(2014) High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy, mAbs, 6:2, 483-492,

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 (ka) rate constant measurements suggest that this is mainly caused by inaccurate ka 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.

(2015) An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies, mAbs, 7:3, 553-561, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: hydrophobic interaction chromatography, HIC, self-interaction nanoparticle spectroscopy, aggregation, developability, manufacturability, self-association, high-throughput screening, solubility, viscosityThe 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 affinitycapture 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.

(2013) High throughput detection of antibody self-interaction by bio-layer interferometry, mAbs, 5:6, 838-841,

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 resulted in a decrease in the maximum rate of polymerization and an increase in the Km 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−1s−1, respectively, representing reductions of 30-, 110- and 1300-fold relative to wild-type enzyme. The fidelity of incorporation was reduced 6- to 130-fold; largely due the significant decrease in the specificity constant for correct dATP:T incorporation. For example, kcat/Km for forming a TTP:T mismatch was decreased tenfold from 0.0002 to 0.00002 μM−1s−1 by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to wild-type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role of 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 dATP incorporation opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.