of a Structurally Defined Chiro-Optical Peptide-Oligothiophene Hybrid Material.

Conducting polymers are routinely used in optoelectronic biomaterials, but large polymer polydispersity and poor aqueous compatibility complicate integration with biomolecular templates and development of discrete and defined supramolecular complexes. Herein, we report on a chiro-optical hybrid material generated by the self-assembly of an anionic peptide and a chemically defined cationic pentameric thiophene in aqueous environment. The peptide acts as a stereochemical template for the thiophene and adopts an α-helical conformation upon association, inducing optical activity in the thiophene π-π* transition region. Theoretical calculations confirm the experimentally observed induced structural changes and indicate the importance of electrostatic interactions in the complex. The association process is also probed at the substrate-solvent interface using peptide-functionalized gold nanoparticles, indicating that the peptide can also act as a scaffold when immobilized, resulting in structurally well-defined supramolecular complexes. The hybrid complex could rapidly be assembled, and the kinetics of the formation could be monitored by utilizing the local surface plasmon resonance originating from the gold nanoparticles. We foresee that these findings will aid in designing novel hybrid materials and provide a possible route for the development of functional optoelectronic interfaces for both biomaterials and energy harvesting applications.

Influence of Surfactant Bilayers on the Refractive Index Sensitivity and Catalytic Properties of Anisotropic Gold Nanoparticles.

Shape-controlled synthesis of gold nanoparticles generally involves the use of surfactants, typically cetyltrimethylammonium (CTAX, X = Cl(-) , Br(-)), to regulate the nucleation growth process and to obtain colloidally stable nanoparticles. The surfactants adsorb on the nanoparticle surface making further functionalization difficult and therefore limit their use in many applications. Herein, the influence of CTAX on nanoparticle sensitivity to local dielectric environment changes is reported. It is shown, both experimentally and theoretically, that the CTAX bilayer significantly reduces the refractive index (RI) sensitivity of anisotropic gold nanoparticles such as nanocubes and concave nanocubes, nanorods, and nanoprisms. The RI sensitivity can be increased by up to 40% by removing the surfactant layer from nanoparticles immobilized on a solid substrate using oxygen plasma treatment. This increase compensates for the otherwise problematic decrease in RI sensitivity caused by the substrate effect. Moreover, the removal of the surfactants both facilitates nanoparticle biofunctionalization and significantly improves their catalytic properties. The strategy presented herein is a simple yet effective universal method for enhancing the RI sensitivity of CTAX-stabilized gold nanoparticles and increasing their potential as transducers in nanoplasmonic sensors, as well as in catalytic and biomedical applications.

Silane-dextran chemistry on lateral flow polymer chips for immunoassays.

The prognosis for patients suffering from cardiovascular and many other diseases can be substantially improved if diagnosed at an early stage. High performance diagnostic testing using disposable microfluidic chips can provide a platform for realizing this vision. Amic AB (Uppsala, Sweden) has developed a new microfluidic test chip for sandwich immunoassays fabricated by injection molding of the cycloolefin-copolymer Zeonor. A highly ordered array of micropillars within the fluidic chip distributes the sample solution by capillary action. Since wetting of the pillar array surface is the only driving force for liquid distribution precise control of the surface chemistry is crucial. In this work we demonstrate a novel protocol for surface hydrophilization and antibody immobilization on cycloolefin-copolymer test chips, based on direct silanisation of the thermoplastic substrate. Dextran is subsequently covalently coupled to amino groups, thus providing a coating with a low contact angle suitable for antibody immobilization. The contact angle of dextran coated chips is stable for at least two months, which enables production of large batches that can be stored for extended periods of time. We demonstrate the utility of the presented platform and surface chemistry in a C-reactive protein assay with a detection limit of 2.6 ng ml(-1), a dynamic range of 10(2) and a coefficient of variance of 15%.