| 1. |
- Al-Khalili, Lubna, et al.
(författare)
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Characterization of Human CD133+Cells in Biocompatible Poly(l-lactic acid) Electrospun Nano-Fiber Scaffolds
- 2016
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Ingår i: Journal of Biomaterials and Tissue Engineering. - : American Scientific Publishers. - 2157-9083 .- 2157-9091. ; 6:12, s. 959-966
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Tidskriftsartikel (refereegranskat)abstract
- CD133+ cells are potential myogenic progenitors for skeletal muscle regeneration to treat muscular dystrophies. The proliferation of human CD133+ stem cells was studied for 14 days in 3D biomimetic electrospun poly-L-lactic acid (PLLA) nano-fiber scaffolds. Additionally, the myogenic differentiation of the cells was studied during the last 7 days of the culture period. The cells were homogeneously distributed in the 3D scaffolds while colony formation and myotube formation occurred in 2D. After a lag phase due to lower initial cell attachment and an adaptation period, the cell growth rate in 3D was comparable to 2D after 7 and 14 days of culture. The expression of the stem cell (SC) marker PAX7 was 1.5-fold higher in 3D than 2D while the differentiation markers MyoG, Desmin and MyoD were only slightly changed (or remain unchanged) in 3D but strongly increased in 2D (12.6, 3.9, and 7.9-fold), and the myotube formation observed in 2D was absent in 3D. The marker expression during proliferation and differentiation, together with the absence of myotubes in 3D, indicates a better maintenance of stemness in 3D PLLA and stronger tendency for spontaneous differentiation in 2D culture. This makes 3D PLLA a promising biomaterial for the expansion of functional CD133+ cells.
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| 2. |
- Brechmann, Nils A., et al.
(författare)
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Antibody capture process based on magnetic beads from very high cell density suspension
- 2021
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Ingår i: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510
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Tidskriftsartikel (refereegranskat)abstract
- Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification.
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| 3. |
- Brechmann, Nils Arnold, et al.
(författare)
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Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture
- 2019
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Ingår i: Biotechnology progress (Print). - : AIChE. - 8756-7938 .- 1520-6033.
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Tidskriftsartikel (refereegranskat)abstract
- High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the developmentof a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarifiedCHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum bindingcapacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for anIgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture stepfrom 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions wereobtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scalepurification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single proteinA capture step, the mAb purity was similar to the one obtained by column chromatography, whilethe host cell protein content was very low, <10 ppm. Our results showed that this magnetic beadmAb purification process, using a dedicated pilot-scale separation device, was a highly efficientsingle step, which directly connected the culture to the downstream process without cell clarification.Purification of mAb directly from non-clarified cell broth without cell separation can providesignificant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.
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| 4. |
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| 5. |
- Larsson, Gen, et al.
(författare)
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Characterisation of the Escherichia coli membrane during fedbatch cultivation
- 2004
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 3, s. 9-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate. Results: During fedbatch cultivation, E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate. Conclusions: The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point.
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| 6. |
- Shokri, Atefeh
(författare)
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Influence of strain, feed profile and cultivation technique on the lipid structure and membrane function in Escherichia coli
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The phospholipid and fatty acids content of the Escherichiacoli membrane were investigated during changes of feed rate,cultivation technique and host strains.During continuous cultivation it was shown that the leakageof periplasmic protein to the medium had an optimum at 0.3 h-1,resulting in transport of 20% of the total recombinant product.This corresponded to the accumulation of high amounts ofphosphatidylglycerol and unsaturated fatty acids, which arethus believed to be responsible for the increased fluidity. Theamount of cyclic fatty acids and cardiolipin were enriched atlower growth rates and marked the transition to conditionsresembling energy and/or carbon starvation.During fed-batch cultivation this pattern was visible butnot as pronounced. The fed-batch cultivation was characterisedby an overall and high level of cardiolipin and a lower degreeof membrane component changes. It was concluded that thedynamic character of this technique did not allow the steadystate membrane performance of continuous cultivation.A relaxed/stringent strain pair was cultivated in fed-batchcultivation and evaluated with respect to different membranestructure and function. The relaxed strain was characterised byheavy foaming during constant feed due to comparatively highercell lysis.During the shift to very low feed rate, the relaxed strainwas able to regulate both phospholipid and fatty acids leadingto a higher flux to saturated fatty acids and neutralphospholipids. The relaxed strain was also characterised by theinability to accumulate cardiolipin at low growth rate. Thedata suggest a more complicated mechanism responsible for theshift in membrane component accumulation at reduced substratefeed rates. This is not based directly on ppGpp, this compoundcould never be detected in these experiments, but is connectedto this formation since the relAﺷ strains have a verydifferent performance characteristics. The differences in lipidaccumulation was obvious for e.g. the performance duringsonication where the opposite behaviour was observed atdifferent feed rate. While the stringent strain showed thecharacteristic and rigid behaviour at low growth rate therelaxed strain was very sensible to sonication and was alsomore subjected to cell lysis.An evaluation of a shift in the model periplasmic protein,from b-lactamase to maltose binding protein, using a weakpromoter from the maltose operon, was investigated. The systemwas designed to operate with both cytoplasmic and periplasmictargeting of the protein. The data showed that the productionwith the periplasmic system, at different growth rates, ishigher at higher growth rate. This agrees with the data for theproduction with b-lactamase where the enrichment of unsaturatedfatty acids and phosphatidylglycerol increases by growth ratefrom 0.3h-1 and above and would thus lead to an increase influidity and transport.Key words:Escherichia coli, Phospholipid and fatty acidregulation, fed-batch, continuous cultivation, leakage, proteintransport.
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| 7. |
- Shokri, Atefeh, et al.
(författare)
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RelA1 gene control of Escherichia coli lipid structure and cell performace during glucose limited fed-batch conditions
- 2006
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 73:2, s. 464-473
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Tidskriftsartikel (refereegranskat)abstract
- At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.
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