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  • 1. Ahlqvist, J.
    et al.
    Kumar, A.
    Sundstrom, H.
    Ledung, E.
    Hornsten, E. G.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mattiasson, B.
    Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 2, p. 216-225Article in journal (Refereed)
    Abstract [en]

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  • 2.
    Backlund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Ignatushchenko, Marina
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, no 1, p. 35-Article in journal (Refereed)
    Abstract [en]

    The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

  • 3.
    Basselet, Pascal
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wegrzyn, Grzegorz
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)2008In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes. Conclusion: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.

  • 4.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Growth rate control of periplasmic product retention in Escherichia coli2008Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium.

    The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention.

    We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate.

    We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm.

    The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions.

    This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment.

  • 5.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Impact of glucose uptake rate on recombinant protein production in Escherichia coli2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli.

    E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.  

    The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation.

    For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm.

    The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.

  • 6.
    Bäcklund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Markland, Katrin
    KTH, School of Biotechnology (BIO).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control2008In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, p. 11-20Article in journal (Refereed)
    Abstract [en]

    A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.

  • 7.
    Bäcklund, Emma
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Markland, Katrin
    KTH, School of Biotechnology (BIO).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Fedbatch design for periplasmic product retention in Escherichia coli2008In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 135, no 4, p. 358-365Article in journal (Refereed)
    Abstract [en]

    The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

  • 8.
    Calles, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures2005Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.

    Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.

  • 9. 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.

  • 10. 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.

  • 11.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, M.
    Stendahl-Andersen, Helle
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes2005In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, no 6, p. 399-406Article in journal (Refereed)
    Abstract [en]

    An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

  • 12.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 1, p. 86-98Article in journal (Refereed)
    Abstract [en]

    Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

  • 13. Charoenrat, Theppanya
    et al.
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process2006In: Biochemical engineering journal, ISSN 1369-703X, E-ISSN 1873-295X, Vol. 30, no 2, p. 205-211Article in journal (Refereed)
    Abstract [en]

    Two strategies to increase the productivity of secreted Thai Rosewood beta-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K(L)a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g(-1) g(-1)) and high-pressure processes (0.035 g g(-1) g(-1)), compared to 0.022 g g(-1) g(-1) in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total beta-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the beta-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.

  • 14. Cheon, Seungwoo
    et al.
    Kim, Hye Mi
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lee, Sang Yup
    Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels2016In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 35, p. 10-21Article in journal (Refereed)
    Abstract [en]

    As climate change has become one of the major global risks, our heavy dependence on petroleum-derived fuels has received much public attention. To solve such problems, production of sustainable fuels has been intensively studied over the past years. Thanks to recent advances in synthetic biology and metabolic engineering technologies, bio-based platforms for advanced biofuels production have been developed using various microorganisms. The strategies for production of advanced biofuels have converged upon four major metabolic routes: the 2-ketoacid pathway, the fatty acid synthesis (FAS) pathway, the isoprenoid pathway, and the reverse β-oxidation pathway. Additionally, the polyketide synthesis pathway has recently been attracting interest as a promising alternative biofuel production route. In this article, recent trends in advanced biofuels production are reviewed by categorizing them into three types of advanced biofuels: alcohols, biodiesel and jet fuel, and gasoline. Focus is given on the strategies of employing synthetic biology and metabolic engineering for the development of microbial strains producing advanced fuels. Finally, the prospects for future advances needed to achieve much more efficient bio-based production of advanced biofuels are discussed, focusing on designing advanced biofuel production pathways coupled with screening, modifying, and creating novel enzymes.

  • 15.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Fed-batch or perfusion for the production of biopharmaceuticals by animal cell cultivation?2009Other (Other academic)
    Abstract [en]

    Fed-batch or perfusion modes are today’s options for the cultivation process development of new candidates drugs. In the cases of unstable proteins, perfusion is the obligatory choice. Otherwise, this choice is dictated by the need of simplicity, the existing technical know-how in the company and the available equipment (e.g. bioreactor size, perfusion device, etc.). The higher risk of contamination and higher technical challenge of perfusion processes lead often to the selection of fed-batch for commercial processes. This is reinforced today in the case of antibody production in established biotech companies by the use of generic fed-batch processes with high yield production and low accumulation of toxic by-products (e.g. lactate, ammonia). However perfusion can be a valuable option due to the usage of smaller bioreactors, today’s availability of scalable low shear force perfusion system (e.g. ATF) and potentially the lower requirement of process development for perfusion. Perfusion can therefore be an attractive choice for instance for the production of glycoproteins, which are not antibody, and/or for small companies, which do not have generic process.

