Passive release of monoclonal antibodies from hybridoma cells

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed.Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.Abbreviations MCAB monoclonal antibodies - PBS phosphate buffered saline - RT room temperature - SDS sodium dodecyl sulphate     

Dilution rate increases production of plant cell-wall degrading enzymes by anaerobic fungi in continuous-flow culture

The gut anaerobic fungi,Neocallimastix hurleyensis and aOrpinomyces sp., were grown in 100 mL batch and continuous-flow cultures on wheat straw at a concentration of 80 g dry matter/L of culture liquid. In batch cultures,N. hurleyensis and Orpinomyces sp. degraded only ca. 9% and 5% of the wheat straw, respectively. In continuous-flow cultures, however, the two fungi degraded 52-56% of the apparent dry matter of wheat straw. Both fungi were able to produce greater quantities (up to x 30) of cell-wall degrading enzymes (CMCase, xylanase, beta-glucosidase and beta-xylosidase) in continuous-flow cultures than in the corresponding batch cultures. Increasing the dilution rate in continuous-flow culture resulted in the production of increased enzyme activity for all the measured cell-wall degrading enzymes, with proportional relationships between dilution rate and the cumulative activities of beta-glucosidase and beta-xylosidase. Dilution rates, however, had no consistent effect on the cumulative production of the fermentation end-products, acetate, formate, D- and L-lactate from both fungi. In addition to acetate and formate,N. hurleyens is produced D- and L-lactate in both batch and continuous-flow cultures, whereas only trace amounts of L-lactate were detected in the Orpinomyces sp. cultures.     

Improved fermentative production of Monascus pigments in roller bottle culture

The quantities and qualities of Monascus pigments produced by the filamentous fungus Monascus anka in batch submerged, agar surface, and roller bottle cultures were compared. In roller bottles, the fungus became attached to the wall of the culture vessels and produced a larger quantity of both intracellular (1508 absorbance units g⁻¹ cell mass) and extracellular (27 absorbance units g⁻¹ cell mass) Monascus red pigments, a yield that was about 10-fold greater than that of batch submerged and agar surface cultures. The optimum time required for maximum pigment production was reduced from 7 days in batch submerged or agar surface cultures to 4 days in roller bottle culture. In the roller bottle culture, the ratio of red to yellow pigments was also greatly increased. The advantage of the rotating vessel might be due to a combination of factors, including better gas exchange, higher medium pH, efficient pigment secretion, solid support for mycelium, and retarded conidiation.     

Inhibition of methicillin-resistant Staphylococcus aureus by an in vitro continuous-flow culture containing human stool microflora

We used an in vitro continuous-flow culture model of human stool microflora to examine the ability of human stool microflora to inhibit growth of two methicillin-resistant S. aureus (MRSA) strains. Continuous-flow cultures consistently eliminated MRSA inocula of 10(6) cfu/mL within 4 days, and addition of continuous-flow culture resulted in elimination of a pre-established MRSA culture ( approximately 10(8) cfu/mL) within 6-8 days. Anaerobic or "aerobic" (i.e., continuous bubbling of room air to eliminate obligate anaerobes) cultures eliminated MRSA at similar rates. The MRSA strains were unable to replicate under anaerobic conditions in sterile filtrates produced from the continuous-flow culture, but rapid growth occurred when glucose was added. These data demonstrate that indigenous stool microflora efficiently eliminate MRSA colonization and obligate anaerobes are not essential for inhibition. Our findings also suggest that inhibition of MRSA in continuous-flow cultures is due to depletion of nutrients rather than production of inhibitory conditions.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

Influence of ethanol on the hopanoid content and the fatty acid pattern in batch and continuous cultures of Zymomonas mobilis

By thin layer chromatographic, gas-liquid chromatographic, and mass spectrometric methods 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) was shown to occur in Zymomonas mobilis. This compound contributed up to 20% to the total lipids.The fatty acid pattern and the content of hopanoids (hopene, hopanol, and THBH) were determined in batch and continuous cultures. In late exponential cells from batch cultures the relative amount of palmitic acid was increased partially at the expense of cis-vaccenic acid, when the initial glucose concentrations were increased. In a batch culture, THBH reached a maximum value in the early exponential growth phase.In an anaerobic continuous culture with a low glucose feed concentration, the THBH content and the relative amount of cis-vaccenic acid were low. The contribution of both compounds increased strongly with increasing glucose feed concentrations (i.e. at higher steady-state ethanol concentrations). The same result was found with aerobic continuous cultures which produced significant amounts of acetaldehyde and acetic acid, in addition to ethanol and carbon dioxide.It was concluded that stability and permeability of the cytoplasmic membrane of the ethanol producing bacterium Z. mobilis was regulated by variations in the distribution of hopanoids and fatty acids.Abbreviations 14:0 myristic acid - 16:0 palmitic acid - 18:1 cisvaccenic acid - THBH 1,2,3,4-tetrahydroxypentane-29-hopane     

Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.     

