Comparative study on quantitation of human myoglobin by both isotope dilution mass spectrometry and surface plasmon resonance based on calibration-free analysis.

The activity of proteins rather than the concentration of proteins in biopharmaceutical and in vitro diagnostics are often the primary focus. Nonetheless, development of a calibration-free concentration analysis (CFCA) approach that accurately quantifies the concentration of proteins based on molecular interactions with specific monoclonal antibodies and without the requirement of external calibrators would be beneficial to diagnostics. Generally, only analytes that interact with the antibody (Ab) are quantified by CFCA. Moreover, protein concentrations measured by CFCA usually vary when different Abs are used, and are lower than those obtained by amino acid analysis because any non-native state population of the target protein is not captured by the Ab. To achieve comparable results between CFCA and traditional amino acid analysis (AAA), an Ab that recognizes the target protein irrespective of its conformation should be used. In this report, three different monoclonal antibodies were used to quantify purified human myoglobin in solution by CFCA. The concentrations obtain by the Abs (i.e., 2.985, 2.912, 3.032 mg mL-1) were comparable with that obtained by AAA. Moreover, isotope dilution mass spectrometry (IDMS) gave a human myoglobin concentration of 2.851 mg mL-1, which is also in agreement with the results from CFCA. The performance of CFCA was evaluated by measuring various parameters, including within-day and between-day precision. The results demonstrated that the active concentration measured by CFCA is comparable with that of IDMS when the appropriate Ab is used. Recommended procedures for performing the new CFCA approach are provided. This study shows that CFCA represents a primary method for accurate protein concentration determination, which should aid the development of certified reference materials. Graphical abstract.

Evaluation of calibration-free concentration analysis provided by Biacore™ systems.

Surface Plasmon Resonance biosensors measure the interaction between a molecule in solution and its interaction partner attached to a sensor surface. Under certain conditions, the observed binding rate can be used directly to obtain the concentration of the molecule in solution, without the use of any standard. This type of assay is referred to as Calibration Free Concentration Analysis, CFCA. By examining experimental conditions, including immobilization levels and temperature, for a range of analytes, and by using global analysis of several sample dilutions, conditions that gave the most robust results were identified. These conditions provided the concentration values that were on average ∼15% lower than those obtained using other methods. The accuracy of the concentration determined may be related to how the analyte is distributed in the dextran matrix and to its distance from the gold surface, and may thereby depend on the conversion of the SPR signal to mass. A good precision of CFCA, ∼8% (n = 21), was demonstrated when this method was used to efficiently guide purification procedures of Interferon α-2a. In this paper, the theory behind CFCA and the future developments, as well as the application of CFCA for absolute and relative concentration measurements (including the assessment of the potency of a biotherapeutic medicine) are discussed, and new evaluation tools that broaden the range of applications, are introduced.

Analysis of the dose-dependent stage-specific in vitro efficacy of a multi-stage malaria vaccine candidate cocktail.

BACKGROUND: The high incidence and mortality rate of malaria remains a serious burden for many developing countries, and a vaccine that induces durable and highly effective immune responses is, therefore, desirable. An earlier analysis of the stage-specific in vitro efficacy of a malaria vaccine candidate cocktail (VAMAX) considered the general properties of complex multi-component, multi-stage combination vaccines in rabbit immunization experiments using a hyper-immunization protocol featuring six consecutive boosts and a strong, lipopolysaccharide-based adjuvant. This follow-up study investigates the effect of antigen dose on the in vitro efficacy of the malaria vaccine cocktail using a conventional vaccination scheme (one prime and two boosts) and a human-compatible adjuvant (Alhydrogel(®)). RESULTS: IgG purified from rabbits immunized with 0.1, 1, 10 or 50 µg doses of the VAMAX vaccine candidate cocktail was analysed for total IgG and antigen-cocktail-specific titers. An increase in cocktail-specific titers was observed between 0.1 and 1 µg and between 10 and 50 µg, whereas no significant difference in titers was observed between 1 and 10 µg. Antigen component-specific antibody titers and stage-specific in vitro efficacy assays were performed with pooled IgG from animals immunized with 1 and 50 µg of the VAMAX cocktail. Here, the component-specific antibody levels showed clear dose dependency whereas the determined stage-specific in vitro IC50 values (as a correlate of efficacy) were only dependent on the titer amounts of stage-specific antibodies. CONCLUSIONS: The stage-specific in vitro efficacy of the VAMAX cocktail strongly correlates with the corresponding antigen-specific titers, which for their part depend on the antigen dose, but there is no indication that the dose has an effect on the in vitro efficacy of the induced antibodies. A comparison of these results with those obtained in the previous hyper-immunization study (where higher levels of antigen-specific IgG were observed) suggests that there is a significant need to induce an immune response matching efficacy requirements, especially for a PfAMA1-based blood stage vaccine, by using higher doses, better adjuvants and/or better formulations.