  • 16.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mastering Oxygen and Carbon Dioxide in Cell Culture2010Other (Other academic)
    Abstract [en]

    Mammalian cells need adequate aeration for the cellular respiration and simultaneously produce carbon dioxide. Aeration in pilot and large scale cultivation is operated by blowing oxygen or oxygen enriched air in the culture, i.e. sparging. The gas bubbles reaching the surface generate foam formation, which is well known to be damageable for the cells. Therefore, it can be preferable to reduce the bubble sparging while maintaining the oxygen concentration in the culture. Carbon dioxide produced by the cells can accumulate in the culture in case its removal rate from the liquid phase is not fast enough. This provokes an acidification of the medium, which has to be compensated by alkali addition. High levels of carbon dioxide and high levels of alkali can be damageable for the process performances. A common way to actively remove the carbon dioxide from the liquid phase is to benefit from the bubbles moving to the liquid surface. Obviously, large scale cultivation can present a dilemma in which reduced sparging is favoured for the aeration strategy in order to avoid cell damage by excessive foaming while increased sparging provide reduced accumulation of carbon dioxide. Typical approaches used in the biopharmaceutical industry for aeration and carbon dioxide removal will be reviewed in the presentation. 

  • 17.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2008In: Proceedings of the Cell Culture Engineering XI Conference, 2008Conference paper (Refereed)
    Abstract [en]

    Achieving satisfying DO and pH controllers are often challenges for pilot and large scale mammalian cultivation. Unsatisfactory DO or pH controls can imply fatal effects for the culture. Large scale bioreactors have long response times due to long mixing times compared to small scale systems where control tuning of DO and pH is not so challenging.

    A method was developed to tune the DO controller PID parameters of a 50 L bioreactor (wv) controlled by a continuous oxygen flow of microbubbles. DO control by continuous flow of pure oxygen microbubbles can oscillate quite widely showing instable behaviour. The method, called lag control here, was based on a lead lag control design by Bode analysis where the prediction part, i.e. ‘lead’ part was omitted. A comparison of this method with a pole placement approach showed the advantage of the lag control. It was decided to omit the derivate part which could lead to instability caused by the long delay observed between the applied oxygen flow and the response detected by the DO probe. Applying the lag control method resulted in a highly satisfactory DO control. In this system, the oxygen microbubbles were almost completely consumed before reaching the liquid surface as demonstrated by the absence of foam. So the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations of this system, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. The method was first tested with the sulphite oxidation method simulating the oxygen consumption with copper as catalyst to establish the PID parameters. Then the selected parameters were successfully used during cell cultivation. Following this, an adaptation of the method was done in order to avoid the sulphite oxidation method, which leaves copper traces in the bioreactor. This was successfully used in a 400 L bioreactor (wv) for the DO controller by continuous oxygen flow of microbbubles. The lag controller method was finally modified to tune the pH controller of the same 400 L bioreactor with control upward by alkali addition or downwards by pulsed carbon dioxide addition.

  • 18.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jiang, Yun
    Biovitrum/SOBI, Sweden.
    westin, Jeanette
    Biovitrum/SOBI, Sweden.
    Dahlenborg, K
    Biovitrum/SOBI, Sweden.
    Sjöblom-Hallén, A
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Öberg, Mikael
    Biovitrum/SOBI, Sweden.
    Development of a fed-batch process for the production of a recombinant protein X in CHO-GS system: Case study from the cell to reactor process ready for pilot scale cultivation2010In: Cells and Culture: Proceedings of the 20th ESACT / [ed] Noll T, Springer Science+Business Media B.V., 2010, p. 723-725Conference paper (Other academic)
    Abstract [en]

    A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.

