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  • 1. Anasontzis, George E.
    et al.
    Pena, Margarita Salazar
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Olsson, Lisbeth
    Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris2014In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 30, no 3, p. 728-735Article in journal (Refereed)
    Abstract [en]

    Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

  • 2.
    Brechmann, Nils Arnold
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Eriksson, Per-Olov
    Eriksson, Kristofer
    Oscarsson, Sven
    Buijs, Jos
    Shokri, Atefeh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Hjälm, Göran
    Chotteau, Véronique
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture2019In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033Article in journal (Refereed)
    Abstract [en]

    High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development

    of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified

    CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding

    capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an

    IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step

    from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were

    obtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scale

    purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,

    where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein

    A capture step, the mAb purity was similar to the one obtained by column chromatography, while

    the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead

    mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient

    single step, which directly connected the culture to the downstream process without cell clarification.

    Purification of mAb directly from non-clarified cell broth without cell separation can provide

    significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

  • 3. Calles, Karin
    et al.
    Eriksson, Ulrika
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 3, p. 653-659Article in journal (Refereed)
    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.

  • 4. Calles, Karin
    et al.
    Svensson, Ingrid
    Lindskog, Eva
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Häggström, Lena
    Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 2, p. 394-400Article in journal (Refereed)
    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.

  • 5.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO).
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO).
    Samani, Puneeth K.
    KTH, School of Biotechnology (BIO).
    Lindskog, Eva
    Fäldt, Eric
    Walsh, Kieron
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactorpart II: Applications for antibody production and cryopreservation2013In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, no 3, p. 768-777Article in journal (Refereed)
    Abstract [en]

    A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 x 108 cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28x more in a 1-month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 x 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing.

  • 6.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO).
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO).
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lindskog, Eva
    Walsh, Kieron
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process2013In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, no 3, p. 754-767Article in journal (Refereed)
    Abstract [en]

    High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor using external hollow fiber filter as cell separation device. Both classical tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20-35 x 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9-1.3 x 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 x 108 cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 x 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 x 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line.

  • 7.
    Doverskog, Magnus
    et al.
    KTH, Superseded Departments, Biotechnology.
    Bertram, Eva.
    KTH, Superseded Departments, Biotechnology.
    Ljunggren, Jan
    Öhman, Lars
    Sennerstam, Roland
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity2000In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 16, no 5, p. 837-846Article in journal (Refereed)
    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.

  • 8. Jahic, Mehmedalija
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Charoenrat, Theppanya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Process technology for production and recovery of heterologous proteins with Pichia pastoris2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 6, p. 1465-1473Article, review/survey (Refereed)
    Abstract [en]

    Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.

  • 9.
    Spens, Erika
    et al.
    KTH, School of Biotechnology (BIO).
    Häggström, Lena
    KTH, School of Biotechnology (BIO).
    Defined protein-free NS0 myeloma cell cultures: stimulation of proloferation by conditioned medium factors2005In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 21, no 1, p. 87-95Article in journal (Refereed)
    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.

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