Monoclonal antibody production using the porous glass bead immobilization technique. Serum-free perfusion.
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Animals↗
Biomedical subjects
H Katinger
Publications and source records attributed to H Katinger.
Amino-acid sequence comparison of nepovirus coat proteins.
The amino acid sequence of the coat proteins of several nepoviruses was determined by a combination of peptide and nucleic acid sequencing (grapevine fanleaf virus, arabis mosaic virus, tomato blackring virus, grapevine chrome mosaic virus). These sequences were compared and showed homologies ranging from 10% to 69%, and 96.7% for the two arabis mosaic virus strains. 10% homology does not reflect any relationship between viruses, and our results implicate, that nepoviruses, considering the homology of the coat protein sequences of viruses as a parameter for virus taxonomy, may be divided into several subgroups.
Amino Acid Sequence↗
Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads.
Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.
Animals↗
Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry.
As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.
Animals↗
Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.
A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.
Animals↗
Stability of von Willebrand factor secretion in different human endothelial hybrid cell lines.
Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. The in vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.
Animals↗
Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.
The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.
Animals↗
Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.
Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.
Antigens, Polyomavirus Transforming↗
HIV-1 gp41 contains two sites for interaction with several proteins on the helper T-lymphoid cell line, H9.
OBJECTIVE: To characterize putative binding sites for HIV-1 gp41 to H9 cells. DESIGN: Based on accumulating evidence in the literature that HIV-1 can bind to cell surfaces independent of CD4, we attempted to clarify whether gp41, the transmembrane HIV-1 protein, contributes to CD4-independent binding. We therefore tested binding of gp41 to cells. METHODS: Using fluorescence-activated cell-sorter analysis, we examined the binding of recombinant gp160 (gp160) and soluble gp41 (sgp41; Env amino acids 539-684) to H9 cells, and located the putative binding site(s) of gp41 by inhibition using 16 HIV-1 Env peptides. Putative HIV-1 receptor proteins in H9 cell lysates were Western blotted and precipitated using sgp41. RESULTS: sgp41 bound to the CD4+ H9 cells and rgp160 bound to H9 cells independent of gp120-binding sites on CD4 molecules. Two gp41 peptides (Env amino acids 591-605 and 651-665) inhibited the binding of sgp41 to H9 cells. Four bands, of 37, 40, 55 and 97 kD, were blotted in H9 cell lysates, and three bands, 40, 97 and 108 kD, were observed in the precipitation analysis using lysates of 125I-surface-labelled H9 cells and sgp41 attached to sepharose beads. CONCLUSIONS: HIV-1 gp41 contains two putative binding sites to H9 cells. These sites may be located within Env amino acids 591-605 (ERYLKDQQLLGIWGC) and 651-665 (TLLEESQNQQEKNEQ). Using two different techniques, five proteins (37, 40, 55, 97 and 108 kD) were identified in H9 lysates as possible candidates for gp41 binding.
Amino Acid Sequence↗
Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain.
The inhibitory effect of Ukrain on malignant cells and on normal cells, in vitro, has been compared. To obtain a 50% inhibition of cell growth, a tenfold concentration had to be used with normal endothelial cells compared to a human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. A laser scanning microscope showed a high uptake of Ukrain in malignant cells, while the content in normal cells under the same experimental conditions was substantially lower.
Alkaloids↗
Cloning and expression of an HIV-1 specific single-chain Fv region fused to Escherichia coli alkaline phosphatase.
We have constructed a single-chain Fv fragment representing the variable domain of the human monoclonal antibody 3D6, binding specifically to HIV-1 gp41. This gene was fused to the coding region of E. coli alkaline phosphatase (EcPhoA) and expressed in E. coli. The EcPhoA signal peptide was used to direct the recombinant fusion protein to the periplasmic space of the bacteria, from where it was purified by hydrophobic interaction chromatography and gel filtration followed by antigen-affinity chromatography using a synthetic HIV-1 peptide as ligand. The purified fusion protein was bifunctional, showing both phosphatase activity as well as antigen-binding specificity identical to that of the original antibody.
Alkaline Phosphatase↗
Expression of a human monoclonal anti-HIV-1 antibody in CHO cells.
The cDNA coding for the light and heavy chains, respectively, of the human monoclonal antibody 3D6 (IgG1, kappa), which binds specifically to human immunodeficiency virus-1 (HIV-1) gp41, was inserted into three different mammalian expression vectors and transfected into Chinese hamster ovary (CHO) cells. Transcription was under the control of Rous sarcoma virus long terminal repeat (RSV LTR), human cytomegalovirus major immediate early (CMV IE) promoter, and mouse mammary tumor virus long terminal repeat (MMTV LTR), respectively. Antibody productivity was monitored in the supernatants of selected clones. The binding characteristics of the CHO-derived antibody to HIV-1 gp41 were found to be identical to that of the original antibody produced by hybridoma cells.
Animals↗
Industrial animal cell reactor systems: aspects of selection and evaluation.
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Animals↗
Modular integrated fluidized bed bioreactor technology.
We describe the design and demonstrate the application of a modular integrated fluidized bed bioreactor system. Basically the system is a reactor vessel equipped with an extending cylinder and a liquid distributor plate. Instead of an external recirculation loop, as used in existing fluidized bed systems, a low shear stress impeller is used as the recirculation pump. The system has several unique features, such as modular exchangeable elements, efficient oxygenation and the option of operating as a stirred tank-, a packed bed- or a fluidized bed reactor. An example of a fluidized bed run using CHO-K1 cells is shown. Under standard culture conditions a 100-fold increase in cell density (up to 1.2 x 10(8) cells/ml) was achieved.
Animals↗
Engineered gene for Escherichia coli alkaline phosphatase for the construction of translational fusions.
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Alkaline Phosphatase↗
Isoelectric focusing in immobilized pH gradients of phosphoglucomutase and esterases from the spiny lobster.
A method is described for detecting polymorphisms of cephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.
Alleles↗
Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients.
Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.
Animals↗
A continuous multistage roller reactor for animal cell culture: 1. Patterns of growth, production and catabolism of a murine hybridoma.
A Tubular Liquid Film Reactor was designed as a model system to transfer a batch culture kinetic to a continuous cascade. Cell density, product formation and substrate consumption rates were followed during fermentation at two dilution rates. In spite of the high dilution rates effective in each segment by itself high cell densities of up to 10(7) cells/ml were achieved due to cell sedimentation. The model character of the reactor was taken to determine critical values of substrate concentrations that influence production rates and result in an adaptation of metabolism.
Alanine↗