Single chain Fab (scFab) fragment

BACKGROUND: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. RESULTS: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. CONCLUSION: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection

Numerical Investigations of Mixed Convection of Incompressible Viscous Fluid in LNG Storage with a Various Locations of Input and Output Mass

The article shows the results of mathematical simulation of mixed convection in the low-temperature storage of liquefied natural gas with a regenerative cooling. The regimes of mixed convection in a closed area with the different arrangement of the input and output sections of the masses are investigated. Two-dimensional nonstationary problem in the model of the Navier-Stokes in dimensionless variables "vorticity - stream function - temperature" was examined. Are obtained distributions of the hydrodynamic parameters and temperatures, characteristic basic laws governing the processes being investigated. Detailed circulating currents and carried out analysis of the mechanism of vortices formation and the temperature distribution in the solution for mixed convection mode with low Reynolds and Grashof numbers (Gr=10{6}, 100<Re<1000). Is established the significant influence of the geometrical arrangement of the input and output mass sections and input stream velocity on the structure of liquid flow and temperature in the low temperature LNG storage tanks

Tools and methods for providing assurance of clonality for legacy cell lines

Over the last several years demonstration of cell line clonality has been a topic of many industry and regulatory presentations and papers. Guidance has been provided by the regulatory authorities, especially the FDA, on a path forward for providing evidence of clonality with high probability. It has been recommended that two-rounds of limiting dilution cloning (LDC) at sufficiently low seeding densities (≤0.5 cells/well) provides sufficient evidence that a cell line is clonal. Furthermore, one-round of LDC may also suffice if supplemental data from a characterized FACS or plate-imaging workflow are also included in the package. Cell lines generated by methods that do not demonstrate high probability of clonal derivation, including legacy cell lines, may require additional studies to provide assurance and/or process control strategies to satisfy regulatory expectations. Within the Biologics function of the IQ Consortium the “Clonality” Working Group is focusing on methods and tools which could be utilized to provide a high assurance of clonality for legacy cell lines. The presentation will outline a three tier approach to address legacy cell line clonality assurance: standard practices already used in industry to support limit of in vitro cell age studies, enhanced control strategies to ensure process consistency, and emerging technologies that could be used to further support cell line clonality

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target

Deletion of a telomeric region on chromosome 8 leads to higher productivity and stability of CHO cell lines

Chinese Hamster Ovary (CHO) cells are widely used for large scale production of recombinant biopharmaceuticals. Although these cells have been extensively used, a demand to further increase the performance i.e. to facilitate the process of clone selection to isolate the highest producing cell lines that maintain stability of production over time is still existing. We compared gene expression profiles of high versus low producing CHO clones to identify regulated genes which can be used as biomarkers during clone selection or for cell line engineering. We present evidence that increased production rates and cell line stability are correlated with the loss of the telomeric region of the chromosome 8. A new parental CHO cell line lacking this region was generated and its capability for protein production was assessed. The average volumetric productivity of cells after gene transfer and selection was found to be several fold improved, facilitating the supply of early drug substance material for e.g. quality determination. In addition, significantly more cell clones with a higher average productivity and higher protein production stability were obtained with the new host cell line after single cell cloning. This allows reduced efforts in single cell sorting, screening of fewer clones and raises the opportunity to circumvent time and labor-intensive stability studies