28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures.

The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling.

The mammalian target of rapamycin (mTOR) pathway plays essential roles in the regulation of translational activity in many eukaryotes. Thus, from a bioprocessing point of view, understanding its molecular mechanisms may provide potential avenues for improving cell culture performance. Toward this end, the mTOR pathway of CHO cells in batch cultures was subjected to rapamycin treatment (inhibition) or nutrient supplementation (induction) and translational activities of CHO cells producing a monoclonal antibody (mAb) were evaluated with polysome profiling technology. Expectedly, rapamycin induced a shift of mRNAs from polysomes towards monosomes, thus reducing maximum cellular growth rate by 30%, while feeding additional nutrients extended mTOR pathway activity during the stationary growth phase in control batch culture, thereby contributing to an increase in global translation activity by up to 2-fold, and up to 5-fold higher specific translation of the heavy and light chains of the recombinant mAb. These increases in translation activity correlated with a 5-day extension in cellular growth and a 4-fold higher final product titer observed upon nutrient feeding. This first study of the relationship between the mTOR pathway and translational activity in CHO cultures provides key insights into the role of translational control in supporting greater productivity, which will lead to further enhancement of CHO cultures.

Translatome analysis of CHO cells to identify key growth genes.

We report the first investigation of translational efficiency on a global scale, also known as translatome, of a Chinese hamster ovary (CHO) DG44 cell line producing monoclonal antibodies (mAb). The translatome data was generated via combined use of high resolution and streamlined polysome profiling technology and proprietary Nimblegen microarrays probing for more than 13K annotated CHO-specific genes. The distribution of ribosome loading during the exponential growth phase revealed the translational activity corresponding to the maximal growth rate, thus allowing us to identify stably and highly translated genes encoding heterogeneous nuclear ribonucleoproteins (Hnrnpc and Hnrnpa2b1), protein regulator of cytokinesis 1 (Prc1), glucose-6-phosphate dehydrogenase (G6pdh), UTP6 small subunit processome (Utp6) and RuvB-like protein 1 (Ruvbl1) as potential key players for cellular growth. Moreover, correlation analysis between transcriptome and translatome data sets showed that transcript level and translation efficiency were uncoupled for 95% of investigated genes, suggesting the implication of translational control mechanisms such as the mTOR pathway. Thus, the current translatome analysis platform offers new insights into gene expression in CHO cell cultures by bridging the gap between transcriptome and proteome data, which will enable researchers of the bioprocessing field to prioritize in high-potential candidate genes and to devise optimal strategies for cell engineering toward improving culture performance.

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.

Rapid characterization of protein productivity and production stability of CHO cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The biopharmaceutical industry has been in pursuit of strategies which can isolate stable and high-producing cell lines. The whole cell mass spectrometry method by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a rapid and simple method for cell characterization based on the differences in the fingerprints of the mass spectra. This work describes how the method was evaluated for the application of screening for stable and high-producing clones from a panel of recombinant Chinese hamster ovary (CHO) cell lines. Detectable m/z values and their relative intensities were collected and processed by partial least squares (PLS). To reduce the errors introduced by the preparation method and spectra noise, high intensity preliminary data was selected and the number of variables introduced was validated by leave-one-out cross-validation. The differences in recombinant protein productivity and titer were revealed by PLS regression with promising results. Partial least-squares discriminant analysis (PLS-DA) was applied to differentiate stable and unstable cell lines as traditional stability testing would require several months involving numerous continuous passages. Results confirmed that the whole cell MALDI-TOF method can be a powerful method for routine monitoring of bioprocesses and study can be further developed by extending the number of the cell lines tested to establish a recombinant cell line database.

Metabolomics-based identification of apoptosis-inducing metabolites in recombinant fed-batch CHO culture media.

A liquid chromatography-mass spectrometry (LC-MS) based metabolomics platform was previously established to identify and profile extracellular metabolites in culture media of mammalian cells. This presented an opportunity to isolate novel apoptosis-inducing metabolites accumulating in the media of antibody-producing Chinese hamster ovary (CHO mAb) fed-batch bioreactor cultures. Media from triplicate cultures were collected daily for the metabolomics analysis. Concurrently, cell pellets were obtained for determination of intracellular caspase activity. Metabolite profiles from the LC-MS data were subsequently examined for their degree of correlation with the caspase activity. A panel of extracellular metabolites, the majority of which were nucleotides/nucleosides and amino acid derivatives, exhibited good (R² > 0.8) and reproducible correlation. Some of these metabolites, such as oxidized glutathione, AMP and GMP, were later shown to induce apoptosis when introduced to fresh CHO mAb cultures. Finally, metabolic engineering targets were proposed to potentially counter the harmful effects of these metabolites.

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-gamma (IFN-gamma). Galactose (+/-uridine), glucosamine (+/-uridine), and N-acetylmannosamine (ManNAc) (+/-cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex ( approximately 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-gamma 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.

Rapid characterization of high/low producer CHO cells using matrix-assisted laser desorption/ionization time-of-flight.

An intact-cell mass spectrometry (ICM) method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was evaluated for the screening of stable recombinant Chinese hamster ovary (CHO) cell lines, an important mammalian cell line in bioprocessing. With rapid and simple cell pretreatments, viabilities of cells could be rapidly distinguished on the different fingerprints of mass spectra. Detectable m/z values on cell surfaces and their relative intensities were processed by two biostatistical methods, principle components analysis (PCA) and partial least squares (PLS), with promising results. Discrimination among cell lines with different expressed recombinant proteins or different productivities could be achieved. The ICM method has the advantage of providing multiple parameters simultaneously and possesses the potential to become a powerful method for routine monitoring of bioprocesses.

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.

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.

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 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.

Enhancing recombinant glycoprotein sialylation through CMP-sialic acid transporter over expression in Chinese hamster ovary cells.

Glycosylation engineering strategies that are currently used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We explored the possibility that over expressing nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT) would improve the sialylation process in Chinese hamster ovary cells (CHO). Our hypothesis was that increasing CMP-SAT in the cells through recombinant means would increase the transport of CMP-sialic acid into the Golgi, resulting in an increased CMP-sialic acid intra-lumenal pool and increased sialylation of the proteins produced. We report the construction of the CMP-SAT expression vector (pcDNA-SAT) using hamster CMP-SAT (GenBank accession number Y12074) and demonstrated its functionality using Lec2 CHO mutant cells. Transfection of pcDNA-SAT into CHO IFN-gamma, a CHO cell line producing recombinant human interferon-gamma (IFN-gamma) resulted in single clones that had 2-20 fold increase in total CMP-SAT expression at the transcript level and 1.8-2.8 fold increase in CMP-SAT at the protein level when compared to untransfected parent CHO IFN-gamma. This resulted in 4%-16% increase in site sialylation of IFN-gamma. There was also a higher proportion of the more sialylated IFN-gamma glycans produced by the clones. We have thus established a novel strategy for sialylation improvement in recombinant protein production that can be considered singly or along with existing glycosylation improvement strategies, including glycosyltransferase over expression and nucleotide sugar feeding. These multiprong approaches can possibly bring us closer toward the goal of maximum and consistent sialylation in glycoprotein production using mammalian cells.