Abstract Cultured hepatocytes have been explored for use in a bioartificial liver. Spheroids formed by cultured hepatocytes exhibit enhanced liver‐specific functions. The kinetics of spheroid formation, using rat hepatocytes, was studied on positively charged surfaces that were either uncoated or coated with collagen or (hydroxyethyl)methacrylate (HEMA). Optimal spheroid formation was obtained on positively charged (Primaria) surfaces at inoculum densities in the range of (3–9) × 10 4 cells/cm 2 . Cells initially attached and spread out on the surface. Subsequent retraction led to the emergence of small clumps of cells attached to the surface, from which spheroids formed and shed off into suspension. The process of spheroid formation took more than 72 h and was accompanied by a decrease in the surface area occupied by attached cells. Optical sectioning of fluorescently stained spheroids using confocal microscopy indicated that most of the cells in the spheroid were viable. Spheroids also maintained a constant albumin synthesis rate for over 7 days in culture. Spheroid formation was evaluated in terms of the changes in spheroid diameter, the surface area covered by attached cells, and the total protein content of the fraction of cells that formed spheroids. The quantitative methodologies developed were used to assess the effect of inoculum cell concentration on spheroid formation and to evaluate the kinetics of spheroid formation on different surfaces both favorable and nonfavorable to spheroid formation.
Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-γ (IFN-γ) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-γ glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDPsugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.
Microarray‐based comparative transcriptomics analysis is a powerful tool to understand therapeutic protein producing mammalian cell lines at the gene expression level. However, an integrated analysis workflow specifically designed for end‐to‐end analysis of microarray data for CHO cells, the most prevalent host for commercial recombinant protein production, is lacking. To address this gap, an automated data analysis workflow in R that leverages public domain analysis modules is developed to analyze microarray based gene expression data. In addition to testing the global transcriptome differences of CHO cells at different conditions, the workflow identifies differentially expressed genes and pathways with intuitive visualizations as the outputs. The utility of this automated workflow is demonstrated by comparing the transcriptomic profiles of recombinant protein expressing CHO cells with and without a temperature shift. Statistically significant differential expression at the gene, pathway, and global transcriptome levels are identified and visualized. An automated workflow like the one developed in this study will enable rapid translation of CHO culture microarray data into biologically relevant information for mechanism‐driven cell line optimization and bioprocess development.
Previous research has implicated mitochondrial physiology and, by extension, respiratory capacity in the initiation and progress of apoptosis of cells in culture and tissue environments. This hypothesis was tested by separating a hybridoma cell population into subpopulations of varying mitochondrial membrane potential (MMP) using Rhodamine 123 stain and fluorescence-activated cell sorter analysis and subjecting them to two apoptosis inducers, rotenone and staurosporin. Apoptotic death was characterized morphologically through the determination of apoptosis-related chromatin condensation and biochemically through the measurement of caspase-3 enzymatic activity. We found dramatic differences in the apoptotic death kinetics for the subpopulations, with the high MMP cells showing higher resistance to apoptotic death. After incubation with 30 microM rotenone, the low MMP cells exhibited one-third of the viability of the high MMP cells and a three-fold increase in the capsase-3 enzymatic activity. No changes were observed in the DNA content or the cell cycle distributions of the two cell subpopulations, which maintained their mean MMP difference after 20 generations. These results suggest that heterogeneity exists in mammalian cell populations with respect to mitochondrial physiology, which correlates with resistance to apoptotic death.
The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon‐labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6‐ 13 C]hexose 6‐phosphate and [4‐ 13 C]erythrose 4‐phosphate carbon enrichment values resulting from [1‐ 13 C]glucose feeding contained more information as compared to those from ribose 5‐phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13 C‐ or 14 C‐enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum , and Escherichia coli supplied with either [2‐ 14 C]glucose or [1‐ 13 C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.
Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.
Abstract Prior research (Follstad, B. D.; Wang, D. I. C.; Stephanopoulos, G. Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur. J. Biochem . 2000 , 267 , 6534–6540.) identified mitochondrial membrane potential (MMP) as a marker of hybridoma subpopulations resistant to apoptosis caused by a variety of apoptosis inducers. In this study, we investigated the viability of hybridoma cell cultures inoculated with cells of varying MMP in regular fed‐batch operation. A hybridoma cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123 (Rh123). These subpopulations showed dramatic differences in their apoptotic death kinetics. Fed‐batches inoculated with a high MMP subpopulation reached higher viable cell concentrations and viabilities that were maintained for prolonged periods of time relative to fed‐batches inoculated with low MMP subpopulations. These results underline the heterogeneous nature of hybridoma cell cultures and suggest that mitochondrial physiology is a critical parameter determining culture performance.
Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account.