SI-traceable calibration-free analysis for the active concentration of G2-EPSPS protein using surface plasmon resonance.

Active proteins play important roles in the function regulation of human bodies and attract much interest for use in pharmaceuticals and clinical diagnostics. However, the lack of primary methods to analyze active proteins means there is currently no metrology standard for active protein measurement. In recent years, calibration-free concentration analysis (CFCA), which is based on surface plasmon resonance (SPR) technology, has been proposed to determine the active concentration of proteins that have specific binding activity with a binding partner without any higher order standards. The CFCA experiment observes the changes of binding rates at totally different two flow rates and uses the known diffusion coefficient of an analyte to calculate the active concentration of proteins, theoretically required, the binding process have to be under diffusion-limited conditions. Measuring the active concentration of G2-EPSPS protein by CFCA was proposed in this study. This method involves optimization of the regeneration buffer and preparation of chip surfaces for appropriate reaction conditions by immobilizing ligands (G2-EPSPS antibodies) on sensor chips (CM5) via amine coupling. The active concentration of G2-EPSPS was then determined by injection of G2-EPSPS protein samples and running buffer over immobilized and reference chip surfaces at two different flow rates (5 and 100µLmin-1). The active concentration of G2-EPSPS was obtained after analyzing these sensorgrams with the 1:1 model. Using the determined active concentration of G2-EPSPS, the association, dissociation, and equilibrium constants of G2-EPSPS and its antibody were determined to be 2.18 ± 0.03 × 106M-1s-1, 5.79 ± 0.06 ×10-3s-1, and 2.65 ± 0.06 × 10-9M, respectively. The performance of the proposed method was evaluated. The within-day precisions were from 3.26% to 4.59%, and the between-day precision was 8.36%. The recovery rate of the method was from 97.46% to 104.34% in the concentration range of 1.5-8nM. The appropriate concentration range of G2-EPSPS in the proposed method was determined to be 1.5-8nM. The active G2-EPSPS protein concentration determined by our method was only 17.82% of that obtained by isotope dilution mass spectrometry, showing the active protein was only a small part of the total G2-EPSPS protein. The measurement principle of the proposed method can be clearly described by equations and the measurement result can be expressed in SI units. Therefore, the proposed method shows promise to become a primary method for active protein concentration measurement, which can benefit the development of certified reference materials for active proteins.

Surrogate potency assays: Comparison of binding profiles complements dose response curves for unambiguous assessment of relative potencies.

Surface plasmon resonance (SPR) systems are widely used for detailed characterization of antibody activities including antigen and Fc-receptor binding. During the later stages of development, where the focus is to ensure that established critical quality attributes (CQAs) are maintained during cell culture, purification and formulation processes, analysis is simplified, and relative potencies are often determined. Here, simulation of binding data revealed that relative potency values, determined via parallel line analysis (PLA) and half maximal effective concentration (EC50) analysis accurately reflect changes in active concentration only if binding kinetics remain unchanged. Changes in the association rate constant shifted dose response curves, and therefore relative potencies, in the same way as changes in analyte concentration do. However, for interactions characterized by stable binding, changes in the dissociation rate constant did not result in any shift, suggesting that this type of change may go unnoticed in the dose response curve. Thus, EC50 and PLA analyses of dose response curves obtained with an anti-TNF-α antibody were complemented with the Biacore functionality for sensorgram comparison analysis, whereby changes in antigen and Fc-receptor binding profiles could be detected. Next, analysis of temperature stressed TNF-α antibody revealed that calibration free concentration analysis (CFCA) data correlated perfectly with relative potency values. Together, these results demonstrate that combinations of SPR based dose response curves, sensorgram comparison and CFCA can be used to strengthen the confidence in relative potency assessments, and suggest that SPR can potentially be used as a surrogate potency assay in the quality control of biotherapeutic medicines.

Biosensor binding data and its applicability to the determination of active concentration.

Protein concentration data are required for understanding protein interactions and are a prerequisite for the determination of affinity and kinetic properties. It is vital for the judgment of protein quality and for monitoring the effect of therapeutic agents. Protein concentration values are typically obtained by comparison to a standard and derived from a standard curve. The use of a protein standard is convenient, but may not give reliable results if samples and standards behave differently. In other cases, a standard preparation may not be available and has to be established and validated. Using surface plasmon resonance (SPR) biosensors, an alternative concentration method is possible. This method is called calibration-free concentration analysis (CFCA); it generates active concentration data directly and without the use of a standard. The active concentration of a protein is defined through its interaction with its binding partner. This concentration can differ from the total protein concentration if some protein fraction is incapable of binding. If a protein has several different binding sites, active concentration data can be established for each binding site using site-specific interaction partners. This review will focus on CFCA analysis. It will reiterate the theory of CFCA and describe how CFCA has been applied in different research segments. The major part of the review will, however, try to set expectations on CFCA and discuss how CFCA can be further developed for absolute and relative concentration measurements.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.