ABSTRACT
The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Collagen/genetics , Endotoxins/antagonists & inhibitors , Etanercept/pharmacology , Receptors, Tumor Necrosis Factor, Type II/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Cell Survival/drug effects , Collagen/metabolism , Endotoxins/metabolism , Endotoxins/toxicity , Etanercept/chemistry , Etanercept/metabolism , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protein Engineering/methods , Protein Multimerization , Receptors, Tumor Necrosis Factor, Type II/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/toxicityABSTRACT
UV spectrophotometric measurement is a widely accepted and standardized routine analysis for quantitation of highly purified proteins; however, the reliability of the results strictly depends on the accuracy of the employed extinction coefficients. In this work, an experimental estimation of the differential refractive index (dn/dc), based on dry weight measurements, was performed in order to determine accurate extinction coefficients for four biotherapeutic proteins and one synthetic copolymer after separation in a size-exclusion ultra-performance liquid chromatograph coupled to an ultraviolet, multiangle light scattering and refractive index (SE-UPLC-UV-MALS-RI) multidetection system. The results showed small deviations with respect to theoretical values, calculated from the specific amino acid sequences, for all the studied immunoglobulins. Nevertheless, for proteins like etanercept and glatiramer acetate, several considerations, such as glycan content, partial specific volume, polarizability, and higher order structure, should be considered to properly calculate theoretical extinction coefficient values. Herein, these values were assessed with simple approximations. The precision of the experimentally obtained extinction coefficients, and its convergence towards the theoretical values, makes them useful for characterization and comparability exercises. Also, these values provide insight into the absorbance and scattering properties of the evaluated proteins. Overall, this methodology is capable of providing accurate extinction coefficients useful for development studies.