Kathy Wong

Transcriptome and Proteome Profiling to Understanding the Biology of High Productivity CHO Cells

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15 K CHO cDNA microarray chip, whereas proteomic analysis was perfomed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.

Low-Glutamine Fed-Batch Cultures of 293-HEK Serum-Free Suspension Cells for Adenovirus Production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low‐glutamine fed‐batch cultures of serum‐free, suspension cells in a 5‐L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell‐exclusion system that allows for aseptic, cell‐free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose‐free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed‐batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed‐batch cultures. In all cases, fed‐batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed‐batch culture using adenovirus‐green fluorescent protein (Ad‐GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed‐batch culture showed that there is an approximately 10‐fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed‐batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Proteomic investigation of metabolic shift in mammalian cell culture

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down‐regulated, and the others were up‐regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down‐regulated, and the precursor of the 23 kDa subunit of NADH‐ubiquinone oxidoreductase, which was up‐regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl‐terminal hydrolase, which is involved in protein turnover and degradation, was also up‐regulated in the metabolically altered cells. The remaining down‐regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up‐regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up‐regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.

Overexpression of heat shock proteins (HSPs) in CHO cells for extended culture viability and improved recombinant protein production

It has been widely reported that CHO cells undergo apoptosis in culture, despite supplementation of nutrients through fed-batch strategies. Improvement of cell viability in culture can effectively improve recombinant protein yield through extension of the cultures production lifespan, especially at high cell densities. Heat shock proteins (HSPs) have been reported to demonstrate anti-apoptotic effects against a wide range of physical and chemical stimuli through their ability to bind and act as antagonists to critical apoptotic molecules. CHO-IFN-gamma cells, expressing recombinant human interferon-gamma (IFN-gamma), were engineered to overexpress two HSPs (HSP27 and HSP70) either individually or in combination. In fed-batch bioreactor cultures, the engineered cell lines exhibited a more gradual viability loss and extension of culture times of 36-72h, with corresponding delays in escalation of caspases 2, 3, 8 and 9 activities, compared to the control cultures utilizing cells transfected with the vector backbone. The extension in culture times translated to a 2.5-fold improvement in IFN-gamma production over controls in fed-batch cultures. These results suggest that overexpression of HSPs represents a promising generic strategy for the development of robust CHO cell lines resistant to apoptotic insults and possessing improved culture characteristics to enhance recombinant glycoprotein yields.

Enhanced IFNγ production in adenosine-treated CHOCells: A mechanistic study

Adenosine causes growth arrest in recombinant mammalian cell cultures, which results in enhanced productivity of the recombinant protein. Adenosine is also known to increase intracellular ATP level when added to mammalian cells. As a cells energy level affects its protein expression capacity, we investigated the factors that contribute to the increase in recombinant protein productivity. Chinese hamster ovary (CHO) cells expressing human interferon‐gamma (IFNγ) were treated with 1 mM adenosine on Day 2 of culture. The growth arrest resulted in 60% reduction in integral viable cell density when compared with control. However, IFNγ titer improved 1.4‐fold alongside a 2.5‐fold increase in average specific productivity. The adenosine‐treated cells also experienced a two‐fold increase in ATP level that sustained for 3 days. Western blot studies revealed a relatively short‐lived but strong activation of the energy sensor AMP‐activated protein kinase (AMPK) in adenosine‐treated cells. Activation of AMPK was probably due to adenosine being temporarily converted to AMP. Activated AMPK should have down‐regulated protein translation by preventing mammalian target of rapamycin (mTOR) from phosphorylating and inactivating 4E‐binding protein 1 (4E‐BP1), a key repressor of protein translation initiation. However, Western blots showed increased phosphorylation of 4E‐BP1 on Day 2 that lasted 3 days. This implied that a high concentration of ATP could keep 4E‐BP1 inhibited, probably by directly modulating mTOR. This corroborated with an earlier in vitro observation (Dennis et al., Science. 2001;294:1102‐1105). Inhibition of translation initiation repression is thus likely to contribute in part to the improvement in IFNγ‐specific productivity and titer.

