PurposeFollowing two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial.MethodsWe undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches.ResultsSoluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein.ConclusionsWe propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.

It was aim of the study to investigate the in vivo potential of a novel insulin-thiomer complex nanoparticulate delivery system. Insulin loaded nanoparticles were obtained by the formation of hydrogen bonds between poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid)-cysteine (PAA-Cys) or poly(acrylic acid) (PAA), respectively, in the presence of insulin. Dissolution behavior of insulin from tablets as well as nanoparticulate suspensions was evaluated in vitro. Serum insulin concentrations and reduction of blood sugar values were determined after oral administration of nanoparticles formulated as enteric coated tablets and suspensions. Results displayed a low serum insulin concentration and pharmacological efficacy in terms of blood sugar reduction after oral administration of enteric coated tablets. On the contrary, nanoparticulate suspensions led to significant serum insulin concentrations. Furthermore a 2.3-fold improvement of the AUC of insulin could be achieved due to the use of thiolated PAA instead of unmodified PAA. In addition, a blood sugar reduction of 22% was observed. Results demonstrate that this novel complex nanoparticulate formulation is an encouraging new attempt toward the noninvasive delivery of peptide drugs.

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In this study the preparation and stabilization of poly(acrylic acid)-cysteine nanoparticles and incorporation of a fluorescence marked model-compound was investigated. Nanoparticles were prepared by ionic gelation of a poly(acrylic acid)-cysteine conjugate with calcium chloride. Poly(acrylic acid)-cysteine nanoparticles display high cohesive properties due to a cross-linking process via calcium bridges in the core and the pervasive formation of disulphide bonds and were 139 ± 34 nm in size. Nanoparticles were loaded with FITC-dextrans (flourescein isothiocyanate-dextrans) of 4, 20 and 40 kDa molecular mass as model-compound via sonication method or via vibration method for 3 and 24 h. In vitro release studies showed an initial burst release followed by an extended release of model-compounds. The lower the molecular mass of the FITC-dextrans, the higher was the amount of incorporated and released model compounds. Vibration seems to be a proper method for the incorporation of hydrophilic and macromolecular drugs in poly(acrylic acid)-cysteine nanoparticles.

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