  • 19.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lindqvist, Anna
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Study of the effect of high pH and alkali addition in a cultivation of Chinese Hamster Ovary cell2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT) / [ed] Jenkins, Nigel; Barron, Niall; Alves, Paula, Springer Science+Business Media B.V., 2012, p. 323-326Conference paper (Refereed)
    Abstract [en]

    This work aimed at studying the impact of alkali addition in a Chinese Hamster Ovary cell culture. Two phenomena were studied, the kinetic rate of direct cell death in presence of high pH and the effect of transitory single contact of high pH on cell viability and growth. Contact with pH 11 or 10 did not provoke immediate cell lysis. The cells survived several minutes to such conditions. Contact with pH 11 during 2 minutes, with pH 10 during 5 minutes, with pH 9 during 5 minutes or 10 minutes did not affect the viability. In these conditions, the growth was not affected except after 5 minutes contact at pH 10 or 10 minutes contact at pH 9 for which the growth was slowed down the first day only. As expected, NaOH addition affected the cells more than Na2CO3 addition. This was due to a higher pH but could be even observed at the same pH (10).

  • 20.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tördahl, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perroud, Philip
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Study of a perfusion process of Chinese Hamster Ovary cells by ATF filtration in bioreactor2009Other (Other academic)
    Abstract [en]

    Perfusion is a mode of operation where a continuous replacement of the conditioned medium by fresh medium is operated. It has the advantage of allowing high cell densities. This mode of operations is also required for some instable proteins since the cell-free supernatant containing the product of interest is immediately stored at low temperature where the proteolysis is not active. The ATF filtration device has been designed to perfuse mammalian cell cultivation process. The cell broth circulation back and forward in the filter prevents the filter clogging and the design ensures a low shear not damageable for the cells.

    The purpose of the present work was to develop and study a perfusion process of Chinese Hamster Ovary cells producing a monoclonal antibody by ATF filtration in a 2 L working volume bioreactor. A serum-free medium was used (Irvine Scientific IS CHO-CD). Cell densities above 40 x 106 cells/mL were obtained. These high cell densities were challenging for the aeration. Pure oxygen aeration by large bubbles from an open tube resulted in satisfying oxygenation until 25 to 30 x 106 cells/mL but became limiting at higher cell densities due to the low kLa of these bubbles and the small liquid height. At higher cell densities, a porous sparger with pure oxygen was used either alone or in combination with the open tube aeration. The performances of the perfusion by ATF filtration, the aeration and the use of anti-foam are presented and discussed.

  • 21.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tördahl, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perroud, Philip
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Unthan, Simon
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Schmidt, Johannes RM
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Doverskog, Magnus
    IMED, AB, Solna, Sweden.
    Study of Alternating Tangential Flow filtration for perfusion and harvest in Chinese Hamster Ovary cells cultivation2010In: Proceedings of the Cell Culture Engineering Conference XII, April 25-30, 2010, Banff, Canada, 2010Conference paper (Other academic)
    Abstract [en]

    Perfusion is a mode of operation where a continuous replacement of the conditioned medium by fresh medium is operated. It has the advantage of allowing high cell densities. This mode of operations is also required for some instable proteins since the cell-free supernatant containing the product of interest is immediately stored at low temperature where the proteolysis is not active. The ATF filtration device, Alternating Tangential Flow, has been designed to perfuse mammalian cell cultivation process and is used (or studied) nowadays for applications like perfusion, medium renewal, harvest, etc. The cell broth circulation back and forward in the filter prevents the filter clogging and the design ensures a low shear not damageable for the cells.

    A perfusion process operated by ATF filtration and using CHO cells producing a monoclonal antibody was developed in a 2 L bioreactor. The medium did not contain animal derived components. Cell densities above 40 x 1E6 cells/mL were obtained with a perfusion rate of 2 reactor volume/day. The highest cell density observed was 48 x 1E6 cells/mL. These high cell densities were challenging for the aeration. Pure oxygen aeration by large bubbles from an open tube resulted in satisfying oxygenation until 25 to 30 x 1E6 cells/mL but became limiting at higher cell densities due to the low kLa of these bubbles and the small liquid height. At higher cell densities, a porous sparger with pure oxygen was used either alone or in combination with the open tube aeration. Automatic delivery of antifoam C and pluronic counteracted the effect of small bubble foam deleterious for the cells. From an operation point-of-view, the perfusion operated by the ATF device was satisfying, without filter fouling, easy to operate and to adjust in comparison with other separation devices by filtration or gravitation.

    Finally harvesting by ATF filtration was evaluated in comparison with ‘one-way’ tangential flow filtration, TFF, on an IgG producing CHO fed-batch process produced in 2 L bioreactor and in 20 L bioreactor. Different settings and filter sizes were compared and the effect of varying the flow rate was studied.

  • 22.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tördal, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perroud, Philip
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Unthan, Simon
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Schmidt, Johannes R.M.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Doverskog, Magnus
    IMED, AB, Solna, Sweden.
    Study of Alternating Tangential Flow filtration for perfusion and harvest in Chinese Hamster Ovary cells cultivation2010Other (Other academic)
    Abstract [en]

    Perfusion is a mode of operation where a continuous replacement of the conditioned medium by fresh medium is operated. It has the advantage of allowing high cell densities. This mode of operations is also required for some instable proteins since the cell-free supernatant containing the product of interest is immediately stored at low temperature where the proteolysis is not active. The ATF filtration device, Alternating Tangential Flow, has been designed to perfuse mammalian cell cultivation process and is used (or studied) nowadays for applications like perfusion, medium renewal, harvest, etc. The cell broth circulation back and forward in the filter prevents the filter clogging and the design ensures a low shear not damageable for the cells.

    A perfusion process operated by ATF filtration and using CHO cells producing a monoclonal antibody was developed in a 2 L bioreactor. The medium did not contain animal derived components. Cell densities above 40 x 106 cells/mL were obtained with a perfusion rate of 2 reactor volume/day. The highest cell density observed was 48 x 106 cells/mL. These high cell densities were challenging for the aeration. Pure oxygen aeration by large bubbles from an open tube resulted in satisfying oxygenation until 25 to 30 x 106 cells/mL but became limiting at higher cell densities due to the low kLa of these bubbles and the small liquid height. At higher cell densities, a porous sparger with pure oxygen was used either alone or in combination with the open tube aeration. Automatic delivery of antifoam C and pluronic counteracted the effect of small bubble foam deleterious for the cells. From an operation point-of-view, the perfusion operated by the ATF device was satisfying, without filter fouling, easy to operate and to adjust in comparison with other separation devices by filtration or acceleration.

     

    Finally harvesting by ATF filtration was evaluated in comparison with ‘one-way’ tangential flow filtration, TFF, on an IgG producing CHO fed-batch process produced in 2 L bioreactor. In both operation modes, ATF and TFF, filter fouling occurred after several minutes and the total process time was comparable but an important difference was that the viability drop obtained when using ATF was 15 % while it was 45 % using the TFF.

  • 23.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wang, Jingjiao
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tolf, Erika
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wicklund, Linn
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Hovatta, Outi
    Karolinska Institutet, Department of Clinical Science.
    Marutle, Amelia
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Comparison of cultivation in Techne spinner, Bellco spinner, shake flask and T-flask of human embryonic stem cells2010In: Proceedings of the SBE's Second International Conference on Stem Cell Engineering, 2010Conference paper (Other academic)
    Abstract [en]

    The recent progress in regenerative medicine indicates that pluripotent human embryonic stem cells (hESCs) may hold great potential providing cellular models for drug development and screening, modelling diseases as well as aid in the development of future cell-based therapies for neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Crucial to the success of generating specialized cell populations, is an understanding of the mechanisms, which influence the control of cell growth and differentiation by extrinsic and intrinsic factors. Nowadays, a limitation for the use of hESCs is the lack of proliferation methods in large scale. The purpose of the present work was to study several cultivation systems, which could potentially provide large-scale cultivation processes suitable for human therapy applications. Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst, were cultivated undifferentiated as embryoids bodies, i.e. large spherical aggregates of cells, in absence of serum and feeder layer. The cell growth and culture behavior in T-falsk, orbitally agitated shake flask, Bellco stirred spinner and Techne stirred spinner were observed. In Bellco spinner, the cells were agitated by a rotating impeller providing a movement comparable to stirred bioreactors. In Techne spinner, a slow and gentle orbital movement provided by a rotating bulb-ended stirrer maintained the cells in suspension. The design of this latter spinner allowed lower shear stress in comparison to Bellco spinner and shake flask. It was observed that the cell growth was fastest in Techne spinner followed by cultivation in T-flask and then cultivation in shake flask. Cultivating in Bellco spinner resulted in embryoid dissociation and viability decrease after 14 days. A larger number of single cells, i.e. cells not growing in aggregates, was observed in the static T-flask culture compared to the agitated systems, i.e. shake flask, Bellco spinner or Techne spinner. Probably the agitation promoted the spontaneous aggregation of the cells in spheres. In particular the Techne spinner allowed the most perfect spherical form among the different compared systems. Finally it was observed that hypoxia with 4 % oxygen concentration improved significantly the growth in Techne spinner or T-flask in comparison with normoxia with 21 % oxygen concentration. It was concluded that cultivation in Techne spinner under hypoxia was the most favorable condition among the ones studied here. The agitation provided by Techne spinner improved the cell growth in comparison with static system (T-flask). However using the other agitated systems, shake flask and Bellco spinner, was not comparably beneficial to the cell growth and viability, probably due to the higher shear stress of these systems compared to Techne spinner.

