Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

Broad epitope coverage of a human in vitro antibody library.

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

Understanding ForteBio's Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant.

Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both purified IgG and IgG supernatant. AHC sensors proved unreliable for kinetic or binning assays at times, whereas AHQ sensors showed poor loading and regeneration abilities. ProA sensors worked well with both purified IgG and IgG supernatant. However, the interaction between ProA sensors and the Fab region of the IgG with VH3 germline limited the application of ProA sensors, especially in the epitope binning experiment. In an attempt to generate a biosensor type that would be compatible with a variety of germlines and sample types, we found that the custom b-AHFc sensors appeared to be robust working with both purified IgG and IgG supernatant, with little evidence of sensor-related artifacts.

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies.

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy.

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.

High throughput detection of antibody self-interaction by bio-layer interferometry.

Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.

High throughput solution-based measurement of antibody-antigen affinity and epitope binning.

Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.

Effect of the Y955C mutation on mitochondrial DNA polymerase nucleotide incorporation efficiency and fidelity.

The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson's disease. In single-turnover kinetic studies, we show that the Y955C mutation results in a decrease in the maximal rate of polymerization and an increase in the K(m) for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 µM(-1) s(-1), respectively, representing reductions of 30-, 110-, and 1300-fold, respectively, relative to the value for the wild-type enzyme. The fidelity of incorporation was reduced 6-130-fold, largely because of the significant decrease in the specificity constant for correct dATP:T incorporation. For example, k(cat)/K(m) for forming a TTP:T mismatch was decreased 10-fold from 0.0002 to 0.00002 µM(-1) s(-1) by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to that of the wild type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, the level of incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role for Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of incorporation of dATP opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.