Miranda G.S. Yap

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. 2009;102: 1182–1196.

Combined In Silico Modeling and Metabolomics Analysis to Characterize Fed-Batch CHO Cell Culture

The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency and yield of these biopharmaceutical products from mammalian cells, which is fully achievable only through proper understanding of cellular functioning. Towards this end, the current study exploited a combined metabolomics and in silico modeling approach to gain a deeper insight into the cellular mechanisms of Chinese hamster ovary (CHO) fed‐batch cultures. Initially, extracellular and intracellular metabolite profiling analysis shortlisted key metabolites associated with cell growth limitation within the energy, glutathione, and glycerophospholipid pathways that have distinct changes at the exponential‐stationary transition phase of the cultures. In addition, biomass compositional analysis newly revealed different amino acid content in the CHO cells from other mammalian cells, indicating the significance of accurate protein composition data in metabolite balancing across required nutrient assimilation, metabolic utilization, and cell growth. Subsequent in silico modeling of CHO cells characterized internal metabolic behaviors attaining physiological changes during growth and non‐growth phases, thereby allowing us to explore relevant pathways to growth limitation and identify major growth‐limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth‐related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance. Biotechnol. Bioeng. 2012; 109:1415–1429.

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (∼20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336.

Engineering mammalian cells in bioprocessing – current achievements and future perspectives

Over the past 20 years, we have seen significant improvements in product titres from 50 mg/l to 5–10 g/l, a more than 100‐fold increase. The main methods that have been employed to achieve this increase in product titre have been through the manipulation of culture media and process control strategies, such as the optimization of fed‐batch processes. An alternative means to increase productivity has been through the engineering of host cells by altering cellular processes. Recombinant DNA technology has been used to over‐express or suppress specific genes to endow particular phenotypes. Cellular processes that have been altered in host cells include metabolism, cell cycle, protein secretion and apoptosis. Cell engineering has also been employed to improve post‐translational modifications such as glycosylation. In this article, an overview of the main cell engineering strategies previously employed and the impact of these strategies are presented. Many of these strategies focus on engineering cell lines with more efficient carbon metabolism towards reducing waste metabolites, achieving a biphasic production system by engineering cell cycle control, increasing protein secretion by targeting specific endoplasmic reticulum stress chaperones, delaying cell death by targeting anti‐apoptosis genes, and engineering glycosylation by enhancing recombinant protein sialylation and antibody glycosylation. Future perspectives for host cell engineering, and possible areas of research, are also discussed in this review.

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.

Metabolomics-driven approach for the improvement of Chinese hamster ovary cell growth: Overexpression of malate dehydrogenase II

We have established a liquid chromatography-mass spectrometry based metabolomics platform to identify extracellular metabolites in the medium of recombinant Chinese hamster ovary (CHO) fed-batch reactor cultures. Amongst the extracellular metabolites identified, malate accumulation was the most significant. The contributing factors to malate efflux were found to be the supply of aspartate from the medium, and an enzymatic bottleneck at malate dehydrogenase II (MDH II) in the tricarboxylic acid cycle. Subsequent metabolic engineering to overexpress MDH II in CHO resulted in increases in intracellular ATP and NADH, and up to 1.9-fold improvement in integral viable cell number.

Metabolomics profiling of extracellular metabolites in recombinant Chinese Hamster Ovary fed-batch culture.

A metabolomics-based approach was used to time profile extracellular metabolites in duplicate fed-batch bioreactor cultures of recombinant Chinese Hamster Ovary (CHO) cells producing monoclonal IgG antibody. Culture medium was collected and analysed using a high-performance liquid chromatography (HPLC) system in tandem with an LTQ-Orbitrap mass spectrometer. An in-house software was developed to pre-process the LC/MS data in terms of filtering and peak detection. This was followed by principal component analysis (PCA) to assess variance amongst the samples, and hierarchical clustering to categorize mass peaks by their time profiles. Finally, LC/MS2 experiments using the LTQ-Orbitrap (where standard was available) and SYNAPT HDMS (where standard was unavailable) were performed to confirm the identities of the metabolites. Two groups of identified metabolites were of particular interest; the first consisted of metabolites that began to accumulate when the culture entered stationary phase. The majority of them were amino acid derivatives and they were likely to be derived from the amino acids in the feed media. Examples included acetylphenylalanine and dimethylarginine which are known to be detrimental to cell growth. The second group of metabolites showed a downward trend as the culture progressed. Two of them were medium components--tryptophan and choline, and these became depleted midway into the culture despite the addition of feed media. The findings demonstrated the potential of utilizing metabolomics to guide medium design for fed-batch culture to potentially improve cell growth and product titer.

