Prediction and verification of centrifugal dewatering of P. pastoris fermentation cultures using an ultra scale-down approach

Recent years have seen a dramatic rise in fermentation broth cell densities and a shift to extracellular product expression in microbial cells. As a result, dewatering characteristics during cell separation is of importance, as any liquor trapped in the sediment results in loss of product, and thus a decrease in product recovery. In this study, an ultra scale-down (USD) approach was developed to enable the rapid assessment of dewatering performance of pilot-scale centrifuges with intermittent solids discharge. The results were then verified at scale for two types of pilot-scale centrifuges: a tubular bowl equipment and a disk-stack centrifuge. Initial experiments showed that employing a laboratory-scale centrifugal mimic based on using a comparable feed concentration to that of the pilot-scale centrifuge, does not successfully predict the dewatering performance at scale (P-value <0.05). However, successful prediction of dewatering levels was achieved using the USD method (P-value ≥0.05), based on using a feed concentration at small-scale that mimicked the same height of solids as that in the pilot-scale centrifuge. Initial experiments used Baker's yeast feed suspensions followed by fresh Pichia pastoris fermentation cultures. This work presents a simple and novel USD approach to predict dewatering levels in two types of pilot-scale centrifuges using small quantities of feedstock (<50 mL). It is a useful tool to determine optimal conditions under which the pilot-scale centrifuge needs to be operated, reducing the need for repeated pilot-scale runs during early stages of process development.     

The scale-down of an industrial disc stack centrifuge

The means are described whereby a disc stack centrifuge may be scaled-down by up to 10-fold of its separation capacity. The centrifuge separation characteristics so measured are suitable for direct scale-up predictions of centrifuge performance where only small volumes of particle suspension are available for study. Such an ability to scale-down is especially important in the processing of biological particles where for example, in the early stage of process development, there is often insufficient fermentation broth for fullscale studies. Scale-down is achieved by the reduction of the number of discs available for separation purposes and by the careful positioning of these discs in the overall disc stack. A combination of dye tracer and particle separation studies are used to optimise the disc stack configuration. The resulting grade efficiency curve is an accurate reflection of the curve for the full-scale centrifuge especially in the critical design region specifying centrifuge throughput for near complete particle recovery.     

Fructose triggers DNA modification and damage in an Escherichia coli plasmid

The nonenzymatic reaction between reducing sugars and amino groups of long-lived macromolecules results in an array of chemical modifications that may account for several physiological complications. The characteristics of the reaction are directly related to the type of the reducing sugars involved, whether aldoses or ketoses, phosphorylated or non-phosphorylated, and these in turn determine the consequences of the induced modifications. So far, most studies have been focused on the nonenzymatic reaction between glucose and proteins, while the reaction with fructose, a faster glycating agent, attracted only a minor attention. We have recently demonstrated that long-term fructose consumption induces age-related changes in collagen from skin and cortical bones faster than glucose. In the present study we provide evidence that fructose and its phosphate metabolites can modify DNA faster than glucose and its phosphate metabolites under in vitro conditions. Incubating the plasmid pBR322 with fructose and glucose phosphate metabolites induced DNA modifications and damage that were verified by gel electrophoresis and transformation capacity of the plasmid into an Escherichia coli host. The intensity of the tested sugars to modified and damage DNA after incubation for 15 days increased significantly in the following order: glucose 1-phosphate < glucose < glucose 6-phosphate < fructose 1-phosphate < fructose < fructose 6-phosphate. The data suggest that fructose should deserve more attention as a factor that may influence glycation and induce physiological complications.     

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 7g/ml to 20 7g/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.     

Analysis of gamma radiation-induced damage to plasmid DNA using dynamic size-sieving capillary electrophoresis

Bacterial plasmids and the chromosomal DNA of many organisms adopt naturally the negatively supercoiled conformation. Therefore, the irradiation of such plasmids could be used to model conformational changes of chromosomal DNA associated with externally-induced damage. We have applied dynamic size-sieving capillary electrophoresis (CE) to monitor the damage of three DNA plasmids, over an unprecedented base pair (bp) size range (2870–27 500 bp), upon exposure to γ-radiation (20–400 Gy). Predominantly, CE with UV absorbance detection in the absence of DNA intercalating dyes was employed to preclude undesirable, induced plasmid conformational changes. Plasmid samples and their enzymatic digestion products were analyzed using both CE and slab gel electrophoresis (SGE) in order to verify the conformation of sample components. Relative to SGE, CE analyses revealed more fine structural features of plasmid degradation.     

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to 相似文献   

Gliotoxin causes oxidative damage to plasmid and cellular DNA

The cytotoxic effects of gliotoxin (Müllbacher, A., and Eichner, R. D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3835-3837), a fungal secondary metabolite, and related epipolythiodioxopiperazines have been investigated using plasmid and eukaryotic DNA. Incubation of the dithiol derivative of these compounds with DNA and Fe3+ is sufficient to cause single- and double-stranded breaks as determined by neutral agarose gel electrophoresis. The disulfide form is inactive except in the presence of a suitable reducing agent, such as reduced glutathione, dithiothreitol, or reduced pyridine coenzymes. The autooxidation of these dithiols produces reducing equivalents as evidenced by (i) the production of H2O2 and (ii) the generation of thiobarbituric acid reactive products when incubated with deoxyribose. The latter process is inhibited by ethanol and desferrioxamine. The DNA damage is abrogated by metal chelators and catalase. We conclude that the antiproliferative action of gliotoxin may be caused by DNA damage effected by reactive oxygen species or other radicals generated through redox cycling.     

