Effects of cysteine,asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.     

Addition of Valproic Acid to CHO Cell Fed-Batch Cultures Improves Monoclonal Antibody Titers

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

Regulation of renal amino acid (AA) transport by hormones,drugs and xenobiotics – a review

Major advances have recently been made in our understanding of the mechanisms and functions of amino acid transport in mammalian cells: - from the whole organism to transporter molecular structure. In this article, we present a brief overview of current knowledge concerning amino acid transporters, followed by a detailed discussion of the relevance of this new information to our broader understanding of the physiological regulation of amino acid handling in the kidney. We focus especially on the influence of hormones and xenobiotics on renal amino acid transport systems. In a growing number of cases, it now seems possible to correlate the effects of hormones, drugs, and xenobiotics with the capacity of renal amino acid transporters. This topic is of clinical relevance for the treatment of many amino acid reabsorption disorders. For example, under suitable conditions glucocorticoids and thyroid hormones stimulate renal reabsorption of amino acids and might therefore be of benefit in the treatment of different kinds of aminoaciduria. Hormonal regulation also underlies the postnatal development of renal amino acid reabsorption capacity, which can be stimulated to mature earlier after exogenous administration of e.g. glucocorticoids. In contrast, many compounds (e.g. heavy metal complexes) selectively damage renal amino acid transporters resulting in urinary amino acid loss. These types of phenomena (stimulation or inhibition of amino acid transporters in the kidney) are reviewed from the perspectives of our new molecular understanding of transport processes and of clinical relevance.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.     

Molecular mechanisms involved in the adaptation to amino acid limitation in mammals

In mammals, metabolic adaptations are required to cope with episodes of protein deprivation and malnutrition. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of the expression of numerous genes. In particular, it has been shown that amino acids by themselves can modify the expression of target genes. This review describes the regulation of amino acids homeostasis and the their role as signal molecules. The recent advances in the understanding of the molecular mechanisms involved in the control of mammalian gene expression in response to amino acid limitation will be described.     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

Artificial induction of chromosome aneuploidy in CHO cells alters their function as host cells

Chinese hamster ovary (CHO) cells are major host cells for biopharmaceuticals. During culture, the chromosome number of CHO cells alters spontaneously. Here, we investigated the effects of artificial changes in the chromosome number on productivity. When cell fusion between antibody-producing CHO-K1-derived cells was induced, we observed a wide range of aneuploidy that was not detected in controls. In particular, antibody productivities were high in clone-derived cell populations that retained a diverse chromosome number distribution. We also induced aneuploid cells using 3-aminobenzamide that causes chromosome non-disjunction. After induction of aneuploidy by 3-aminobenzamide, cells with an increased chromosome number were isolated, but cells with a decreased chromosome number could not be isolated. When antibody expression vectors were introduced into these isolated clones, productivity tended to increase in cells with an increased chromosome number. Further analysis was carried out by focusing on clone 5E8 with an average chromosome number of 37. When 5E8 cells were used as host, the productivity of multiple antibodies, including difficult-to-express antibodies, was improved compared with CHO-K1 cells. The copies of exogenous genes integrated into the genome were significantly increased in 5E8 cells. These findings expand the possibilities for host cell selection and contribute to the efficient construction of cell lines for recombinant protein production.     

The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.     

Changes of shikimate pathway in glyphosate tolerant alfalfa cell lines with reduced embryogenic ability

Glyphosate tolerant cell lines were selected from highly embryogenic cell suspension culture ofMedicago sativa L. Resistant cell lines showed significant reduction of embryogenic ability and during long-term culture in the presence of glyphosate gradual loss of this ability was observed. After glyphosate treatment the increased activity of 5-enolpyruvylshikimate-3-phosphate synthase in tolerant cell lines overcame the block in aromatic amino acid synthesis which was observed in control cell lines. Glyphosate caused marked increase in the content of shikimic acid in both control and tolerant cell lines but the accumulation of shikimic acid was considerably lower in tolerant calli. Significant qualitative and quantitative differences were found in the content of individual phenolic acids. The considerable decrease in the amount of cinnamic acid derivates and broader spectrum of hydroxybenzoic acids suggest in tolerant cell lines the activation of alternative pathway not regulated by phenylalanine ammonia lyase. The possible role of altered pool of phenolic acids on the embryogenic ability is discussed.     

Molecular regulation of amino acid biosynthesis in plants

Summary Our understanding of amino acid biosynthesis in plants has grown by leaps and bounds in the last decade. It appears that most of the amino acid biosynthesis takes place in the chloroplast. Recent demonstration of glutamine synthetase and DAHP synthase in the vascular tisuue has added a new dimension in the complexity of the nitrogen cycle in plants. Isolation of various genes and transformation of plants with the modified forms of the genes are providing tools for understanding the regulation of various pathways. Plant transformation approaches are also going to provide the food of the future with an improved amino acid composition.     

Molecular cloning and functional analysis of Chinese sturgeon (Acipenser sinensis) growth hormone receptor

A full length cDNA encoding the growth hormone receptor (GHR) of Chinese sturgeon was cloned in order to investigate the mechanism of growth hormone in regulating the growth of Chinese sturgeon. The open reading frame of the cloned Chinese sturgeon growth hormone receptor (csGHR) cDNA encodes a trans-membrane protein of 611 amino acids containing all the characteristic motifs of GHR. By sequence alignment, substitutions of amino acid residues highly conserved in other species were identified. Using the CHO cell culture system, the function of csGHR and the biological significance of the amino acid substitution in csGHR were examined. The promoter of serine protease inhibitor 2.1 (Spi2.1) was trans-activated upon stimulation of seabream GH (sbGH) in the csGHR-expressing CHO cells. Furthermore, CHO cells stably expressing csGHR were stimulated to proliferate by sbGH. In agreement with our previous report, Chinese sturgeon growth hormone-binding protein (csGHBP) was detected in the culture medium of CHO cells stably expressing csGHR. Mutation of Asp residue in the ligand binding motif in csGHR to Glu significantly enhanced csGHR’s biological function, whereas mutation of Asp residue to Ala decreased its biological function. The results demonstrated that the cloned csGHR was of full biological function and the csGHBP could be generated through proteolysis of csGHR. These findings might provide new insights into thoroughly understanding the regulatory mechanism of Chinese sturgeon growth.