Adenovirus Vector Production in 293 Fed-Batch Cultyres

Adenovirus vector production kinetics using 293 cells were investigated in serum-free fedbatch cultures. The monitoring of infection progress and vector production was facilitated by the use of a recombinant adenovirus expressing greenfluorescence protein (GFP). The metabolic activities at different stages of the culture were monitored with online measurements of oxygen uptake rate (OUR), which allowed for the controlling of glucose and glutamine levels in a fed-batch mode. The metabolism of 293 cells shifted from a high lactate producing state to a low lactate producing state gradually. As a result the maximum viable cell density reached was higher than that achieved in a batch culture. Comparing cell metabolism before and after the metabolic shift, the specific consumption rates of glucose and all amino acids in cells in low lactate producing state were significantly reduced. The extension of such strategy enabled us to perform adenovirus infection at a high cell density without medium replacement. It is envisaged that the above studies will aid the development of production process that delivers the required level of viral vectors for gene therapy.

TRANSCRIPTIONAL PROFILING OF BATCH AND FED-BATCH PROTEIN-FREE 293-HEK CULTURES USING DNA MICROARRAY

Dynamic nutrient feeding to control glutamine at low levels in protein-free fed-batch cultures of 293-human embryonic kidney (HEK) cells achieved cell concentrations of 6 x 10(6) cells/ml. This represented a 4-fold improvement in cell concentration compared to batch cultures. Reduction in glutamine and glucose consumption, as well as lactate and ammonia production, were also observed in these fed-batch cultures. High virus production titers of 3 x 10(11) pfu/ml were achieved in fed-batch cultures which were 10,000-fold higher than batch cultures. An investigation of the transcriptional regulation of the metabolic changes associated with the batch and the low-glutamine fed-batch cultures using DNA microarray was conducted. This analysis provides better understanding of the transcriptional regulatory mechanism resulting in the observed physiological changes. Transcriptional profiling of cells from the mid-exponential, late exponential and stationary phases of both the batch and fed-batch were undertaken using an 18,000 element human chip. Transcriptional profiles were ontologically classified to provide a global view of the genetic changes. Furthermore, a pathway-oriented analysis focusing on cellular metabolism was conducted to reveal the dynamic regulation of genes related to amino acid metabolism, tRNA synthetases, TCA cycle, electron transport chain and glycolysis.

Elucidating apoptotic cell death in cho cell batch & fed-batch cultures

Chinese Hamster Ovary (CHO) cells are regarded as one of the industrial ‘work-horses’ for complex biotherapeutics production. In these processes, loss in culture viability occurs primarily via apoptosis, a genetically controlled form of cellular suicide. By applying microarray technology using our ‘in-house’ developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis, the genetic circuitry that regulates and executes apoptosis induction can be carried out rapidly. We found that in both batch and fed-batch cultures, receptorand mitochondrial-mediated apoptosis pathways play important roles in apoptosis induction. There are also several other minor pro-apoptotic genes that appear to be upregulated during apoptosis induction in CHO cells. However, although these other genes had been implicated in apoptosis induction, their exact role in apoptosis induction has yet to be elucidated. By having a greater understanding of the regulatory circuitry of apoptosis relevant to cell culture processes, future effective strategies could be developed towards cell death prevention.

Enhancing recombinant glycoprotein yield and quality using gene targeted CHO cells lines

Background It has been widely reported that CHO cells undergo apoptosis in culture, despite nutrient supplementation through fed-batch strategies. An understanding of apoptosis signaling can thus enable the identification of key genetic targets for the engineering of cell lines that could prolong culture viability and attain higher cell densities to effectively improve recombinant glycoprotein yield and quality.

Identifying key signatures of highly productive CHO cells from transcriptome and proteome profiles

Address: 1Bioprocessing Technology Institute, Biomedical Sciences Institutes, 20 Biopolis Way, #06-01 Centros, Singapore 138668, 2Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 and 3Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455-0132, USA * Corresponding author