Investigation of the unit operations involved in the continuous flow isolation of β‐galactosidase from <i>Escherichia coli</i>

Higgins, Joseph; Lewis, D.; Daly, William H.; Mosqueira, F. G.; Dunnill, P.; Lilly, M. D.

doi:10.1002/bit.260200202

Cited by 52 publications

(17 citation statements)

References 18 publications

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“…The protein and DNA release analysis confirmed increased cell lysis in samples processed with the disc‐stack centrifuge as observed by other researchers [11]. In addition, agarose‐gel analysis indicated that released DNA was subjected to degradation.…”

Section: Resultssupporting

confidence: 81%

“…Results shown above indicated that the methods used at the cell harvest stage can have a substantial impact on SC plasmid yield and the level of contaminant DNA (chromosomal and degraded plasmid isoforms) entering the product stream. Previous studies with a disc‐stack centrifuge in the context of enzyme recovery have shown that decreases in product yield could be associated with premature cell lysis [10,11]. A series of experiments were performed with the dual objective of confirming that the decreased yield and increased contamination of disc‐stack‐centrifuge‐derived samples could be attributed to cell lysis and to establish a technique to characterize cells before proceeding to the lysis stage (Scheme 1).…”

Section: Resultsmentioning

confidence: 99%

“…Centrifugation methods are commonly applied for the harvesting of large volumes of culture in the bioprocessing sector. Previous studies have shown that Escherichia coli cells harvested with an intermittent‐discharge, continuous flow disc‐stack centrifuge are subjected to stresses that are sufficiently high to cause lysis [10–12]. The magnitude of the effect has been shown to be related to the cell culture medium used, with cells grown in a defined medium being more susceptible to damage [10,13].…”

Section: Introductionmentioning

confidence: 99%

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Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

Kong¹,

Rock²,

Booth³

et al. 2008

Biotech and App Biochem

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The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

Kong¹,

Rock²,

Booth³

et al. 2008

Biotech and App Biochem

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“…The DNA concentrations in all of the process streams were predicted using a mass balance approach. Using data from a previous study (Higgins et al;[15]) it was assumed that the DNA remained soluble under the conditions used in the study. Figure 6 shows the average experimental and simulated DNA results for a number of small scale trials.…”

Section: Resultsmentioning

confidence: 99%

Computer-based simulation of the recovery of intracellular enzymes and its pilot-scale verification

Bulmer

Clarkson

Titchener-Hooker

et al. 1996

Bioprocess Engineering

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Accurate and reliable models are required for a range of unit operations if simulations are to be used for accelerating the design and optimisation of bioprocesses. This paper presents results of pilot-plant studies that have been used to verify process simulations for a sequence of operations comprising of cell disruption, fractional protein precipitation and centrifugal separation. These have been tested using the purification of the enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae as being representative of the recovery of a labile intracellular enzyme. Comparison of pilot-plant against simulated data highlights where improvements to the models are required and has resulted in increased confidence in the simulations for a wide range of conditions including the operational scale and the nature of the starting material. Results demonstrate the effectiveness of the verification approach for the development of reliable predictive models to assess the feasibility of process designs and performance of a train of bioprocess operations. List of symbols

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“…Moreover, PEI has been used for the selective separation of nucleic acids from microbial cell extracts by precipitation [12][13][14]. Various other cationic agents had been used for the precipitation of nucleic acids: spermine, spermidine, cobalthexamine [15][16][17], divalent metal ions [18], and protamine sulfate (PS) [19,20].…”

Section: Introductionmentioning

confidence: 99%

DNA Depletion by Precipitation in the Purification of Cell Culture‐Derived Influenza Vaccines

et al. 2010

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A pilot study for the purification of cell culture-derived human influenza virus is presented, which focuses on the early removal of DNA by precipitation. Strains of influenza virus were propagated using Madin Darby canine kidney cells as a host. A harvesting time of about 72 h postinfection was chosen to minimize the level of impurities (host cell DNA and protein). Cell culture supernatant was clarified by centrifugation and the performance of this operation was characterized on the basis of Sigma theory. An average clarification efficiency of 93 % (based on turbidity) and a product yield of 85 % (based on hemagglutination activity) were obtained at a load of 1.6 · 10 -8 m s -1. Furthermore, the applicability of Sigma theory for scale-up studies using two different laboratory centrifuges was verified. Selective precipitation of DNA was achieved by the addition of polyethyleneimine (PEI). Full factorial design was applied to optimize selectivity considering pH, ionic strength, and the concentration and molecular weight of PEI. Under optimized conditions, treatment with PEI resulted in a reduction of DNA to 15 % of the initial amount, while 86 % of virions (based on neuraminidase activity) were recovered. The subsequent concentration of virions was realized by tangentialflow ultrafiltration. Recovery based on hemagglutination activity was determined to 63 % on average. Including the previous precipitation step, overall reduction in DNA after tangential-flow ultrafiltration was 500-fold. These results indicate that the suggested unit operations are suited for the early depletion of DNA in cell culture-derived influenza vaccine production.

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Investigation of the unit operations involved in the continuous flow isolation of β‐galactosidase from Escherichia coli

Cited by 52 publications

References 18 publications

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

Computer-based simulation of the recovery of intracellular enzymes and its pilot-scale verification

DNA Depletion by Precipitation in the Purification of Cell Culture‐Derived Influenza Vaccines

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