The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions.
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ABSTRACT:
SUBMITTER: Nam JH
PROVIDER: S-EPMC2698439 | biostudies-other | 2009 Mar
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:Hypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. However, death and disability rates in HIE infants treated with TH remain high. Although the cellular mechanism of the neuroprotective effect of TH remains unclear, astrocytic erythropoietin (EPO) is known to be a key mediator of neuroprotection under hypoxic conditions. In the present study, we investigated the hypothermia effect on EPO expression in astrocytes and determined whether hypothermia attenuates neuronal damage via EPO signaling. METHODS:Astrocytes derived from rat cerebral cortex were cultured under oxygen/glucose deprivation (OGD). The expression of EPO and hypoxia-inducible factor (HIF), a transcription factor of EPO, was assessed. After OGD, astrocytes were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions, and then EPO and HIF expression was assessed. After OGD, rat cortical neurons were cultured in astrocyte-conditioned medium (ACM) derived from the hypothermic group, and neuronal apoptosis was evaluated. RESULTS:OGD induced EPO mRNA and protein expression, although at lower levels than hypoxia alone. HIF-1? and HIF-2? protein expression increased under hypoxia alone and OGD, although OGD increased HIF-2? protein expression less than hypoxia alone. EPO gene and protein expression after OGD was significantly higher under hypothermia. Moreover, expression of HIF-1? and HIF-2? protein was enhanced under hypothermia. In the presence of ACM derived from hypothermic astrocytes following OGD, the number of cleaved caspase 3 and TdT-mediated dUTP nick-end labeling-positive apoptotic neurons was lower than in the presence of ACM from normothermic astrocytes following OGD. Blockade of EPO signaling using anti-EPO neutralization antibody attenuated the anti-apoptotic effect of ACM derived from hypothermic astrocytes following OGD. CONCLUSIONS:Hypothermia after OGD stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. Investigating the neuroprotective effect of EPO from astrocytes under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE.
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Project description:Alkylation of 2-bromoaniline with benzyl bromide under ostensibly basic N-alkylation conditions resulted in migration of bromine from the 2- to the 4-aryl position. Herein we report our studies to elucidate the mechanism of this rearrangement with the objective of suppressing this unexpected outcome. We find that careful choice of reagents is critical, and that this behavior may be extrapolated to alkylation reactions of electron-rich bromo- and iodoanilines in general.
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Project description:Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume-almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.
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Project description:Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection.
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Project description:Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.
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Project description:In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM?+?A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM?+?A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3?×?106 cells/ml vs 4.2?×?106 cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM?+?A (2.2?×?106 cells/ml vs 3.2?×?106 cells/ml, day 5). Supplementation with copper at 13.7-20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures.
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Project description:The isomerization of estragole to trans-anethole is an important reaction and is industrially performed using an excess of NaOH or KOH in ethanol at high temperatures with very low selectivity. Simple Ru-based transition-metal complexes, under homogeneous, ionic liquid (IL)-supported (biphasic) and "solventless" conditions, can be used for this reaction. The selectivity of this reaction is more sensitive to the solvent/support used than the ligands associated with the metal catalyst. Thus, under the optimized reaction conditions, 100% conversion can be achieved in the estragole isomerization, using as little as 4 × 10-3 mol % (40 ppm) of [RuHCl(CO)(PPh3)3] in toluene, reflecting a total turnover number (TON) of 25 000 and turnover frequencies (TOFs) of up to 500 min-1 at 80 °C. Using a dimeric Ru precursor, [RuCl(μ-Cl)(η3:η3-C10H16)]2, in ethanol associated with P(OEt)3, a TON of 10 000 and a TOF of 125 min-1 are obtained with 100% conversion and 99% selectivity. These two Ru catalytic systems can be transposed to biphasic IL systems by using ionic-tagged P-ligands such as 1-(3-(diphenylphosphanyl)propyl)-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide immobilized in 1-(3-hydroxypropyl)-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl) imide with up to 99% selectivity and almost complete estragole conversion. However, the reaction is much slower than that performed under solventless or homogeneous conditions. The use of ionic-tagged ligands significantly reduces the Ru leaching to the organic phase, compared to that in reactions performed under homogeneous conditions, where the catalytic system loses catalytic performance after the second recycling. Detailed kinetic investigations of the reaction catalyzed by [RuHCl(CO)(PPh3)3] indicate that a simplified kinetic model (a monomolecular reversible first-order reaction) is adequate for fitting the homogeneous reaction at 80 °C and under biphasic conditions. However, the kinetics of the reaction are better described if all of the elementary steps are taken into consideration, especially at 40 °C.
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