Brian Christopher Bales

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant “flu binders” for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Estimating amounts of iron oxide from gradient echo images

Rat legs directly injected with superparamagnetic iron oxide (SPIO) were studied by dual‐echo, gradient‐echo imaging. The amount of iron injected was estimated using a point dipole model for the SPIO injection site. Saturation magnetization of 6:1 PEG/amino modified silane‐coated iron oxide particles with 5‐ to 6‐nm core and 20–25 hydrodynamic diameter was ∼110 emu/g of iron. Estimates of the amount of iron injected made from signal void volumes surrounding SPIO centers yielded erroneous results varying with sample orientation in the scanner and echo time (TE). For example, a 10 μL, 3‐μg iron injection produced signal void volumes of 80 and 210 μL at TE of 9.8 and 25 ms, respectively, giving apparent iron contents of 6 ± 1 and 10 ± 2 μg respectively. A more effective approach uses the phase difference between two gradient recalled echo images. To estimate iron content, this approach fits the expected (3 cos2θ − 1)/|r|3 spatial phase distribution to the observed phase differences. Extraneous phase effects made fitting phase at a single TE ineffective. With the dual echo method, 18 independent estimates were 2.48 ± 0.26 μg std, independently of sample orientation. Estimates in empty control regions were −90 and −140 ng. A 1‐μg injection indicated 0.5, 1.2, and 1.2 μg. Magn Reson Med, 2009.

Performance of Redox Active and Chelatable Iron Assays to Determine Labile Iron Release From Intravenous Iron Formulations

Emerging data from global markets outside the United States, where many generic iron sucrose formulations are available, have revealed that non‐US generic intravenous (i.v.) iron formulations may have iron release profiles that differ from the reference listed drug (RLD). The first generic i.v. iron approved in the United States was sodium ferric gluconate complex in 2011. We evaluated chelatable and redox labile iron assay methods to measure the amount of labile iron released from i.v. iron formulations in biorelevant matrices in vitro. The majority of published labile iron assays evaluated were not suitable for use in vitro due to overwhelming interference by the presence of the i.v. iron products. However, an optimized high‐performance liquid chromatography (HPLC)‐based method performed well for use in vitro labile iron detection in a biorelevant matrix. Application of this method may enhance bioequivalence evaluation of generic i.v. iron formulations in the future.

In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations

ABSTRACT Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre‐clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration‐time profiles (t0–240min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2=0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model. HIGHLIGHTSIntravenous iron formulations are complex drugs that are colloidal suspensions of iron oxide nanoparticles.Small formulation changes that are not discerned by physicochemical characterization may allow for more labile iron release.These characteristics of IV iron formulations make bioequivalence evaluation challenging.An in vitro to in vivo correlation model for labile iron release could improve bioequivalence evaluation.