Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis.

In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.

Assessment of immunogenicity and drug activity in patient sera by flow-induced dispersion analysis.

Biopharmaceuticals have revolutionized the treatment of many diseases such as diabetes, cancer, and autoimmune disorders. These complex entities provide unique advantages like high specificity towards their target. Unfortunately, biopharmaceuticals are also prone to elicit undesired immunogenic responses (immunogenicity), compromising treatment efficacy as well as patient safety due to severe adverse effects including life threatening conditions. Current immunogenicity assays are hampered by immobilization procedures, complicated sample pre-treatment, or rely on cell-based methods which all prevent reliable and continuous monitoring of patients. In this work, we present Flow Induced Dispersion Analysis (FIDA) for assessment of immunogenicity and drug activity in serum samples from arthritis patients receiving adalimumab. FIDA is a first principle technique for size-based characterization of biomolecules and their complexes under biologically relevant conditions. The FIDA methodology rely on an absolute and quantitative readout (hydrodynamic radius) thus reducing the need for positive and negative controls. Here, FIDA is applied for evaluating active adalimumab in serum by studying the interaction with its target tumor necrosis factor alpha (TNF-α). We report proof of principle for a quantitative approach for stratifying patients exhibiting presence of neutralizing and non-neutralizing antibodies based on their individual drug activity pattern. Further, it can be applied to any biopharmaceutical having soluble drug targets and it holds potential in a companion diagnostics setting.

Size-based characterization of adalimumab and TNF-α interactions using flow induced dispersion analysis: assessment of avidity-stabilized multiple bound species.

The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. Often, protein interactions involve multiple binding, avidity, oligomerization, and are dependent on the local environment. Current analytical methodologies are unable to provide a detailed mechanistic characterization considering all these parameters, since they often rely on surface immobilization, cannot measure under biorelevant conditions, or do not feature a structurally-related readout for indicating formation of multiple bound species. In this work, we report the use of flow induced dispersion analysis (FIDA) for in-solution characterization of complex protein interactions under in vivo like conditions. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. Here, the FIDA technology is utilized for a size-based characterization of the interaction between TNF-α and adalimumab. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, thus providing essential information on the TNF-α-adalimumab binding mechanism. Furthermore, it is shown that the avidity stabilized complexes involving formation of multiple non-covalent bonds are formed on a longer timescale than the primary complexes formed in a simple 1 to 1 binding event.

In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis.

Biopharmaceuticals such as protein and peptide-based drugs are often produced by fermentation processes where it is necessary to monitor the amount and quality of the product expressed during fermentation and for release testing of the final drug product. Standard procedures involve surface-based ligand binding technologies such as enzyme-linked immunosorbent assay and biolayer interferometry, or extensive purification using, e.g., preparative chromatography followed by spectrophotometric protein quantification. The multistep nature of these methodologies leads to lengthy protocols and renders real-time process control impractical. Recently, flow-induced dispersion analysis (FIDA) was introduced as a novel in-solution ligand binding technology, requiring only nano/microliter sample volumes. FIDA is based on Taylor dispersion analysis in narrow fused silica capillaries and provides the hydrodynamic radius of the binding ligand and complex in addition to the detailed binding characterization. Here, we demonstrate the use of FIDA for quantification of monoclonal IgG antibodies (rituximab) directly in mammalian cell fermentation broth with only 4 min of analysis time. The FIDA assay utilizes a small anti-IgG affibody, conjugated to a fluorophore, as a selective rituximab binder. The apparent change in the hydrodynamic radius of the affibody, as it interacts with known concentrations of rituximab, is used for generating a binding curve in a blank fermentation medium, and hence determining the dissociation constant and complex size. Finally, the binding curve is utilized for quantifying the rituximab titer concentration in clarified fermentation broth samples.

Electromembrane Extraction of Unconjugated Fluorescein Isothiocyanate from Solutions of Labeled Proteins Prior to Flow Induced Dispersion Analysis.

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 µL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 µL 0.1% (w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 µL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (Kd) and hydrodynamic radius of the complex were determined to be 0.8 µM and 5.87 nm, respectively, which was in concordance with previously reported values.

Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

Flow-Induced Dispersion Analysis (FIDA) for Protein Quantification and Characterization.

Flow-Induced Dispersion Analysis (FIDA) enables characterization and quantification of proteins under native conditions. FIDA is based on measuring the change in size of a ligand as it selectively interacts with the target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), which increase in the presence of the analyte due to binding to the analyte. The Kd of the interaction may be obtained in a titration experiment and the measurement of the apparent ligand size in an unknown sample forms the basis for determining the analyte concentration. The apparent molecular size is measured by Taylor dispersion analysis (TDA) in fused silica capillary capillaries. FIDA is a "ligand-binding" assay and has therefore certain features in common with Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), and Biolayer Interferometry (BLI) based techniques. However, FIDA probes a single in-solution binding event and thus makes assay development straightforward, and the absolute size measurement enables built-in assay quality control. Further, as FIDA does not involve surface chemistries, complications related to nonspecific adsorption of analyte and assay components are minimized enabling direct measurement in, e.g., plasma and serum.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.