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.     

Degradation of supercoiled plasmid DNA within a capillary device

Supercoiled plasmid DNA is susceptible to fluid stress in large-scale manufacturing processes. A capillary device was used to generate controlled shear conditions and the effects of different stresses on plasmid DNA structure were investigated. Computational fluid dynamics (CFD) analysis was employed to characterize the flow environment in the capillary device and different analytical techniques were used to quantify the DNA breakage. It was found that the degradation of plasmid DNA occurred at the entrance of the capillary and that the shear stress within the capillary did not affect the DNA structure. The degradation rate of plasmids was well correlated with the average elongational strain rate or the pressure drop at the entrance region. The conclusion may also be drawn that laminar shear stress does not play a significant role in plasmid DNA degradation.     

Repair of UV damage in plasmid DNA by human fibroblasts

Summary Plasmid DNA from Bacillus subtilis was introduced into monolayers of human fibroblasts by means of a modification of the calcium phosphate coprecipitation technique, comprising centrifugation of the coprecipitate onto the cells and treatment with polyethyleneglycol. The amount of DNA resistant to removal from the monolayers ranged from 10% to 15% of the input DNA. By determination of the biological activity of the plasmid DNA, re-extracted after various periods following entry into the fibroblasts and subsequently used as donor for B. subtilis protoplasts, it was shown that the activity of the plasmid DNA was gradually lost. When ultraviolet light-inactivated plasmid DNA was used as donor, reactivation of the plasmid was observed, which was completed within 2 h. The dose-dependent incorporation of [¹⁴C]-thymidine suggests that DNA repair processes were involved in reactivation of the plasmid DNA.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80 °C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

The efficiency of transformation by electroporation has been known to be compromised by strain dependency. A high efficiency protocol is still lacking for distinct two-hybrid yeast strains of diverse genetic features. Here, we used 0.5 M lithium acetate (LiAc) and 50 mM Tris-HCl with 5 mM EDTA (pH 7.5), i.e., fivefold the standard concentrations, and voltage at 1.0 to develop a protocol which, for the first time, is able to effect an average efficiency of 1.84 × 10⁶ transformants/μg DNA for three commonly used yeast strains committed to two-hybrid screening experiments.     

Heavy ion-induced plasmid DNA damage in aerated or deaerated conditions

Using the plasmid relaxation assay, the induction of single strand breaks (SSB) and base damages was investigated in air-dried plasmid DNA irradiated under air or under vacuum, with two high LET particles. We first observed that an irradiation with 12C5+ ion produced less of both damages when performed in a vacuum rather than in the presence of air. This could be due to the presence of O2 which increases the primary radicalar effects in the latter case. Another explanation is a difference in the degree of hydration of the DNA molecules. Indeed, under vacuum only the water molecules tightly bound to DNA will persist. In contrast, in the presence of air, the outer hydration shell enhances the amount of hydroxyl radicals available for the radiolytic attack. However, no difference in the SSB induction was observed when DNA was irradiated with 36S16+ ion in the presence of air or under vacuum. This is likely due to the LET effect which partly cancels the production of radicals by recombination and increases the formation of superoxide anions in the track. Similarly, the lower induction of damage by 36S16+ irradiation in comparison with the 12C5+ ion is a consequence of the higher ionizing density for 36S16+ than for 12C5+ ions. Meanwhile, for both ions, base damages are not detected when DNA is irradiated under vacuum, whereas they are as frequent as SSB when irradiation is performed in the presence of air. Altogether, these observations support the idea that SSB and base damage are not formed by the same mechanism.     

Effect of phenol on ultra structure and plasmid DNA of Xanthomonas oryzae pv. oryzae

Most phenolic substances of plant origin are toxic to microorganisms and they confer some degree of protection to plants against phytopathogens. Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice (Oryza sativa) was treated with phenol (monohydroxy benzene) and its effects on the morphology and cytological changes of the bacterium were studied. Total lysis of cells occurred with 5 mM conc of phenol while at 2 mM conc, the cell walls became rough and cell contents started shrinking. Plasmids isolated from both treated (2 mM) and control cells did not show any marked difference under electron microscope except that they differed in their quantity and might influence pathogenicity.     

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.     

Purification of supercoiled plasmid DNA using chromatographic processes

The interest in purifying injectable-grade plasmid DNA has increased with the development of gene therapy and DNA vaccination technologies. In this paper we develop a method for purifying a 4.8 kb plasmid based on chromatographic processes. An NaCl gradient was optimized on a Q Sepharose column and plasmid was eluted at 800-820 mM NaCl in a broad peak. Supercoiled plasmid was isolated after a final Sepharcryl S1000 SF gel filtration step. Final plasmid preparation was depleted of proteins and RNA, as revealed by the BCA assay and 1% agarose gel electrophoresis.     

Separation of plasmid DNA isoforms using centrifugal ultrafiltration

Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms.