Method development for the clearance study of the Pluronic F-68 nonionic surfactant used in the upstream process of monoclonal antibody production.

Pluronic F-68 is a nonionic surfactant, which is often used in the upstream process of biopharmaceutical production. However, the number of analytical methods developed for determination of Pluronic F-68 in the in-process and drug substance samples of biological drug production process is quite low. The lack of chromophore groups on the molecule and the interference caused by the high protein content of the samples hamper analysis. In this paper the development and qualification of a mixed-mode (MM) HPLC method with charged aerosol detection is reported. The method enables the analysis of samples with up to 85 g/L protein concentration. The range of the method was set to 250-500 µg/mL, where it was found to be accurate (89-111 % recovery) and precise (0.8-3.2 % relative standard deviation). The high sensitivity of the method indicates that even lower concentration range can be feasible. The novel method successfully demonstrates Pluronic F-68 clearance during the downstream process of the monoclonal antibody production.

Toxicity mitigation by N-acetylcysteine and synergistic toxic effect of nano and bulk ZnO to Panagrellus redivivus.

To better understand the nanosize-relevant toxic effects and underlying mechanisms, N-acetylcysteine (NAC), as a mitigation agent, an ionic form of Zn (ZnCl2), and the binary mixture of ZnO with different particle sizes (15 nm and 140 nm), was used in toxicity assays with the nematode Panagrellus redivivus. The ZnCl2 concentrations were applied to show the amount of dissolved Zn ions present in the test system. Reactive oxygen species (ROS) measuring method was developed to fit the used test system. Our studies have shown that NAC can mitigate the toxic effects of both studied particle sizes. In the applied concentrations, ZnCl2 was less toxic than both of the ZnO particles. This finding indicates that not only ions and ROS produced by the dissolution are behind the toxic effects of the ZnO NPs, but also other particle size-dependent toxic effects, like the spontaneous ROS generation, are also relevant. When the two materials were applied in binary mixtures, the toxic effects increased significantly, and the dissolved zinc content and the ROS generation also increased. It is assumed that the chemical and physical properties of the materials have been mutually reinforcing to form a more reactive mixture that is more toxic to the P. redivivus test organism. Our findings demonstrate the importance of using mitigation agent and mixtures to evaluate the size-dependent toxicity of the ZnO.

Toxicity and uptake of nanoparticulate and bulk ZnO in nematodes with different life strategies.

Despite the increasing number and quantity of nanomaterials released in the environment, our knowledge on their bioavailability and possible toxicity to organisms is rather limited. Thus, we know quite little about sensitivity of various nematode feeding types and life strategies to treatments with nano metal oxides. The toxicity of zinc oxide nanoparticles (nano-ZnO) (with a particle size of 25 nm) and the bulk counterpart was investigated in two free-living nematode species of different life strategies: Xiphinema vuittenezi, a K-strategist plant-feeder nematode and Panagrellus redivivus, an r-strategist bacterivor nematode. The internal zinc concentration and the concentration of minor and trace elements were determined by total reflection X-ray fluorescence spectrometry. Concentration-dependent mortality in both nematode species was observed following a 24-h exposure both to nano-ZnO and bulk ZnO. The zinc concentration of the treating suspension had a significant effect on the internal zinc content of the animals in both cases. Particle size did not influence the internal zinc content. Our results show that nano and bulk ZnO have a similar dose-response effect on mortality of the bacterivor P. redivivus. In contrast, the nano-ZnO has stronger toxic effect on the mortality of X. vuittenezi. In general, X. vuittenezi did not react more sensitively to the treatments than P. redivivus, but appeared sensitive to the nano-ZnO treatment compared to bulk ZnO.

The role of stabilization centers in protein thermal stability.

The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins.

Uptake and toxicity of nano-ZnO in the plant-feeding nematode, Xiphinema vuittenezi: the role of dissolved zinc and nanoparticle-specific effects.

Nanoparticulate ZnO is one of the most commonly applied nanomaterials. As ZnO is more soluble than many other oxide nanoparticles, its toxicity beyond the nanoparticle-specific effects can be attributed to the dissolved ionic zinc. The investigation of uptake and toxicity of nano-ZnO in the plant-feeding nematode, Xiphinema vuittenezi, which was used in previous studies as a biological model organism, was aimed. The establishment of the role of dissolved zinc and nanoparticle-specific effects in the toxicity was also the objective of our study. Zn uptake was found to be significantly higher for bulk and nano-ZnO than for ZnSO4 solution; however, treatments caused loss of potassium in the worms in a dissolved-zinc-dependent manner. The toxicity was the lowest for bulk ZnO, and it was very similar for nano-ZnO and ZnSO4 solution. Accordingly, the toxicity of ZnO nanoparticles is a combination of dissolved-zinc-caused toxicity and nanoparticle-specific effects.