A detailed understanding of the enhanced hypothermic productivity of interferon-γ by Chinese-hamster ovary cells

Culturing CHO (Chinese‐hamster ovary) cells at low temperature leads to growth arrest in the G0/G1 phase of the cell cycle and, in many cases, causes an increase in the specific productivity of recombinant protein. Controlled proliferation is often used to increase CHO specific productivity, and thus there is speculation that the enhanced productivity at low temperature is due to G0/G1‐phase growth arrest. However, we show that the positive effect of low temperature on recombinant protein production is due to elevated mRNA levels and not due to growth arrest and that a cell line can still exhibit growth‐associated productivity at low temperatures. Using a CHO cell producing recombinant human IFN‐γ (interferon‐γ), we show that productivity increases as the percentage of cells in the S phase of the cell cycle increases, at both 32 and 37°C. The increased productivity is due to higher recombinant IFN‐γ mRNA levels. We also show that, for a given cell‐cycle distribution, specific productivity increases as the temperature is lowered from 37 to 32°C. Thus specific productivity is maximized when cells are actively growing (high percentage of S‐phase cells) and also exposed to low temperature. These findings have important implications for cell‐culture optimization and cell‐line engineering, providing evidence that a CHO cell line capable of actively growing at low temperature would provide improved total protein production relative to the current growth strategies, namely 37°C active growth or low‐temperature growth arrest.

Comparison of acetate inhibition on growth of host and recombinantE. coli K12 strains

SummaryWe investigated the extent of acetate inhibition on growth rate of different host and recombinantE. coli K12 strains, cultivated in defined and complex media. A mathematical model was proposed to describe this inhibition. Our results show that acetate inhibition is more significant for recombinant cells than for host cells, and for cells cultured in defined medium than in complex medium.

Hyper-stimulation of monoclonal antibody production by high osmolarity stress in eRDF medium.

An earlier study (Chua et al., 1994) showed that hybridoma 2HG11 cultivated in a basal medium called eRDF, which is enriched in amino acids, enabled higher immunoglobulin (Ig) production with and without serum, when compared to two other traditional media RPMI and DMEM/F12. A further enhancement of Ig productivity was achieved when the osmolarity of the culture medium was increased from 300 mOsm to 350 mOsm (Oh et al., 1993). To determine whether the eRDF media was indeed better, three other cell lines, two IgG producers (TB/C3 and I13/17) and an IgM producer (B10), were tested. The results showed that maximum viable cell densities in eRDF medium were up to 3-times higher than in RPMI and maximum Ig titres were 2-8-times higher than in DMEM/F12 and RPMI. The three cell lines were similarly subjected to osmotic increases from 300 mOsm to 350 and 400 mOsm by addition of NaCl. There was an increase in Ig titres of between 30% to 100% compared to the control medium, although cell growth was reduced. Thus, hyper-stimulation by osmolarity stress was found to be generally effective in eliciting higher Ig production; the extent of enhancement being more pronounced for certain cell lines. Other osmolytes such as sucrose and KCl demonstrated similar effects of increasing Ig productivity. Study on the mechanism of action of osmotic stress on hybridoma 2HG11 revealed that hyper-stimulation of Ig productivity was fundamentally related to a greater availability of amino acids to cells as the cells actively accumulated more salt and amino acids to compensate for the higher medium osmolarity. Uptake of the amino acid analogues 14C-aminoisobutyric acid and 3H-methylaminoisobutyric acid into cells increased to 2.34 x 10(3) cpm per cell per min and 6.35 x 10(3) cpm per cell per min, respectively, under osmotic stress. This corresponds to an 85% increase in uptake via the Na(+)-dependent symport and a 50% increase in uptake via the Na(+)-independent and Na(+)-dependent symports. In the 350 mOsm medium, hybridomas also demonstrated an increase in metabolic activities of 5-10% compared to the control. This, together with the reduced specific growth rate in cells under osmotic stress, suggests that more energy was channelled into the biosynthetic pathway of Ig production.