  • 24.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wåhlgren, Caroline
    Biovitrum/SOBI, Sweden.
    Pettersson, Helena
    Biovitrum/SOBI, Sweden.
    Effect of Peptones and Study of Feeding Strategies in a CHO Based Fed-batch Process for the Production of a Human Monoclonal Antibody2007In: Cell Technology for Cell Products: Proceedings of the 19th ESACT Meeting, Harrogate, UK, June 5-8, 2005 / [ed] Smith R, Dordrecht, The Netherlands: Springer Netherlands, 2007, p. 371-374Conference paper (Other academic)
    Abstract [en]

    Eight commercial peptones, derived from plants, were studied for their ability of improving the cell growth and the productivity of a CHO cell line producing a human monoclonal antibody. They were also compared to yeast, lactalbumin and meat derived peptones. Seven plant peptones were selected and further studied in combination by Design of Experiment. The best three peptones were then tested in combinations in fed-batch cultivation. The fed-batch process was based on low concentrations of glucose and glutamine with feeding of amino acids, peptones and feed medium including vitamins, metal traces and biosynthesis precursors. This process was based on Biovitrum protein-free proprietary medium for the base medium and the feeding medium. Different feeding strategies, different peptone combinations and phosphate feeding were studied for their ability to improve the cell density, the cell specific productivity and the cultivation longevity

  • 25.
    Chotteau, Véronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control. KTH, School of Electrical Engineering (EES), Centres, ACCESS Linnaeus Centre.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), 2012, p. 327-330Conference paper (Refereed)
    Abstract [en]

    A method has been developed to tune the DO and pH controller PID parameters for pilot / large scale mammalian cultivation. Our approach is to identify a model of the variable to be controlled (e.g. DO, pH) and to design several possible PID controllers based on this model. The controllers were first tested in computer simulations, followed by wet simulation and finally the best controller was tested on the real process. The approach is developed for the tuning of the DO controller of a 50 L bioreactor using microbubble continuous oxygen flow. The method, called lag control here, is based on a lead lag control design using Bode analysis where the prediction part is omitted. Experiments show that the approach results in a highly satisfactory DO control. The oxygen microbubbles were almost completely consumed before reaching the liquid surface so the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. This approach was also successfully used for the tuning of a 400 L bioreactor DO controller and pH controller.

  • 26.
    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.

  • 27.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Samani, Puneeth K
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Conference paper (Other academic)
    Abstract [en]

    Perfusion of an IgG producing CHO cell line was performed in a WAVE Bioreactor™ using either Alternating Tangential Flow or Tangential Flow Filtration. The properties and performances obtained with both filtration systems were compared. Very high cell densities were achieved and could be stably maintained. Then the cell density could be significantly further increased showing the capacity of the system set-up.

  • 28.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Samani, Puneeth K.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Conference paper (Other academic)
    Abstract [en]

    Perfusion of an IgG producing CHO cell line was performed in a WAVE Bioreactor™ using either Alternating Tangential Flow or Tangential Flow Filtration. The properties and performances obtained in this bioreactor with both filtration systems were studied.

    • Very high cell densities were achieved and could be stably maintained at high viability indicating of a healthy process suitable for instance for efficient cell banking.

    Cell density could be significantly further increased showing the capacity of the system set-up.

  • 29.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor™2011In: BMC Proceedings, 2011, Volume 5, Supplement 8, P105, BioMed Central, 2011, p. 105-Conference paper (Refereed)
    Abstract [en]

    Major advantages of perfusion are high cell numbers and high total production in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the product yield is low. On the other hand, disadvantages are for example technical challenges originating from non-robust cell separation devices as well as sterility concerns from the more complex set-up needed.

