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Genzel Y.,Max Planck Institute for Dynamics of Complex Technical Systems | Vogel T.,Max Planck Institute for Dynamics of Complex Technical Systems | Vogel T.,Esslingen University of Applied Sciences | Buck J.,Max Planck Institute for Dynamics of Complex Technical Systems | And 7 more authors.
Vaccine | Year: 2014

High cell densities in animal cell culture can be obtained by continuous perfusion of fresh culture medium across hollow fiber membranes that retain the cells. Careful selection of the membrane type and cut-off allows to control accumulation of target molecules and removal of low molecular weight compounds. In this report, perfusion with the scalable ATF (alternating tangential filtration, Refine Technology) system was evaluated for two suspension cell lines, the avian cell line AGE1.CR and the human cell line CAP. Both were cultivated in chemically defined media optimized for batch cell growth in a 1. L stirred tank bioreactor connected to the smallest ATF unit (ATF2) and infected with cell line-adapted human influenza A virus (A/PR/8/34 (H1N1), typical diameter: 80-100. nm).At concentrations of about 25 million cells/mL three different membrane cut-offs (50. kDa, 0.2. μm and 0.5. μm) were tested and compared to batch cultivations performed at 5 million cells/mL. For medium and large cut-offs no cell-density effect could be observed with cell-specific virus yields of 1428-1708 virions/AGE1.CR cell (infected with moi 0.001) and 1883-4086 virions/CAP cell (moi of 0.025) compared to 1292 virions/AGE1.CR cell and 3883 virions/CAP cell in batch cultures. Even at a concentration of 48 million AGE1.CR cells/mL (cut-off: 0.2. μm) a cell-specific yield of 1266 virions/cell was reached. Only for the small cut-off (50. kDa) used with AGE1.CR cells a decrease in cell-specific yield was measured with 518 virions/cell. Surprisingly, the ratio of infectious to total virions seemed to be increased in ATF compared to batch cultures. AGE1.CR cell-derived virus particles were present in the permeate (0.2 and 0.5. μm cut-off), whereas CAP cell-derived virions were not, suggesting possible differences in morphology, aggregation or membrane properties of the virions released by the two cell lines.To our knowledge, this is the first study that illustrates the potential of ATF-based perfusion of chemically defined media across cell-retaining membranes for production of an influenza A vaccine. © 2014 Elsevier Ltd.


Genzel Y.,Max Planck Institute for Dynamics of Complex Technical Systems | Behrendt I.,Max Planck Institute for Dynamics of Complex Technical Systems | Rodig J.,Max Planck Institute for Dynamics of Complex Technical Systems | Rapp E.,Max Planck Institute for Dynamics of Complex Technical Systems | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log10 HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log10 HA units/100 μL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production. © 2012 Springer-Verlag.


Fischer S.,Novartis | Fischer S.,Institute of Pharmaceutical Biotechnology | Charara N.,Novartis | Gerber A.,Novartis | And 4 more authors.
Biotechnology and Bioengineering | Year: 2012

The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult-to-express proteins. For this reason, we evaluated the more recently established novel CAP-T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP-T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. © 2012 Wiley Periodicals, Inc.


Patent
CEVEC Pharmaceuticals GmbH | Date: 2014-12-19

The present invention relates to a method for the production of a permanent human cell line, wherein isolated primary human cells are transfected simultaneously with a sequence allowing the expression of at least one cell transforming factor and a sequence allowing the expression of at least one recombinant polypeptide.


The present invention relates to recombinant glycoproteins having highly or fully sialylated O-linked GalNAc glycans (GalNAc O-glycans), preferably core 1 GalNAc O-glycans, as well as cell lines that are genetically modified to overexpress a -galactoside -2,3-sialyltransferase 1 (ST3Gal1), preferably human ST3Gal1, which can be used for the production of said recombinant glycoproteins. Further, the present invention relates to respective methods of expressing recombinant glycoproteins, methods of increasing the degree of sialylation of recombinant glycoproteins, and methods of decreasing the micro-heterogeneity of GalNAc O-glycans. Finally, the present invention relates to respective uses of the above cell lines for the production of recombinant glycoproteins, for increasing the degree of sialylation of recombinant glycoproteins, and for decreasing the micro-heterogeneity of O-linked GalNAc glycans of recombinant glycoproteins.


Patent
CEVEC Pharmaceuticals GmbH | Date: 2013-10-18

The present invention relates to a method for the production of human Cytomegalovirus (HCMV) particles, the method including the steps of: (a) contacting and thereby infecting a permanent human amniocyte cell with HCMV, (b) incubating the amniocyte cell, (c) allowing expression of HCMV particles, and (d) isolating of the HCMV particles, wherein the permanent human amniocyte cell expresses the adenoviral gene products E1A and E1B and wherein the amniocyte cells are cultured in serum free medium. Furthermore, the present invention relates to HCMV particles produced by the method of the present invention as well as to a HCMV based vaccine comprising the HCMV particles, the use of the HCMV particles for use in the preparation of a HCMV based vaccine and the HCMV particles for use in the preparation of a therapeutic or diagnostic agent for the prevention or treatment of a HCMV related disease.


