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Since a culture increases in cell number when dividing cells separate into two newborn cells, the fraction of mitotic cells in a growing cell population directly reflects the overall growth behavior of a cell culture. To rapidly assess the effects of growth conditions on the fraction of mitotic cells we have employed an antibody specific for the phosphorylated form of histone H3 for the identification of mitotic cells using flow cytometry. The phosphorylation of histone H3 closely correlates with the chromosomal condensation that accompanies the onset of mitosis, and, therefore, it represents a convenient marker for dividing cells. We have optimized the protocol for the staining of mitotic cells for both Chinese hamster ovary and hybridoma cell cultures. Fluorescence micrographs taken of stained cells show that cells in the various stages of mitosis can be detected based on the morphological characteristics of the chromosomes. The variation in the mitotic cell fraction has been determined throughout the batch growth phases of cultures under different growth conditions. The dynamics of the mitotic index show that balanced growth was never truly reached and that the growth rate is in fact quite variable for these cultures since large variations in the mitotic index are observed. In addition, a large increase in the fraction of mitotic cells just prior to the exponential growth phase for all cultures indicates that they are partially synchronized at the exit from the lag phase. According to a two-staged, age structured population balance model, the mitotic index is directly proportional to the growth rate of a culture. The proportionality constant for this case is shown to be the time required for cells to progress through mitosis. This time is believed to be constant for a particular cell line, as shown by experimental data. Thus, growth rates can be determined solely by measurement of the fraction of cells in mitosis. The mitotic index measurements were then used to calculate the growth in cell number of the cultures, and these simulations accurately reflect observed cell counts. Other simulations also show that changes in cell growth can be predicted before they are reflected in the cell count data. This technique can be used as a sensitive indicator of cell growth and could be useful as a process monitoring technique and for developing better feeding strategies for animal cell cultures. [Copyright &y& Elsevier]

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product. [ABSTRACT FROM AUTHOR]

Glycosylation is a critical quality attribute for many therapeutic glycoproteins. Numerous studies in academia and industry have sought to understand and control protein glycosylation in order to obtain consistent glycoform profiles in production. In many cases, the resulting enhancement of protein glycosylation comes at the expense of protein productivity. Here we examine the impact of insulin-like growth factor-I analogue, LongR 3 (LR3), on an Fc-fusion producing Chinese Hamster Ovary (CHO) cell process. We observe that LR3 improves protein productivity and significantly increases protein N-linked glycosylation. The beneficial effects can be seen at low concentrations (micrograms/L), which allows LR3 to be a cost-efficient additive for improving cell culture performances. The recombinant protein examined in this study has both higher sialic acid content and lower percent of asialylated species of N-linked glycans in the presence of LR3. Finally, CHO-specific glycosylation Real-Time Reverse Transcription (RT 2 ) PCR Arrays combined with an enzymatic assay demonstrate that LR3 significantly down-regulates cytosolic sialidase gene Neu2 and hexosaminidase-D gene Hexdc , decreases extracellular sialidase activity, and therefore prevents recombinant glycoprotein desialylation. [ABSTRACT FROM AUTHOR]

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability. [ABSTRACT FROM AUTHOR]

Abstract: Dextran sulfate has been reported to reduce cell aggregation and increase culture viability in suspension cultures of CHO cells. However, only a few of these reports have focused on the effects of dextran sulfate at the cellular level, especially with respect to cell viability. The goal of this study was to examine whether dextran sulfate inhibits CHO cell death through interference with the cellular apoptotic pathway. Apoptosis was induced using staurosporine, and the addition of dextran sulfate was shown to prevent apoptosis, as evident by a reduction in DNA laddering. Dextran sulfate also affected other events in the apoptotic pathway following the addition of staurosporine. Specifically, dextran sulfate decreased pro-caspase-9 activation, cleaved caspase-3 levels, cytochrome c release into the cytosol and mitochondrial transmembrane potential collapse. This data indicates that the mechanism by which dextran sulfate decreases staurosporine-induced apoptosis involves the mitochondrial pathway as early as cytochrome c release, but without caspase-8 activation. This study advances the understanding of the anti-apoptotic action of dextran sulfate on CHO cells and provides a rationale for the application of dextran sulfate in CHO cell culture processes. [ABSTRACT FROM AUTHOR]

Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 μm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible “fingerprint” of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability. [Copyright &y& Elsevier]