    In the present work, the use of a WAVE Bioreactor™ system 20/50 in perfusion mode with10 L disposable Cellbag™ bioreactors customized with two dip tubes in combination with disposable hollow fiber filters as external cell separating devices were investigated. A comparison between Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) was performed using a recombinant CHO cell line producing a monoclonal antibody (mAb) as a model system. 

  • 30.
    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.

  • 31.
    Eriksson, Ulrika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Hassel, Jenny
    Lüllau, Elke
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, no 1, p. 76-86Article in journal (Refereed)
    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.

  • 32.
    Eriksson, Ulrika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Identification of autocrine factors influencing proliferation in serum-free cultures of Trichoplusia ni cells2005In: Animal Cell Technology Meets Genomics / [ed] Godia, F; Fussenegger, M, 2005, p. 133-135Conference paper (Refereed)
    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.

  • 33.
    Eriksson, Ulrika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Yeast extract from express five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells2005In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 27, no 20, p. 1623-1627Article in journal (Refereed)
    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.

  • 34.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Developing nucleic acid-based electrical detection systems2006In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 5Article in journal (Refereed)
    Abstract [en]

    Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.

  • 35.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Liu, Yanling
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gene-based identification of bacterial colonies with an electric chip2005In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 2, p. 270-276Article in journal (Refereed)
    Abstract [en]

    A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.

  • 36.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

     

    A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

     

    Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

  • 37.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.

      

    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.

  • 38.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Optimisation of surface expression using the AIDA autotransporter2011In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

    Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

    Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

  • 39. Jahic, M.
    et al.
    Knoblechner, J.
    Charoenrat, T.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Interfacing Pichia pastoris cultivation with expanded bed adsorption2006In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 93, no 6, p. 1040-1049Article in journal (Refereed)
    Abstract [en]

    For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L (.) h.

  • 40. 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.

  • 41.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Improved detection and performance of surface expression from the AIDA-I autotransporter2013Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield.

    In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications.

  • 42.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Do, Thi-Huyen
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli2012In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, article id 118Article in journal (Refereed)
    Abstract [en]

    Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

  • 43. Kepka, C.
    et al.
    Collet, E.
    Roos, F.
    Tjerneld, F.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography2005In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1075, no 02-jan, p. 33-41Article in journal (Refereed)
    Abstract [en]

    In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to > 95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process. © 2005 Elsevier B.V. All rights reserved.

  • 44.
    Kuttuva Rajarao, Gunaratna
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Shokri, Atefeh
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Bhattacharya, Prosun
    KTH, School of Architecture and the Built Environment (ABE), Land and Water Resources Engineering (moved 20130630).
    Jacks, Gunnar
    KTH, School of Architecture and the Built Environment (ABE), Land and Water Resources Engineering (moved 20130630).
    Bundschuh, Jochen
    KTH, School of Architecture and the Built Environment (ABE), Land and Water Resources Engineering (moved 20130630).
    Von Brömssen, M.
    Microbial characterization of Holocene alluvial sediments in the Meghna Flood Plain of Matlab Upazila, Bangladesh2010In: Arsenic in Geosphere and Human Diseases, As 2010 - 3rd International Congress: Arsenic in the Environment, 2010, p. 140-142Conference paper (Refereed)
  • 45.
    Lakshmanan, Ramnath
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Okoli, Chuka
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology. KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Boutonnet, Magali
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology.
    Järås, Sven
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology.
    Kuttuva Rajarao, Gunaratna
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Effect of Magnetic Iron Oxide Nanoparticles for Surface Water Treatment: Trace Minerals and MicrobesManuscript (preprint) (Other academic)
  • 46.
    Larsdotter, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Microalgae for Phosphorus Removal from Wastewater in a Nordic Climate2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    As part of a research project aiming to develop and evaluate a hydroponic system for wastewater treatment in Sweden, extended nutrient removal by microalgae was tested. The hydroponic/microalgal wastewater treatment system was built in a greenhouse in order to improve growth conditions for plants and algae. Studies on the treatment step with microalgae showed that phosphorus removal could be successfully accomplished owing to the cmbined effect of phosphorus assimilation and biologically mediated chemical precipitation of calcium phosphates. This precipitation was mainly induced by the increased pH in the algal cultures, and the pH increase was in turn a result of the inorganic carbon assimilation by the algae. The results showed that the algal growth was mainly light limited which resulted in higher algal biomass density and also lowe residual nutrients in the water at longer hydraulic retention times (HRT). In contrast the phosphorus removal rate was load limited, i.e. shorter HRT gave higher removal rates. This load dependency was due to the chemical precipitation, whereas the phosphorus assimilation was dependent on algal growth. Furthermore, results from an intensive study during summer showed that culture depths of 17 cm gave higher removal efficiencies (78% - 92%) than cultures of 33 cm (66% - 88%). On the other hand, the removal rate per area was higher in the deeper cultures, which implies that these may be preferred if area is of concern.