Patent
CEVEC Pharmaceuticals GmbH | Date: 2014-04-23

The present invention relates to a method for the production of HCMV particles, the method including the steps of: (a) contacting and thereby infecting a permanent human amniocyte cell with HCMV, (b) incubating the amniocyte cell, (c) allowing expression of HCMV particles, and (d) isolating of the HCMV particles, wherein the permanent human amniocyte cell expresses the adenoviral gene products E1A and E1B. Furthermore, the present invention relates HCMV particles produced by the method of the present invention as well as to a HCMV based vaccine comprising the HCMV particles, the use of the HCMV particles for use in the preparation of a HCMV based vaccine and the HCMV particles for use in the preparation of a therapeutic or diagnostic agent for the prevention or treatment of a HCMV related disease.


Trademark
CEVEC Pharmaceuticals GmbH | Date: 2011-10-11

Chemical, biological and biochemical additives, preparations and reagents for use in the manufacture of chemicals, biologicals and biochemicals, namely, preparations and reagents for non medical use, in particular for scientific, research, laboratory, industrial and commercial purposes; chemical, biological and biochemical additives, preparations and reagents for research and characterisation of diseases for non-medical purposes; chemical, biological and biochemical additives, preparations and reagents, namely, cell lines, cells, proteins and polypeptides, for scientific and medical research and development of pharmaceuticals; chemical, biological and biochemical additives, preparations and reagents for use in the manufacture of medicines and pharmaceuticals; cultures of microorganisms other than for medical and veterinary use; cell lines, cells and amino acids for laboratory or research use; nucleic acid sequences, proteins and polypaptides for other than medical and veterinary use; diagnostic reagents and preparations comprising proteins, polypeptides, nucleotides, nucleic acids, cell lines and cells for scientific, research, laboratory, and commercial purposes for the development of medicines and for use in the manufacture of medicines and pharmaceuticals. Medicines, chemical, biological and biochemical preparations and proteins for the treatment of cancer, tumourous diseases, proliferative diseases, allergies, auto-immune diseases, inflammation, inflammatory diseases, infectious diseases, osteoporosis, graft versus host and host versus graft diseases, transplant rejection, coronary heart diseases, minimal residual diseases, restenosis, metabolic disorders, neurological disorders, diabetes, B cell malignancy, rheumatoid arthritis, immunological disorders, parasitic reactions, skin diseases, neurodegenerative diseases and cardiovascular diseases, except thyreostatica and cough and cold medicines; pharmaceutical and veterinary preparations and reagents for use in the treatment of cancer, tumorous diseases, proliferative diseases, allergies, auto-immune diseases, inflammation, inflammatory diseases, infectious diseases, osteoporosis, graft versus host and host versus graft diseases, transplant rejection, coronary heart diseases, minimal residual diseases, restenosis, metabolic disorders, neurological disorders, diabetes, B cell malignancy, rheumatoid arthritis, immunological disorders, parasitic reactions, skin diseases, neurodegenerative diseases and cardio-vascular diseases, except thyreostatica and cough and cold medicines; chemical, biological and biochemical reagents for medical and veterinary purposes, except thyreostatica and cough and cold medicines; diagnostic and analytic preparations for medical and veterinary purposes; cell lines, cells, diagnostic proteins and polypeptides for medical or clinical use; nucleic acid sequences, diagnostic proteins, polypeptides and chemical reagents for medical and veterinary purposes; amino acids for medical or veterinary purposes; diagnostic reagents and preparations, namely, microorganisms, proteins, polypeptides, nucleotides, nucleic acids, cell lines and cells for medical, veterinary and diagnostic purposes, except thyreostatica and cough and cold medicines. Scientific and industrial research, namely, in the fields of medicine, pharmaceuticals, molecular biology, biotechnology, immunology and genetics; research and development of medicines, diagnostic and analytic preparations, methods of therapy and expression systems; scientific and technological consulting services, namely, in the fields of medicine, pharmaceuticals, molecular biology, biotechnology, immunology and genetics; industrial consulting services, namely, consultancy pertaining to pharmacology; breeding, recovery, optimisation, synthesis and modification of cell culture lines, cells, microorganisms, polypeptides, proteins, nucleotides, nucleic acids for scientific, research, laboratory, industrial and commercial purposes, namely, for use in the fields of medicine, pharmaceuticals, molecular biology, biotechnology, immunology and genetics, for the development of medicines and for use in the manufacture of medicines and pharmaceuticals.


Patent
CEVEC Pharmaceuticals GmbH | Date: 2011-08-16

The present invention relates to a method for the production of an influenza virus-based vaccine using permanent human amniocyte cells, as well as the use of a permanent human amniocyte cell for the production of a influenza virus-based vaccine.


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