How to stop disproportionation of a hydrochloride salt of a very weakly basic compound in a non-clinical suspension formulation.

Our objectives were to stabilize a non-clinical suspension for use in toxicological studies and to develop methods to investigate the stability of the formulation in terms of salt disproportionation. The compound under research was a hydrochloride salt of a practically insoluble discovery compound ODM-203. The first of the three formulation approaches was a suspension prepared and stored at room temperature. The second formulation was stabilized by pH adjustment. In the third approach cooling was used to prevent salt disproportionation. 5 mg/mL aqueous suspension consisting of 20 mg/mL PVP/VA and 5 mg/mL Tween 80 was prepared for each of the approaches. The polymer was used as precipitation inhibitor to provide prolonged supersaturation while Tween 80 was used to enhance dissolution and homogeneity of the suspension. The consequences of salt disproportionation were studied by a small-scale in vitro dissolution method and by an in vivo pharmacokinetic study in rats. Our results show that disproportionation was successfully suppressed by applying cooling of the suspension in an ice bath at 2-8 °C. This procedure enabled us to proceed to the toxicological studies in rats. The in vivo study results obtained for the practically insoluble compound showed adequate exposures with acceptable variation at each dose level.

Improved surface stability and biotin binding properties of streptavidin coating on polystyrene.

The ultimate nature of streptavidin to bind biotin tightly is widely utilized in many solid-phase based applications to provide a universal binding surface for biotinylated molecules. However, the preparation of the streptavidin coatings by passive adsorption may heavily alter the binding properties of native streptavidin and may not result in the best possible capture surface for demanding solid-phase assays. By introducing sulphydryl groups through primary amines in the protein, we have activated and conjugated native streptavidin into larger protein polymers resulting in high local binding density when coated on polystyrene. This thiolated streptavidin formed through chemical modification has improved adsorption properties and biotin binding capability, compared to the native streptavidin. When this thiolated streptavidin is coated on polystyrene, a dense surface is formed, which provides up to 3-fold increase in the biotin binding efficiency and improves the surface stability by minimizing the desorption of the adsorbed protein from the surface during incubation. Furthermore, this high-capacity surface is resistant to harsh chemical treatments, such as denaturing conditions or mild reducing conditions. The improved adsorption properties of the thiolated streptavidin allow the coating process to be performed with shorter incubation times (15min), still providing enhanced solid-phase properties, compared to a reference streptavidin surface.

Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout.

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well's bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu-biotin binding densities of 0.080 and 0.47 pmol/mm(2), respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay.

Utilization of recombinant Fab fragments in a cTnI immunoassay conducted in spot wells.

OBJECTIVES: To evaluate the performance of a new cTnI immunoassay utilizing site-specifically biotinylated recombinant Fab fragments on recently established spot wells. DESIGN AND METHODS: Two different cTnI-specific recombinant site-specifically biotinylated Fab fragments were produced. The performance of the new sandwich-type cTnI immunoassay in spot wells was evaluated in terms of binding capacity, assay kinetics and assay sensitivity and compared with a cTnI immunoassay carried out in conventional microtitration wells. Furthermore, the functionality of the recombinant Fab fragments was compared to the corresponding monoclonal antibodies in assay with one, two or three capture antibodies. RESULTS: The signal-to-background level was improved, providing an analytical detection limit of 0.002 microg/l with a surface of two capture Fab fragments. The spot wells increased the signal levels 2-fold and a further 4-fold improvement was detected with the Fab fragments already after 5 min assay time. CONCLUSIONS: The spot-concept in combination with site-oriented capture Fab fragments carries great promise as a very useful approach to improve the immunoassay performance of future point-of-care cTnI assays.