    Nitrogen removal was achieved mainly by the assimilation of nitrate to algal biomass, and removal efficiencies of around 40% (nitrate) could be reached for most parts of the year although the nitrogen removal performance was quite uneven. Up to 60% - 80% could however be reached during summer in the shallow cultures. A net removal in total nitrogen of up to 40% was observed in the shallow cultures during summer, which was most probably a consequence of grazing zooplankton and subsequent urea excretion and ammonia volatilisation as a reslt of the high pH values.

    Over the year, there were large fluctuations in algal growth and removal efficiency as a result of the seasonal variations in light and tempeature. During winter, phosphorus removal efficiencies lower than 25% were observed in the shallow tanks and lower than 10% in the deep tanks. Additional illumination during winter improved the phosphorus removal in the shallow cultures but did not have a significant efect on the deep cultures. Such additional illumination increases the total energy demand of the system, and hence alternative methods for phosphorus removal during winter would probably be more economical unless the algal biomass roduced had great commercial value.

  • 47.
    Larsdotter, Karin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jansen, Jes La Cour
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Biologically mediated phosphorus precipitation in wastewater treatment with microalgae2007In: Environmental technology, ISSN 0959-3330, E-ISSN 1479-487X, Vol. 28, no 9, p. 953-960Article in journal (Refereed)
    Abstract [en]

    A lab-scale continuous microalgal culture was grown on sterile-filtered wastewater in order to clarify the phosphorus removing mechanisms in a microalgal treatment step that treats residual phosphorus from a hydroponic wastewater treatment pilot plant. The phosphorus assimilation was dependent on algal biomass production, whereas the chemical precipitation was dependent on phosphorus load, i.e. an increase in average precipitation rate with decreased hydraulic retention time was observed. The chemical precipitation was mainly a result of the increased pH, which was biologically mediated by the photosynthesising algae. The precipitate was composed of a calcium phosphate with magnesium included, magnesium hydroxide and calcite. A significant nitrogen removal was also experienced, which implies that the microalgal wastewater treatment is appropriate both for phosphorus and nitrogen removal.

  • 48.
    Larsdotter, Karin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jansen, Jes La Cour
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Microalgae as a phosphorus trap after hydroponic wastewater treatmentManuscript (Other academic)
  • 49.
    Larsdotter, Karin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jansen, Jes la Cour
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO).
    Phosphorus removal from wastewater by microalgae in Sweden: a year-round perspective2010In: Environmental technology, ISSN 0959-3330, E-ISSN 1479-487X, Vol. 31, no 2, p. 117-123Article in journal (Refereed)
    Abstract [en]

    The phosphorus and nitrogen removing capacity of a microalgal treatment step in Sweden was studied during an annual cycle. The treatment step had been constructed for extended phosphorus removal in a hydroponic wastewater treatment system, which had been built in a greenhouse. Two culture depths (17 and 33 cm) were compared as well as the effect of additional illumination during winter. The results showed large fluctuations in algal biomass production and phosphorus removal as a result of season. The phosphorus removal efficiency showed a clear correlation with pH, and the shallow cultures generally had higher phosphorus removal efficiencies than the deeper cultures. The efficiencies were between 60% and 100% during summer but mostly lower than 25% during winter, except in the shallow culture with extra illumination where efficiencies of 60-80% were recorded even during winter. A nitrogen removal efficiency of around 40% was reached for most parts of the year, and efficiencies of up to 60-80% were achieved during summer in the shallow cultures. In conclusion, the results showed that a large proportion of the phosphorus could be removed on a year-round basis, hence reducing the need for chemical precipitation, and also that significant nitrogen removal is possible.

  • 50.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Challenges in production of recombinant proteins2008In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, p. 1-2Article in journal (Refereed)
12 1 - 50 of 71
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