Development of a generic transient transfection process at 100 L scale.

Abstract:

:We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L(-1) IgG.

journal_name

Cytotechnology

journal_title

Cytotechnology

authors

Tuvesson O,Uhe C,Rozkov A,Lüllau E

doi

10.1007/s10616-008-9135-2

subject

Has Abstract

pub_date

2008-02-01 00:00:00

pages

123-36

issue

2

eissn

0920-9069

issn

1573-0778

journal_volume

56

pub_type

杂志文章
  • Comparative cytogenetics between two species of the family Pseudopimelodidae (Siluriformes): occurrence of natural triploidy and supernumerary chromosomes.

    abstract::The Pseudopimelodidae family comprises 35 species however, cytogenetic studies have been performed in only six species. This study uncovered karyotypic data on Pseudopimelodus pulcher and Microglanis cottoides. Both species possessed 2n = 54, with 20m + 16sm + 10st + 8a and FN = 100 for P. pulcher and 30m + 14sm + 6st...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-013-9676-x

    authors: Gouveia JG,de Moraes VP,Pires LB,da Rosa R,Dias AL

    更新日期:2015-03-01 00:00:00

  • Impact of chicken-origin cells on adaptation of a low pathogenic influenza virus.

    abstract::Understanding the growth dynamics of influenza viruses is an essential step in virus replication and cell-adaptation. The aim of this study was to elucidate the growth kinetic of a low pathogenic avian influenza H9N2 subtype in chicken embryo fibroblast (CEF) and chicken tracheal epithelial (CTE) cells during consecut...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-012-9495-5

    authors: Shahsavandi S,Ebrahimi MM,Mohammadi A,Zarrin Lebas N

    更新日期:2013-05-01 00:00:00

  • Relative survivability of human osteoblasts is enhanced by 39 °C and ascorbic acid after exposure to photopolymerization ingredients.

    abstract::Photopolymerizable hydrogels offer great potential in cartilage tissue engineering due to their ability to conform to irregular defect shapes and be applied in a potentially minimally invasive manner. An important process requirement in the use of photopolymerizable hydrogels is the ability of the suspended cells to w...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-012-9512-8

    authors: Dua R,Ramaswamy S

    更新日期:2013-08-01 00:00:00

  • Liver stem cells.

    abstract::The capacity of hepatocytes and cholangiocytes to contribute to their own maintenance has long been recognized. More recently, studies have indicated the presence of both intra-hepatic and extra-hepatic stem/progenitor cell populations. The intraorgan compartment probably derives primarily from the biliary tree, most ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1023/A:1024826823194

    authors: Theise ND

    更新日期:2003-03-01 00:00:00

  • Isolation, culture and identification of pulmonary arterial smooth muscle cells from rat distal pulmonary arteries.

    abstract::The culture of pulmonary arterial smooth muscle cells (PASMCs) is one of the most powerful tools for exploring the mechanisms of pulmonary hypertension (PH). Both pulmonary vasoconstriction and remodeling occur predominantly in distal pulmonary arteries (PA). In this study, we provide our detailed and standardized pro...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-017-0081-8

    authors: Peng G,Xu J,Liu R,Fu Z,Li S,Hong W,Chen J,Li B,Ran P

    更新日期:2017-10-01 00:00:00

  • Anti-cancer effects of celecoxib on nasopharyngeal carcinoma HNE-1 cells expressing COX-2 oncoprotein.

    abstract::Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor with antitumor and antiangiogenic activities. To investigate the effects of celecoxib on nasopharyngeal carcinoma (NPC), HNE-1 cells were treated with celecoxib at various concentrations. MTT assay, migration assay and invasion assay were performed to observe...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-010-9296-7

    authors: Chen J,Ran Y,Hong C,Chen Z,You Y

    更新日期:2010-10-01 00:00:00

  • Immunocytochemistry assay in BHK-21 cell line infected with Porcine Sapelovirus.

    abstract::The present study describes an immunocytochemistry (ICC) assay with self-raised hyperimmune sera and a Baby Hamster Kidney-21 (BHK-21) cell line infected with Porcine Sapelovirus (PSV). Sapelovivus/IVRI/SPF-c-6/2015 strain Indian PSV was isolated from the porcine IBRS-2 cell line and investigated for growth on non-por...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-019-00315-4

    authors: Kumari S,Singh R,Desingu PA,Ray PK,Taru Sharma G,Saikumar G

    更新日期:2019-06-01 00:00:00

  • Influence of aeration-homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism.

    abstract::This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-013-9612-0

    authors: Núñez EG,Leme J,de Almeida Parizotto L,Chagas WA,de Rezende AG,da Costa BL,Monteiro DC,Boldorini VL,Jorge SA,Astray RM,Pereira CA,Caricati CP,Tonso A

    更新日期:2014-08-01 00:00:00

  • Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium.

    abstract::Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00749813

    authors: Hashimoto K,Shimizu M

    更新日期:1993-01-01 00:00:00

  • Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells.

    abstract::The light and truncated heavy chains of human factor VIII, expressed separately in baculovirus-infected insect cells, exhibited different secretory behaviour when compared with each other and with a biologically active fusion molecule of the truncated heavy and light chains. The light chain was very efficiently secret...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF02215666

    authors: Webb E,Tkalcevic J,Edwards S,Hocking D,Nisbet I

    更新日期:1996-01-01 00:00:00

  • Anti-hyperuricemic effect of isorhamnetin in cultured hepatocytes and model mice: structure-activity relationships of methylquercetins as inhibitors of uric acid production.

    abstract::Hyperuricemia is an important risk factor for gout. Isorhamnetin (3'-O-methylquercetin) is an O-methylated flavonol, which occurs in onion, almond and sea buckthorn. It is also one of the metabolites of quercetin in mammals. In the present study, we investigated anti-hyperuricemic effect of isorhamnetin adopting both ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-018-0275-8

    authors: Adachi SI,Kondo S,Sato Y,Yoshizawa F,Yagasaki K

    更新日期:2019-02-01 00:00:00

  • β-mercaptoethanol promotes osteogenesis of human mesenchymal stem cells via sirt1-ERK pathway.

    abstract::Human umbilical cord-derived mesenchymal stem cells (hUMSCs) hold strong self-renewal capacity and low immunogenicity, which have attracted attention as potential candidates for bone repair and regeneration. However, insufficient osteogenic differentiation markedly hinders the clinical applications of hUMSCs. In the p...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-020-00412-9

    authors: Liu J,Wang H,Ren W,Zhou Y,Ye Z,Tan WS

    更新日期:2020-10-01 00:00:00

  • Generation of lymphokine activated killer cells in a new high density dialyzing culture apparatus.

    abstract::In order to establish an efficient culture system for the generation of lymphokine activated killer (LAK) cells, we have developed a new device which is essentially based on a continuous dialyzing culture vessel. LAK cells grown in such a system showed higher cytotoxicity than those grown under conventional culture co...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00365414

    authors: Kohgo Y,Sakamaki S,Kanisawa Y,Nojiri S,Ueno Y,Itoh Y,Takahashi M,Sasagawa Y,Hosoi S,Sato S,Niitsu Y

    更新日期:1989-02-01 00:00:00

  • Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene.

    abstract::The presence of a highly purified recombinant interleukin-2 (rIL-2) increased the production of immunoglobulin (IgM or IgG) by human-human hybridomas to 1.5-2.0 times the production by untreated cells. However, these cells did not react with anti-Tac (IL-2 receptor alpha) antibody. To enhance the response of the hybri...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00148808

    authors: Iwamoto K,Shintani Y,Sasada R,Honjo T,Kitano K

    更新日期:1990-07-01 00:00:00

  • Three-dimensional co-culture of hepatocytes and stellate cells.

    abstract::Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts a...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-004-7996-6

    authors: Abu-Absi SF,Hansen LK,Hu WS

    更新日期:2004-07-01 00:00:00

  • Gingival fibroblasts "in vitro" and Down's syndrome.

    abstract::Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of chromosome 21 could alter growth regulation and biochemical mechanisms, so we...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:

    authors: Solmi R,Rossetti A,Talassi O,Tomasini GL,Fato R,Estornell E,Lucarini G,Lenaz G,Simonelli L,Brunelli MA

    更新日期:1993-01-01 00:00:00

  • Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells.

    abstract::There is an unmet medical need for the improvement of pancreatic islet maintenance in culture. Due to restricted donor availability it is essential to ameliorate islet viability and graft engraftment. The aim of this study was to compare the standard tissue culture techniques with the advanced Real Architecture For 3D...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-017-0067-6

    authors: Szebeni GJ,Tancos Z,Feher LZ,Alfoldi R,Kobolak J,Dinnyes A,Puskas LG

    更新日期:2017-04-01 00:00:00

  • Heterohybridomas that secrete high levels of pseudomonas-specific therapeutic human monoclonal antibodies: their generation and large scale growth in an automated hollow fiber cell culture system.

    abstract::Fusion of lymphoblastoid cell lines that produce human monoclonal antibodies against Pseudomonas aeruginosa with the human/mouse heteromyeloma SHM-D33 generated heterohybrids that were stable and secreted antibody in the range of 20 to 300 micrograms/ml. One of the hybridoma cell lines ws adapted to serum-free medium ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00365266

    authors: Gammon MC,Banas MH,Boccumini LE,Sigal NH,Zweerink HJ

    更新日期:1990-01-01 00:00:00

  • Evaluating the bottlenecks of recombinant IgM production in mammalian cells.

    abstract::Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-014-9693-4

    authors: Chromikova V,Mader A,Steinfellner W,Kunert R

    更新日期:2015-03-01 00:00:00

  • Dielectric spectroscopy of mammalian cells. 2. Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3.

