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  • 1. Calles, Karin
    et al.
    Eriksson, Ulrika
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.2006Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, nr 3, s. 653-659Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.

  • 2. Calles, Karin
    et al.
    Svensson, Ingrid
    Lindskog, Eva
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity2006Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, nr 2, s. 394-400Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages > 100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.

  • 3. Doverskog, M.
    et al.
    Jacobsson, U.
    Chapman, B. E.
    Kuchel, P. W.
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective H-1/N-15 NMR in vitro assay2000Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, nr 1, s. 87-97Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This is the second of two papers [Drews, M., Doverskog, M., Qhman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Haggstrom, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified H-1/N-15 spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-N-15] glutamine was selectively incorporated into 2-oxoglutarate forming [2-N-15] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-N-15] glutamate min (-1) (mg total protein)(-1) in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.

  • 4.
    Doverskog, Magnus
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Bertram, Eva.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ljunggren, Jan
    Öhman, Lars
    Sennerstam, Roland
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity2000Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 16, nr 5, s. 837-846Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (mu(N,max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining mu(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase mu(N,max), decrease the time to reach mu(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean;population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

  • 5. Drews, M.
    et al.
    Doverskog, M.
    Ohman, L.
    Chapman, B. E.
    Jacobsson, U.
    Kuchel, P. W.
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR2000Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 78, nr 1, s. 23-37Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutamine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. H-1/N-15 NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorporated into alanine. (NH4+)-N-15 was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that the [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

  • 6.
    Eriksson, Ulrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Hassel, Jenny
    Lüllau, Elke
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures2005Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, nr 1, s. 76-86Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B 1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45 kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25 kDa, as well as precursor forms above 48 kDa. Metalloproteinase bands below the main band at 48 kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor DL-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10 kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.

  • 7.
    Eriksson, Ulrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Identification of autocrine factors influencing proliferation in serum-free cultures of Trichoplusia ni cells2005Ingår i: Animal Cell Technology Meets Genomics / [ed] Godia, F; Fussenegger, M, 2005, s. 133-135Konferensbidrag (Refereegranskat)
    Abstract [en]

    The aim of this study is to understand how proliferation of Trichoplusia ni cells is regulated in serum-free cultures. The hypothesis is that T ni (Hi5) cells produce extracellular factors, which influence growth and productivity. To study this, the effect of conditioned medium (CM) on cell growth was investigated. Addition of 10 - 20% CM shortened the lag phase and increased the maximum cell density. CM was further concentrated and fractionated on a gel filtration column. Fractions which either inhibit or stimulate proliferation have been identified. These results suggest that extracellular protein factors are involved in regulation of proliferation.

  • 8.
    Eriksson, Ulrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Yeast extract from express five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells2005Ingår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 27, nr 20, s. 1623-1627Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The yeast extract (of unknown origin) present in the commercially available serum-free medium 'Express Five' contains factors ('yeast extract factors') up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors. The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components, which seem to be generally present in yeast extracts, are also required for T. ni proliferation.

  • 9. Han, L.
    et al.
    Doverskog, M.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by C-13 NMR spectroscopy2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 92, nr 3, s. 237-249Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia colt from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610)(.)3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO2 produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-C-13] labelled glycine, and the redistribution of label in the [1-C-13], [2-C-13], and [1,2-C-13] isotopomeres of excreted acetate was analysed by C-13 NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-C-13] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.

  • 10. Han, L.
    et al.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch2002Ingår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 24, nr 6, s. 483-488Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l(-1) (Han L, Doverskog M, Enfors S-O, Haggstrom L, 2002, J. Biotechnol. 92: 237-249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.

  • 11. Han, L.
    et al.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Häggström, Lena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components2003Ingår i: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 25, nr 4, s. 205-212Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO2 and acetate was similar to90% at the end of the culture (25 h, 27 g L-1 dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at similar to1 g L-1) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.

