The geochemistry of two hard water rivers,the Rhine and the Rhone,Part 4: The determination of the solubility product of 10750oxy-apatite

The ionic product of 10750oxy-apatite has been calculated from data sets from the hard water rivers Rhine and Rhone. An overall value of 10^–50 has been obtained, but this value has an uncertainty due to the uncertainty of the third ionisation constant of phosphoric acid. Implicitly it has been shown that these rivers are saturated with respect to 10750oxy-apatite and that thus calcium controls the solubility of o-phosphate.     

Insight into the process of product expulsion in cellobiohydrolase Cel6A from Trichoderma reesei by computational modeling

Glycoside hydrolase cellulase family 6 from Trichoderma reesei (TrCel6A) is an important cellobiohydrolase to hydrolyze cellooligosaccharide into cellobiose. The knowledge of enzymatic mechanisms is critical for improving the conversion efficiency of cellulose into ethanol or other chemicals. However, the process of product expulsion, a key component of enzymatic depolymerization, from TrCel6A has not yet been described in detail. Here, conventional molecular dynamics and steered molecular dynamics (SMD) were applied to study product expulsion from TrCel6A. Tyr103 may be a crucial residue in product expulsion given that it exhibits two different posthydrolytic conformations. In one conformation, Tyr103 rotates to open the ?3 subsite. However, Tyr103 does not rotate in the other conformation. Three different routes for product expulsion were proposed on the basis of the two different conformations. The total energy barriers of the three routes were calculated through SMD simulations. The total energy barrier of product expulsion through Route 1, in which Tyr103 does not rotate, was 22.2 kcal·mol^?1. The total energy barriers of product expulsion through Routes 2 and 3, in which Tyr103 rotates to open the ?3 subsite, were 10.3 and 14.4 kcal·mol^?1, respectively. Therefore, Routes 2 and 3 have lower energy barriers than Route 1, and Route 2 is the thermodynamically optimal route for product expulsion. Consequently, the rotation of Tyr103 may be crucial for product release from TrCel6A. Results of this work have potential applications in cellulase engineering.     

Product quality of a recombinant fusion protein expressed in immobilised baby hamster kidney cells grown in protein-free medium

Baby hamster kidney cells producing a recombinant IgG-IL2 fusion protein were grown as spinner batch cultures in a protein-free medium, with cells immobilised in porous and non-porous supports at different support concentrations and agitation rates. Product quality, i.e., integrity, decreases up to 60% with the increase in agitation rate or support concentration and from porous to non-porous supports. LDH activity is a good indicator for the monitoring of product degradation, and thus product quality. Optimal conditions regarding titre of intact product were achieved at 60 rpm with 2 g porous support l^–1.     

Crystallization and liquid‐liquid phase separation of monoclonal antibodies and fc‐fusion proteins: Screening results

Crystallization holds the potential to be used for protein purification and low‐viscosity drug substance and drug product formulations. Twenty‐two different proteins (20 monoclonal antibodies and two Fc‐fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG₂s examined, at least four formed diffraction‐quality crystals and four others formed crystal‐like particles. At least three of the IgG₂s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high‐yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid‐liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc‐fusions, for example, was concentrated by LLPS to a self‐buffering solution at 150 g/L. Crystallization and LLPS in the salting‐in region were shown to be feasible. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Physicochemical characterization of Remsima®

Remsima® (infliximab) was recently approved as the world's first biosimilar monoclonal antibody (mAb) in both the European Union and Korea. To achieve this, extensive physicochemical characterization of Remsima® in relation to Remicade® was conducted in order to demonstrate the highly similar properties between the two molecules. A multitude of state-of-the-art analyses revealed that Remsima® has identical primary as well as indistinguishable higher order structures compared with the original product. Monomer and aggregate contents of Remsima® were also found to be comparable with those of Remicade®. In terms of charge isoforms, although Remsima® was observed to contain slightly less basic variants than the original antibody, the difference was shown to be largely due to the presence of C-terminal lysine. On the other hand, this lysine was found to be rapidly clipped inside serum in vitro and in vivo, suggesting it has no effect on the biological potency or safety of the drug. Analysis of the glycan contents of the antibodies showed comparable glycan types and distributions. Recent results of clinical studies have further confirmed that the two antibody products are highly similar to each other. Based on this research as well as previous clinical and non-clinical comparability studies, Remsima® can be considered as a highly similar molecule to Remicade® in terms of physicochemical properties, efficacy, and safety for its final approval as a biosimilar product to Remicade®.     

Review: In vivo and postmortem effects of feed antioxidants in livestock: a review of the implications on authorization of antioxidant feed additives

The pivotal roles of regulatory jurisdictions in the feed additive sector cannot be over-emphasized. In the European Union (EU), antioxidant substances are authorized as feed additives for prolonging the shelf life of feedstuffs based on their effect for preventing lipid peroxidation. However, the efficacy of antioxidants transcends their functional use as technological additives in animal feeds. Promising research results have revealed the in vivo efficacy of dietary antioxidants for combating oxidative stress in production animals. The in vivo effect of antioxidants is significant for enhancing animal health and welfare. Similarly, postmortem effect of dietary antioxidants has been demonstrated to improve the nutritional, organoleptic and shelf-life qualities of animal products. In practice, dietary antioxidants have been traditionally used by farmers for these benefits in livestock production. However, some antioxidants particularly when supplemented in excess could act as prooxidants and exert detrimental effects on animal well-being and product quality. Presently, there is no exclusive legislation in the EU to justify the authorization of antioxidant products for these in vivo and postmortem efficacy claims. To indicate these efficacy claims and appropriate dosage on product labels, it is important to broaden the authorization status of antioxidants through the appraisal of existing EU legislations on feed additives. Such regulatory review will have major impact on the legislative categorization of antioxidants and the efficacy assessment in the technical dossier application. The present review harnesses the scientific investigations of these efficacy claims in production animals and, proposes potential categorization and appraisal of in vivo methodologies for efficacy assessment of antioxidants. This review further elucidates the implication of such regulatory review on the practical application of antioxidants as feed additives in livestock production. Effecting these regulatory changes will stimulate the innovation of more potent antioxidant products and create potential new markets that will have profound economic impacts on the feed additive industry. Based on the in vivo efficacy claims, antioxidants may have to contend with the legislative controversy of either to be considered as veterinary drugs or feed additives. In this scenario, antioxidants are not intended to diagnose or cure diseases as ascribed to veterinary products. This twisted distinction can be logically debated with reference to the stipulated status of feed additives in Commission Regulation (EC) No 1831/2003. Nonetheless, it is imperative for relevant stakeholders in the feed additive industry to lobby for the review of existing EU legislations for authorization of antioxidants for these efficacy claims.