Analytical similarity assessment of rituximab biosimilar CT-P10 to reference medicinal product

CT-P10 (Truxima?) was recently approved as the world's first rituximab biosimilar product in the European Union (EU) and South Korea. To demonstrate biosimilarity of CT-P10 with the reference medicinal product (RMP), extensive 3-way similarity assessment has been conducted between CT-P10, EU-Rituximab and US-Rituximab, focusing on the physicochemical and biological quality attributes. A multitude of state-of-the-art analyses revealed that CT-P10 has identical primary and higher order structures compared to the original product. Purity/impurity profiles of CT-P10 measured by the levels of aggregates, fragments, non-glycosylated form and process-related impurities were also found to be comparable with those of RMPs. In terms of the post-translational modification, CT-P10 contains slightly less N-terminal pyro-glutamate variant, which has been known not to affect product efficacy or safety. Oligosaccharide profiling has revealed that, although CT-P10 contains the same conserved glycan species and relative proportion with the RMPs, the content of total afucosylated glycan in CT-P10 was slightly higher than in EU- or US-Rituximab. Nevertheless, the effect of the observed level of afucosylation in CT-P10 drug product on Fc receptor binding affinity or antibody-dependent cell-mediated cytotoxicity was found to be negligible based on the spiking study with highly afucosylated sample. Arrays of biological assays representative of known and putative mechanisms of action for rituximab have shown that biological activities of CT-P10 are within the quality range of RMPs. Recent results of clinical studies have further confirmed that the CT-P10 exhibits equivalent clinical efficacy and safety profiles compared to EU- and US-Rituximab. The current 3-way similarity assessment together with clinical study results confidently demonstrate that CT-P10 is highly similar with EU- and US-Rituximab in terms of physicochemical properties, biological activities, efficacy, and safety for its final approval as a biosimilar product.     

Insights into phytase-containing transgenic <Emphasis Type="Italic">Lemna minor</Emphasis> (L.) as a novel feed additive

This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n?=?75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P–Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

<Emphasis Type="Italic">Luteimonas aestuarii</Emphasis> sp. nov., isolated from tidal flat sediment

A novel bacterium B9^T was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9^T is associated with the genus Lysobacter (≤ 97.2%), Xanthomonas (≤ 96.8%), Pseudomonas (≤ 96.7%), and Luteimonas (≤ 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255^T (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9^T and Luteimonas mephitis B1953/27.1^T. The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C_11:0, iso-C_15:0, iso-C_16:0, iso-C_17:0, iso-C_17:0 ω9c, and iso-C_11:0 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9^T was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9^T (= KCTC 22048^T, DSM 19680^T).     

Purification and characterization of a cold-active lipase from Pichia lynferdii Y-7723: pH-dependant activity deviation

Lipases with abnormal functionalities such as high thermostability and optimal activity at extreme conditions gain special attentions because of their applicability in the restricted reaction conditions. In particular, coldactive lipases have gained special attentions in various industrial fields such as washer detergent, pharmaceutical catalyst, and production of structured lipid. However, production of cold-active lipase is mostly found from psychrophilic microorganisms. Recently we found a novel cold-active lipase from Pichia lynferdii Y-7723 which is mesophilic yeast strain. In this study, we purified the cold active lipase and the enzyme was further characterized in several parameters. The enzyme was purified with 33 purification fold using chromatographic techniques and the purified lipase represented maximum lipolytic activity at 15°C and the maximum activity was highly dependent on pH.     

Functional analysis of the invariant residue G791 of Escherichia coli 16S rRNA

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.     

Modulation of TNFSF expression in lymphoid tissue inducer cells by dendritic cells activated with Toll-like receptor ligands

Toll-like receptors (TLRs), which recognize structurally conserved components among pathogens, are mainly expressed by antigen-presenting cells such as dendritic cells (DCs), B cells, and macrophages. Recognition through TLRs triggers innate immune responses and influences antigen-specific adaptive immune responses. Although studies on the expression and functions of TLRs in antigen-presenting cells have been extensively reported, studies in lymphoid tissue inducer (LTi) cells have been limited. In this study, we observed that LTi cells expressed TLR2 and TLR4 mRNA as well as TLR2 protein and upregulated OX40L, CD30L, and TRANCE expression after stimulation with the TLR2 ligand zymosan or TLR4 ligand LPS. The expression of tumor necrosis factor superfamily (TNFSF) members was significantly upregulated when cells were cocultured with DCs, suggesting that upregulated TNFSF expression may contribute to antigen-specific adaptive immune responses.     

Detection of insulin-antibody binding on a solid surface using imaging ellipsometry

Imaging ellipsometry (IE) was used to detect the binding of insulin to its antibody on a solid surface. The modification of a gold surface with 11-mecaptoundecanoic acid (11-MUA), the adsorption of protein G, and antibody immobilization onto the protein G layer were confirmed by surface plasmon resonance. Ellipsometric images and ellipsometric angles of the surface antibody were acquired using the IE system by off-null ellipsometry. Ellipsometric images of antigen binding to the antibody were acquired, and their mean optical intensities estimated. Changes in mean optical intensity indicated that the detection range for insulin was from 10 ng/ml to 100 microg/ml.