The effects of material properties and fluid flow intensity on plasmid DNA recovery during cell lysis

This article describes how a combination of an ultra scale-down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Sigma(M)) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell-plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO-S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development.

Section: Theoretical Considerations Centrifugation Scale-downmentioning

confidence: 99%

Section: Rotating Disc Devicementioning

confidence: 99%

Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

Tait

Aucamp

Bugeon

et al. 2009

“…However, the prevailing fluid flow stresses normally associated with intense mixing may cause damage to the large macromolecules in the solution (Davison, 1959;Levy et al, 1999aLevy et al, , 1999bNguyen and Kausch, 1992). An important process engineering question that arises is what influence this kind of stress has on the yield and purity of the supercoiled plasmid DNA, and if the impact is detrimental, what mixing strategy may be adopted to minimize it.…”

Section: Introductionmentioning

confidence: 99%

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

Meacle

Lander

Shamlou

et al. 2004

Self Cite

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 F 0.03 M (pH 12.9 F 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 À10 5 s À1) 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 À105 s À1). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

“…In addition, the shear device was provided with a top cover that provided an air-tight seal. Shearing of the liquid inside the chamber was achieved by the rotation of a¯at disk in the chamber and the pro®le of the prevailing energy dissipation rates was obtained using CFD simulations as described previously (Levy et al, 1999b). In the present study, the shear device was operated over a range of energy dissipation rates corresponding to those used in the two ®ltering centrifuges and the ultra scaledown unit.…”

Section: Resultsmentioning

confidence: 99%

Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth

Boulding

Yim

Keshavarz-Moore

et al. 2002

Self Cite

Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates.