A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer?


:Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.


Biotechnol Bioeng


Chusainow J,Yang YS,Yeo JH,Toh PC,Asvadi P,Wong NS,Yap MG




Has Abstract


2009-03-01 00:00:00












  • Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast.

    abstract::In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the ac...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Smith JD,Tang BC,Robinson AS

    更新日期:2004-02-05 00:00:00

  • Numerical simulation of a fully baffled biological reactor: the differential circumferential averaging mixing plane approach.

    abstract::A modified mixing plane approach for steady state simulation of flow field in fully baffled biological reactor is presented and discussed. Without requiring any experimental input, this approach of dividing the vessel into suitable number of connected and disconnected zones; solving steady state equation separately in...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Dubey H,Das SK,Panda T

    更新日期:2006-11-05 00:00:00

  • High-yield method for immobilization of enzymes.

    abstract::Two types of polyethylenimine-coated glass microbeads (13-44 micrometer) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were per...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Wasserman BP,Hultin HO,Jacobson BS

    更新日期:1980-02-01 00:00:00

  • Microbial granulation for lactic acid production.

    abstract::This work investigated the formation of microbial granules to boost the productivity of lactic acid (LA). The flocculated form of LA-producing microbial consortium, dominated by Lactobacillus sp. (91.5% of total sequence), was initially obtained in a continuous stirred-tank reactor (CSTR), which was fed with 2% glucos...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Kim DH,Lee MK,Hwang Y,Im WT,Yun YM,Park C,Kim MS

    更新日期:2016-01-01 00:00:00

  • Cultivation of hybridoma cells in an inclined bioreactor.

    abstract::Murine hybridoma cells were grown in a bubble column that was inclined up to 45 degrees from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Lu GZ,Gray MR,Thompson BG,Suresh MR

    更新日期:1995-01-20 00:00:00

  • The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study.

    abstract::At the laboratory scale, sudden step increases from 30 to 42 degrees C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decrea...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Caspeta L,Flores N,Pérez NO,Bolívar F,Ramírez OT

    更新日期:2009-02-01 00:00:00

  • Analysis of E. coli phoA-lacZ fusion gene expression inserted into a multicopy plasmid and host cell's chromosome.

    abstract::The production of beta-galactosidase from the E. coli phoA-lacZ fusion gene was studied to compare the gene expression behavior of two cloning methods: insertion to multicopy plasmids and integration into host cell's chromosome. The chromosome-integrating strain showed more tight control of fusion gene expression leve...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Shin PK,Seo JH

    更新日期:1990-12-20 00:00:00

  • Removal of color from kraft mill wastewaters with cultures of white-rot fungi and with immobilized mycelium of Coriolus versicolor.

    abstract::Screening fifteen strains of white-rot fungi for their ability to decolorize combined bleached kraft effluent showed that Coriolus versicolor in liquid culture removed over 60% of the color of the effluent within six days in the presence of sucrose. Treatment of the same effluent with this fungus, immobilized in beads...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Livernoche D,Jurasek L,Desrochers M,Dorica J,Veliky IA

    更新日期:1983-08-01 00:00:00

  • The role of pH on the resistance of resting- and active anammox bacteria to NO2- inhibition.

    abstract::The anaerobic oxidation of ammonium (anammox) uses nitrite as terminal electron acceptor. The nitrite can cause inhibition to the bacteria that catalyze the anammox reaction. The literature shows a great divergence on the levels of NO2 (-) causing inhibition. Moreover, the conditions influencing the resistance of anam...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Carvajal-Arroyo JM,Puyol D,Li G,Sierra-Álvarez R,Field JA

    更新日期:2014-10-01 00:00:00

  • Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas.

    abstract::A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous m...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Mancuso A,Beardsley NJ,Wehrli S,Pickup S,Matschinsky FM,Glickson JD

    更新日期:2004-09-30 00:00:00

  • Understanding mild acid pretreatment of sugarcane bagasse through particle scale modeling.

    abstract::Sugarcane bagasse is an abundant and sustainable resource, generated as a by-product of sugarcane milling. The cellulosic material within bagasse can be broken down into glucose molecules and fermented to produce ethanol, making it a promising feedstock for biofuel production. Mild acid pretreatment hydrolyses the hem...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Greenwood AA,Farrell TW,O'Hara IM

    更新日期:2013-12-01 00:00:00

  • Biotransformation of D-methionine into L-methionine in the cascade of four enzymes.

    abstract::D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Findrik Z,Vasić-Racki D

    更新日期:2007-12-01 00:00:00

  • Mobile phase modifier effects in multimodal cation exchange chromatography.

    abstract::This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corrobora...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Holstein MA,Parimal S,McCallum SA,Cramer SM

    更新日期:2012-01-01 00:00:00

  • Classification, error detection, and reconciliation of process information in complex biochemical systems.

    abstract::Bioprocess identification starts with collection of process information. Usually there is a variety of information available, consisting of actual measurement data, historical data, empirical kinetic and yield correlations, and general knowledge available from literature. A central problem is to find out how the vario...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Noorman HJ,Romein B,Luyben KC,Heijnen JJ

    更新日期:1996-02-20 00:00:00

  • A mathematical description of recombinant yeast.

    abstract::A model was formulated to examine specific experimental data of growth and heterologous product formation with recombinant Saccharomyces cerevisiae while incorporating available literature. The model simulated dry cell weight, glucose, ethanol, dissolved oxygen, human Epidermal Growth Factor (hEGF) production, fractio...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Coppella SJ,Dhurjati P

    更新日期:1990-02-20 00:00:00

  • Fundamental questions in optimizing ion-exchange chromatography of proteins using computer-aided process design.

    abstract::The major objectives for preparative protein chromatography are maximal loading and increased flow rate while maintaining defined resolution. Conventionally a series of chromatographic experiments are performed and the optimal conditions are selected according to the separation criteria. Computer-aided process design ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Jungbauer A,Kaltenbrunner O

    更新日期:1996-10-20 00:00:00

  • Simulation of penicillin production in fed-batch cultivations using a morphologically structured model.

    abstract::A mathematical model is formulated to describe trends in biomass and penicillin formation as well as substrate consumption for fed-batch cultivations. The biomass is structured into three morphological compartments, and glucose and corn steep liquor are considered as substrates for growth. Penicillin formation is assu...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Zangirolami TC,Johansen CL,Nielsen J,Jørgensen SB

    更新日期:1997-12-20 00:00:00

  • Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus.

    abstract::Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density biore...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Schlatmann JE,Moreno PR,Vinke JL,Ten Hoopen HJ,Verpoorte R,Heijnen JJ

    更新日期:1994-08-05 00:00:00

  • Survival and apoptotic pathways initiated by TNF-alpha: modeling and predictions.

    abstract::We present a mathematical model which includes TNF-alpha initiated survival and apoptotic cascades, as well as nuclear transcription of IkappaB. These pathways play a crucial role in deciding cell fate in response to inflammation and infection. Our model incorporates known specific protein-protein interactions as iden...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Rangamani P,Sirovich L

    更新日期:2007-08-01 00:00:00

  • A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system.

    abstract::A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Zhang J,Kalogerakis N,Behie LA,Iatrou K

    更新日期:1993-07-01 00:00:00

  • Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5'-coding region variant of the cadA gene.

    abstract::Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Jeon HG,Cheong DE,Han Y,Song JJ,Choi JH

    更新日期:2016-07-01 00:00:00

  • Production of micronic particles of biocompatible polymer using supercritical carbon dioxide.

    abstract::Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 mum were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents....

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Benedetti L,Bertucco A,Pallado P

    更新日期:1997-01-20 00:00:00

  • Resting cells of recombinant E. coli show high epoxidation yields on energy source and high sensitivity to product inhibition.

    abstract::Metabolically active resting (i.e., nongrowing) bacterial cells have a high potential in cofactor-dependent redox biotransformations. Where growing cells require carbon and energy for biomass production, resting cells can potentially exploit their metabolism more efficiently for redox biocatalysis allowing higher spec...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Julsing MK,Kuhn D,Schmid A,Bühler B

    更新日期:2012-05-01 00:00:00

  • CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.

    abstract::Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to in...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Landberg J,Wright NR,Wulff T,Herrgård MJ,Nielsen AT

    更新日期:2020-08-18 00:00:00

  • Lewis cell studies to determine reactor design data for two-liquid-phase bacterial and enzymic reactions.

    abstract::Substrate transfer rates from organic to aqueous phases were measured in the presence and absence of biocatalyst in the reaction medium, using modified Lewis cells. These measurements, in combination with intrinsic aqueous phase biocatalytic reaction kinetics, were used to confirm that benzyl acetate hydrolysis by pig...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Woodley JM,Brazier AJ,Lilly MD

    更新日期:1991-01-20 00:00:00

  • Development of a kinetic model for the alcoholic fermentation of must.

    abstract::We Propose a kinetic expression which accounts for the temperature dependence of ethanol yield losses in batch alcoholic fermentation. Moreover, the characteristic parameters of the microbial growth equation have been calculated for Saccharomyces cerevisiae under typical wine industry conditions. A substrate consumpti...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Caro I,Pérez L,Cantero D

    更新日期:1991-10-05 00:00:00

  • Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli.

    abstract::L-Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l-threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l-threonine productivity...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Liu J,Li H,Xiong H,Xie X,Chen N,Zhao G,Caiyin Q,Zhu H,Qiao J

    更新日期:2019-01-01 00:00:00

  • New approaches for verification of kinetic parameters of immobilized concanavalin A: invertase preparations investigated by flow microcalorimetry.

    abstract::In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as stan...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Gemeiner P,Docolomansky P,Nahálka J,Stefuca V,Danielsson B

    更新日期:1996-01-05 00:00:00

  • Development of waxy cassava with different Biological and physico-chemical characteristics of starches for industrial applications.

    abstract::The quality of cassava starch, an important trait in cassava breeding programs, determines its applications in various industries. For example, development of waxy (having a low level of amylose) cassava is in demand. Amylose is synthesized by granule-bound starch synthase I (GBSSI) in plants, and therefore, down-regu...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Zhao SS,Dufour D,Sánchez T,Ceballos H,Zhang P

    更新日期:2011-08-01 00:00:00

  • Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator.

    abstract::Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin bi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Andersen DC,Bridges T,Gawlitzek M,Hoy C

    更新日期:2000-10-05 00:00:00