  • 12.
    Lindskog, Eva
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Eriksson, Ulrika
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Extracellular proteolytic activity innon-infected Sf9 and Trichoplusia ni Hi5 insect cell cultures: Implications for proliferation.Manuskript (preprint) (Övrigt vetenskapligt)
  • 13.
    Lindskog, Eva
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Svensson, Ingrid
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells2006Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 71, nr 4, s. 444-449Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

  • 14.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Cell-derived proteins and their role in proliferatin of protein-free NS0 cell culturesArtikel i tidskrift (Refereegranskat)
  • 15.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Conditioned medium factors in protein-free cultures of NSO cells2005Ingår i: Animal Cell Technology Meets Genomics / [ed] Godia, F; Fussenegger, M, 2005, s. 143-145Konferensbidrag (Refereegranskat)
    Abstract [en]

    An NS0 myeloma cell line has successfully been adapted to a protein-free medium without animal-derived components. The antibody yield in this medium is almost three-fold that in a traditional serum-medium. Kinetic studies of specific growth rate and specific product formation show that the antibody production in the serum-medium is strongly coupled to cell division while in the protein-free medium it is not. We propose that the large difference in growth and productivity profiles is due to the difference in supply and origin of growth factors. In the serum-medium proliferation is dependent on and related to factors present in the serum while in the protein-free medium the cells have adapted to an environment without serum-derived factors. Instead, the cells have started to produce their own factors through which they control their proliferation. We are currently trying to identify and characterise such conditioned medium factors and so far we have discovered at least one factor at about 20 kDa with a clear positive effect on cell growth.

  • 16.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Defined protein and animal component-free NS0 fed-batch culture2007Ingår i: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 98, nr 6, s. 1183-1194Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a stepwise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for nonmeasurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L-1 was obtained as compared to 2.3 X 10(6) cells mL(-1) and 70 mg antibody L-1 in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6-fold increase in total cell yield, a 10.5-fold increase 6.2 in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not i significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.

  • 17.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO).
    Defined protein-free NS0 myeloma cell cultures: stimulation of proloferation by conditioned medium factors2005Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 21, nr 1, s. 87-95Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, β-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 × 106 cells mL -1 in this medium. The antibody yield was 195 mg L-1 in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 × 10 6 cells mL-1, had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.

  • 18.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Proliferation of NSO cells in protein-free medium: The role of cell-derived proteins, known growth factors and cellular receptors2009Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 141, nr 3-4, s. 123-129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    NSO cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to Support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may Open for the novel approaches to improve animal cell processes. The following proteins were identified in NSO conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6,TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11. IL-25, MIF, TGF-beta and TNF-beta as well as the known growth factors EGF, IGF-1/11, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NSO cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NSO cell membranes since TGF-beta(1) caused cell death, and IGF-1/11 and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp 130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NSO cells as demonstrated by siRNA gene silencing.

  • 19.
    Spens, Erika
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Protease activity in protein-free NSO myeloma cell cultures2005Ingår i: In vitro Cellular & Developmental Biology-Animal, ISSN 1071-2690, E-ISSN 1543-706X, Vol. 41, nr 10, s. 330-336Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that thiscell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identifiedas aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular massesof 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using anenzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid andcysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product oraffecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, butaddition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibodyproduct was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acidproteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM,one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotininsignificantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminalamino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitorand cystatin C.

  • 20.
    Svensson, Ingrid
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Calles, Karin
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Lindskog, Eva
    KTH, Skolan för bioteknologi (BIO).
    Henriksson, Hongbin
    KTH, Skolan för bioteknologi (BIO).
    Eriksson, Ulrika
    KTH, Skolan för bioteknologi (BIO).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells2005Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 69, nr 1, s. 92-98Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

  • 21.
    Svensson, Marie
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Han, Ling
    KTH, Skolan för bioteknologi (BIO).
    Silfversparre, Gustav
    KTH, Skolan för bioteknologi (BIO).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO).
    Enfors, Sven-olof
    KTH, Skolan för bioteknologi (BIO).
    Control of endotoxin release in Escherichia coli fed-batch cultures2005Ingår i: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, nr 2, s. 91-97Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 degrees C at specific growth rates approaching 0.05 h(-1). Endotoxin analyses from a 20 degrees C GLFB culture gave similar results. An alternative fermentation technique, the temperature-limited fed-batch (TLFB) technique, reduced the endotoxin concentration in a culture with a biomass concentration of 30 g l(-1) from the 850 mg l(-1) in traditional GLFB cultures to about 20 mg l(-1). The TLFB technique uses the temperature to regulate the dissolved oxygen tension, while all substrate components are unregulated. It appears to be severe glucose limitation that triggers the extensive release of endotoxins rather than a low growth rate. Furthermore, it is not the low temperature that stabilizes the OM when using the TLFB technique. Simulations and experimental data show that this technique results in the same biomass productivity as the GLFB technique.

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