首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 °C to 32 °C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 °C, at 32 °C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

2.
The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.  相似文献   

3.
The effect of different cell culture conditions on N‐glycosylation site‐occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t‐PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N‐glycosylation site that is variably occupied. Different environmental factors that affect the site‐occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t‐PA (type I t‐PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site‐occupancy of t‐PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site‐occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site‐occupancy of t‐PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N‐glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site‐occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N‐glycan site‐occupancy within a specific range. An increase in culture pH correlated with a decrease in site‐occupancy. Similarly, delaying the timing for butyrate addition also decreased site‐occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N‐glycan site‐occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.  相似文献   

4.
The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1‐checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2015;112: 141–155. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.  相似文献   

5.
Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.  相似文献   

6.
Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q P with increasing cell densities up to a threshold of >1 × 108 cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q P any further. A positive linear correlation (r 2 = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 106 to 3 × 108 cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.  相似文献   

7.
Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.  相似文献   

8.
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-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma 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 UDP-sugar 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.  相似文献   

9.
Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.  相似文献   

10.
Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β‐IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β‐IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL‐supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein‐assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5–1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β‐IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:584–593, 2014  相似文献   

11.
Effects of amino acid additions on ammonium stressed CHO cells   总被引:7,自引:0,他引:7  
Ammonium is a toxic and inhibitory byproduct of mammalian cell metabolism. At the end of a typical recombinant protein production campaign, the ammonium concentration can be as high as 10 mM, mainly due to glutamine metabolism. Intracellular pH (pH(i)) levels are sensitive to ammonium, which negatively impacts both cell growth and recombinant protein productivity. Ammonium also negatively affects the recombinant protein glycosylation profile, thus altering quality. Many strategies have been adopted to reduce ammonium accumulation, with limited results. This study investigated the addition of amino acids to the growth media for Chinese hamster ovary (CHO) cell cultures as a means of mitigating the negative effects of ammonium. Threonine, proline, and glycine additions improved CHO cell growth and recombinant protein levels. Further, the threonine, proline, and glycine additions positively impacted important metabolic parameters, including glucose consumption, lactate production, glutamine utilization, and final ammonium levels. Additionally, threonine, proline, and glycine increased the level of alpha(2,3)-linked sialic acid, galactose-beta(1,4)-N-acetylglucosamine, and alpha(2,6)-linked sialic acid residues on the recombinant tissue plasminogen activator (t-PA). Thus, threonine, proline, and glycine can be used to mitigate some of the toxic effects of ammonium on cell growth, recombinant protein productivity, and protein quality.  相似文献   

12.
We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.  相似文献   

13.
A kinetic-metabolic model approach describing and simulating Chinese hamster ovary (CHO) cell behavior is presented. The model includes glycolysis, pentose phosphate pathway, TCA cycle, respiratory chain, redox state and energetic metabolism. Growth kinetic is defined as a function of the major precursors for the synthesis of cell building blocks. Michaelis–Menten type kinetic is used for metabolic intermediates as well as for regulatory functions from energy shuttles (ATP/ADP) and cofactors (NAD/H and NADP/H). Model structure and parameters were first calibrated using results from bioreactor cultures of CHO cells expressing recombinant t-PA. It is shown that the model can simulate experimental data for all available experimental data, such as extracellular glucose, glutamine, lactate and ammonium concentration time profiles, as well as cell energetic state. A sensitivity analysis allowed identifying the most sensitive parameters. The model was then shown to be readily adaptable for studying the effect of sodium butyrate on CHO cells metabolism, where it was applied to the cases with sodium butyrate addition either at mid-exponential growth phase (48 h) or at the early plateau phase (74 h). In both cases, a global optimization routine was used for the simultaneous estimation of the most sensitive parameters, while the insensitive parameters were considered as constants. Finally, confidence intervals for the estimated parameters were calculated. Results presented here further substantiate our previous findings that butyrate treatment at mid-exponential phase may cause a shift in cellular metabolism toward a sustained and increased efficiency of glucose utilization channeled through the TCA cycle.  相似文献   