    abstract::Low-frequency dielectric spectroscopy has been used in situ, i.e. while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells. This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is cha...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00749815

    authors: Degouys V,Cerckel I,Garcia A,Harfield J,Dubois D,Fabry L,Miller AO

    更新日期:1993-01-01 00:00:00

  • Cytoglobin: a potential marker for adipogenic differentiation in preadipocytes in vitro.

    abstract::Obesity, mainly characterized by the excess fat storage, is a global health problem resulting in serious morbidity and mortality. Identification of molecular mechanisms in adipogenic differentiation pathway might lead to development of new strategies for diagnosis, prevention and therapy of obesity and associated dise...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-016-0047-2

    authors: Doğan A,Demirci S,Kıratlı B,Şahin F

    更新日期:2017-02-01 00:00:00

  • Accuracy of the endpoint assay for virus titration.

    abstract::The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF02522040

    authors: Nielsen LK,Smyth GK,Greenfield PF

    更新日期:1992-01-01 00:00:00

  • Metabolic rates of vascular endothelial cellsin vitro.

    abstract::Cultures of endothelial cells and cell lines of endothelial origin were maintained at confluence without medium exchange for a period of 72 h. During this time period the concentration of nutrients - amino acids and glucose - and metabolic waste products - lactate and ammonium - was determined as well as cell vitality...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/BF00761778

    authors: Schrimpf G,Schröder M,Weitnauer E,Friedl P

    更新日期:1994-01-01 00:00:00

  • The role of recombinant proteins in the development of serum-free media.

    abstract::Early developments in serum-free media led to a variety of formulations in which components normally provided in serum and required for growth (insulin, transferrin, lipid supplements, trace elements) and poorly defined components (extracts, hydrolysates) were added to defined basal media. These additives were mostly ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-006-9002-y

    authors: Keenan J,Pearson D,Clynes M

    更新日期:2006-03-01 00:00:00

  • The enhancement of growth and differentiation of rat adrenal nerve cells by the addition of conditioned medium from human fibroblast cultures.

    abstract::The growth of rat adrenal nerve cells was remarkably enhanced by supplementing the cultured medium from the human fibroblast cell line, Hs 68. Maximum specific growth rate and length of the neurites were observed as 0.076 (1/hr) and 0.026 mm, respectively in 20% supplement of five day old medium. In adding more than 2...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1023/A:1007942619463

    authors: Hong JS,Kim DS,Kim SH,Choi DH,Lee JH,Lee HY

    更新日期:1998-03-01 00:00:00

  • Lithium chloride promotes neuronal differentiation of rat neural stem cells and enhances neural regeneration in Parkinson's disease model.

    abstract::Parkinson's disease (PD) is one of the most common neural degenerative disease, affecting millions of people globally. Great progress has been made in the PD treatment, and one of the most promising one is the stem cell-based therapy. Thus, studies on the differentiation of neural stem cells (NSCs) are important to th...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-016-0056-1

    authors: Qi L,Tang Y,He W,Pan H,Jiang W,Wang L,Deng W

    更新日期:2017-04-01 00:00:00

  • Culture of major pelvic ganglion neurons from adult rat.

    abstract::Successful culturing of neurons from adult animals has been historically difficult for a relatively long time. In this study, we reported the development of a novel method for the isolation and the culture of major pelvic ganglion (MPG) neurons from adult rat. The cultured cells were identified by neuron morphology an...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-012-9515-5

    authors: Cheng S,Yang X,Zhang Y,Xiao C

    更新日期:2013-08-01 00:00:00

  • Cytotoxic and genotoxic effects of dioxacarb by human peripheral blood lymphocytes CAs and Allium test.

    abstract::Dioxacarb (Elecron, Famid) is a phenyl methylcarbamate insecticide and in vitro cytotoxic and genotoxic effects of this pesticide on human peripheral blood lymphocytes and Allium root meristematic cells were investigated by chromosomal aberrations (CAs) and Allium test. Human lymphocytes were treated with 62.5, 125, 2...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-014-9741-0

    authors: Eren Y,Erdoğmuş SF,Akyıl D,Özkara A,Konuk M,Sağlam E

    更新日期:2015-12-01 00:00:00

  • Regulation of hTERT transcription: a target of cellular and viral mechanisms for immortalization and carcinogenesis.

    abstract::A hallmark of human cancer cells is immortal cell growth, which is associated with telomere maintenance by telomerase. The transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is a major mechanism that negatively and positively controls telomerase activity in normal and cancer cells, r...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-004-5122-4

    authors: Horikawa I,Michishita E,Barrett JC

    更新日期:2004-06-01 00:00:00

  • Real-time transfer of lentiviral particles by producer cells using an engineered coculture system.

    abstract::Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and ...

    journal_title:Cytotechnology

    pub_type: 杂志文章

    doi:10.1007/s10616-019-00343-0

    authors: Timmins LM,Patel RS,Teryek MS,Parekkadan B

    更新日期:2019-10-01 00:00:00