Inhibition of caspase activity delays apoptosis in a transfected NS/0 myeloma cell line

The productivity of NS/0 myeloma batch cultures is often compromised by the premature induction of apoptosis, now established to be the predominant method of cell death during culture decline. Caspase proteases have recently been shown to play a major role in the transmission of signals for apoptotic cell death. Using a specific inhibitor that targets a range of caspases (Z-VAD-fmk) we assessed whether inhibition of caspase activity could prolong the viability of NS&vbar;h=0 cells under conditions that cause apoptotic cell death in batch cultures. Z-VAD-fmk was found to significantly reduce apoptotic cell death (by approximately 50%) induced by cytotoxins and to preserve membrane integrity to a similar extent. In conditions of low serum, Z-VAD-fmk reduced certain features of apoptosis (e.g., DNA fragmentation), but only marginally improved viability. In medium-depleted batch cultures, Z-VAD-fmk afforded a delay of between 24 and 48 h in both the induction of apoptosis and loss of viability. Despite an apparent increase in viability in Z-VAD-fmk-treated NS&vbar;h=0 cultures, no improvement in productivity could be demonstrated, suggesting that at least some normal pathways for protein production are shut down upstream of caspase activation. An examination of mitochondrial membrane potential (Deltapsim) in Z-VAD-fmk-treated and untreated NS&vbar;h=0 cells revealed only a small initial difference (5%) in the levels of Deltapsim depolarization. Similar levels of mitochondrial dysfunction, despite caspase inactivity, may therefore be responsible for the comparable productivity in untreated and Z-VAD-fmk-treated cultures. Thus, this study suggests that, while a delay in cell death due to caspase inhibition may reduce problems associated with cellular disintegration, it does not permit productivity improvements in this type of culture.     

Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture

Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.     

Production of L- AND D-lactate by Pediococcus pentosaceus in batch and steady-state continuous culture

The fast growth and acid production of a strain of Pediococcus pentosaceus , used as a starter culture in the production of dry sausages, was dependent on the presence of acetate. In a batch culture on a mixture of glucose and sucrose both sugars were consumed simultaneously. Similar growth rates and product yields were obtained on glucose and sucrose, d - AND l -lactate were produced via a D- and L-lactate dehydrogenase (LDH), respectively, and no racemase was present. In batch cultures about 15% of the lactic acid produced was the D-isomer, whereas in a sucrose-limited, continuous culture the fraction of D-lactic acid increased with decreasing dilution rate. The results are discussed in relation to the two LDH activities.     

Enhancement of productivity of recombinant α-amidating enzyme by low temperature culture

We have produced a recombinant C-terminal α-amidating enzyme (799BglIIα-AE) derived from Xenopus laevis by culturing a CHO cell line named 3μ-1S. Recently, we demonstrated that culturing 3μ-1S cells at a temperature below 37 °C led to the following phenomena: inhibited cell growth with high viability, enhanced cellular productivity (maximally at 32 °C), and suppressed medium consumption and release of impurities from the cells. Therefore, it is suggested that the 799BglIIα-AE production will be increased by culturing a sufficient number of the cells at a low temperature (especially at 32 °C). To assess this effect on batch and perfusion cultures, the culture temperature was shifted from 37 to 32 °C in the mid-exponential phase in the case of batch culture and from 37 to 34 °C when the cell density became high enough in the case of perfusion culture. Application of the low temperature culture to batch and perfusion cultures was effective in comparison with the culture at 37 °C: the productivity per medium and the productivity per time were increased severalfold with enhanced cellular productivity at a low culture temperature. The low temperature culture also increased the relative content of 799BglIIα-AE in the supernatant and reduced the glucose consumption. The method presented here would contribute to production of bioactive proteins using other recombinant cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

The purification and characterization of wheat-germ agglutinin

The purification of wheat-germ agglutinin by precipitation with ammonium sulphate and by chromatography on Sephadex G-75, Sepharose-ovomucoid and CM-cellulose is described. This procedure gave agglutinin preparations which were homogeneous on polyacrylamide gels under a variety of conditions. Purified wheat-germ agglutinin formed colourless solutions and was relatively insoluble at neutral pH; maximum solubility in 1mm-tris-HCl buffer, pH7.4, was approx. 1mg/ml. The agglutinin was a glycoprotein containing a single polypeptide chain with an approximate molecular weight of 23000. The N-terminus of the oxidized agglutinin was cysteic acid and the C-terminus was glycine. The amino acid composition showed that the protein was extremely rich in cysteine and cystine; there were 15-17 free SH groups/mol. The absorption maximum for the protein was at 272nm and the molar extinction coefficient at 280nm was 1.09x10(5) litre.mol(-1).cm(-1). Equilibrium dialysis indicated that there was only one binding site per molecule for N-acetylglucosamine.     

Vero细胞微载体不同培养方法的评价

对三种不同培养方法进行细胞生长速度、密度、营养及代谢产物浓度的比较分析,以优化和筛选最佳培养条件与方式。用同体积生物反应罐,基本培养条件相同,采用批培养、再循环培养、灌流培养三种方式进行了Vero细胞微载体（CytodexI）的周期培养。三种培养方法均达到预期效果,最终细胞密度分别为每毫升2.09×10     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2

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.     

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO₂ into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO₂ sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO₂ sparging.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.