14.
The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.  相似文献   

15.
A primary objective of cell line development and process optimisation in animal cell culture is the improvement of culture performance as indicated by desirable properties such as high cell concentration, viability, productivity and product quality. The inefficient energy metabolism of mammalian cells in culture is still a major limiting factor for improvements in process performance. It results in high uptake rates of glucose and glutamine and the concomitant accumulation of waste products which in turn limits final cell concentrations and growth. To avoid these negative side effects, a CHO host cell line was established recently which is able to grow in completely glutamine free medium (Hernandez Bort et al., 2010). To determine the influence of this adaptation on productivity and product quality, the same procedure was repeated with a recombinant CHO cell line producing an erythropoietin-Fc fusion protein (CHO-EpoFc) for this publication. After adaptation to higher cell densities and glutamine free medium, culture performance was monitored in batch bioprocesses and revealed comparable growth properties and EpoFc product formation in both cell lines. The level of reactive oxygen species was elevated in the adapted cells, reflecting a higher level of oxidative stress, however, at the same time the level of the oxido-protective glutathione was also higher, so that cells seem adequately protected against cellular damage. Analysis of nucleotides and nucleotide sugars revealed elevated UDP-sugars in cells grown in the absence of glutamine. Furthermore, the antennarity of N-glycans was moderately higher on the Epo part of the protein produced by the adapted cell line compared to the parental cell line. Except for this, the glycosylation, with respect to site occupancy, degree of sialylation and glycoform structure, was highly comparable, both for the Epo and the Fc part of the protein.  相似文献   

16.
The use of a temperature switch to control the growth and productivity of temperature-sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39 degrees C to select mutagenized CHO-K1 cells, which resulted in the isolation of 31 temperature-sensitive mutants that were growth inhibited at 39 degrees C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1-B1) that maintains the ts phenotype.Continuous exposure to the nonpermissive temperature (npt) of 39 degrees C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34 degrees C and 39 degrees C arrested the 5G1-B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth-arrested cells was 3-4 times that of control cultures maintained at a constant 34 degrees C over the crucial 72-130-h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1-B1 cells arrested in the G(1) or G(0) phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime.These results demonstrate that growth-arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. (c) 1993 John Wiley & Sons, Inc.  相似文献   

17.
Shear stress in suspension culture was investigated as a possible manipulative parameter for the control of glycosylation of the recombinant tissue-type plasminogen activator protein (r-tPA) produced by recombinant Chinese hamster ovary (CHO) cell culture, grown in protein-free media. Resulting fractions of partially glycosylated, Type II, and fully glycosylated, Type I, r-tPA protein were monitored as a direct function of the shear characteristics of the culture environment. The shear-induced response of CHO culture to levels of low shear stress, where exponential growth was not obtained, and to higher levels of shear stress, which resulted in extensive cell death, were examined through manipulation of the bioreactor stirring velocity. Both apparent and intrinsic cell growth, metabolite consumption, byproduct and r-tPA production, and r-tPA glycosylation, from a variable site-occupancy standpoint, were monitored throughout. Kinetic analyses revealed a shear-stress-induced alteration of cellular homeostasis resulting in a nonlinear dependency of metabolic yield coefficients and an intrinsic cell lysis kinetic constant on shear stress. Damaging levels of shear stress were used to investigate the shear dependence of cell death and lysis, as well as the effects on the intrinsic growth rate of the culture. Kinetic models were also developed on the basis of the intrinsic state of the culture and compared to traditional models. Total r-tPA production was maximized under moderate shear conditions, as was the viable CHO cell density of the culture. However, Type II r-tPA production and the fraction of Type II glycoform production ratio was maximized under damaging levels of shear stress. Analyses of biomass production yield coefficients coupled with a plug-flow reactor model of glycan addition in the endoplasmic reticulum (ER) were used to propose an overall mechanism of decreased r-tPA protein site-occupancy glycosylation with increasing shear stress. Decreased residence time of r-tPA in the ER as a result of increased protein synthesis related to shear protection mechanisms is proposed to limit contact of site Asn184 with the membrane-bound oligosaccharyltransferase enzyme in the ER.  相似文献   

18.
Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.  相似文献   

19.
Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half-life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed-batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high-mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2-F-peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.  相似文献   

20.
The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号