ANALISIS DEL ESTADO DE SITUACION FINANCIERA

The aim of this study was to predict the effectiveness of adding flocculant to a large-scale process to aid the removal of cells and debris. Having ascertained the optimum dosage of

PEI to cause aggregation, an experiment was devised to simulate the addition of PEI to a large-scale cell culture by reproducing, on a small scale, the conditions the broth would encounter during cell separation by disc-stack centrifugation. Cells (cell line #2) were cultured in roller bottles for several days until the viable cell count had increased fifteen to twenty-fold from inoculation. A daily sample was then taken and treated in the following manner: the sample was divided into six 2 0 ml measures, each of which had a volume of either 0.01% v/v PEI or distilled water added to it, as shown in Table 2. The samples were inverted ten times to mix them thoroughly, then examined under a microscope. Where possible, cell counts were made. Certain samples were then exposed to conditions of shear as described in Section 6.13.1, to mimic the forces cells would encounter in the feed zone of a disc- stack centrifuge. Other samples were not sheared and acted as controls for the experiment. After shearing the samples were again examined microscopically and cell counts were made where possible. Solids were then removed from the samples by centrifugation in 14 ml test-tubes (MSE (Cat. #34411-122, Code 1286), MSE Scientific Instruments Ltd., Crawley, Sussex.) in a bench-top centrifuge with a swing-out rotor (MSE Centaur 11). The revolutions per minute and centrifugation time were calculated to give the same separation capability as the disc- stack centrifuge, as described in Section 6.13.2. At all stages, cell counts were made and photographs of the samples were taken, and the final supernatant was assayed for extracellular IgM, DNA and protein.

6.13.1 Mimic of centrifuge feed zone.

Cells entering the disc-stack centrifuge encounter a shear force as they are accelerated to the speed of the centrifuge. Hoare and Dunnill (1986) note that the effects of such forces on protein precipitates is significant, and it is reasonable to assume that floes of cells and debris could also be affected. The aim of this part of the experiment was to simulate on a small scale the shear experienced by the cells,

to ascertain whether or not the floes, or indeed the cells themselves, were strong enough to survive the shear without breaking. This was done by shearing the samples in a capillary rheometer system based on an Instron Food Testing Instrument, Table Model 1140 (Instron Ltd.).

It was difficult to quantify the shear forces experienced by the cells in the feed zone of the disc-stack centrifuge, as little information was available on the subject. However, the diameter of the feed line to the centrifuge was much larger than the largest capillary diameter for the Instron, which was 0.575 mm, approximately forty times the diameter of a viable mammalian cell. Since clumps of one hundred or more cells can form on the addition of PEI, there was deemed to be a risk of a smaller capillary becoming blocked and the floes being broken by this mechanism rather than by applied shear, a situation that would not arise in a large-scale situation because of the larger diameter feed line used. The level of shear in this experiment was therefore limited to that attainable with the largest bore capillary. By varying the length of capillary used the residence time of the samples could be altered. Two lengths of capillary were used in this study; Table 2 shows the capillary length used on each sample. Shear was applied to both flocculated and non-flocculated cells. Capillaries of varying lengths and internal diameters could be affixed to the barrel of the Instron. The sample to be sheared was then placed in the barrel and driven through the capillary of choice by the action of a piston travelling through the barrel at a fixed speed. The sheared sample was collected from the end of the capillary. The sample size used was 20 ml.

The shear applied was calculated using the following equation:-

Y = VB^ 15 d^

where : -

V = average shear rate (s^)

V = crosshead speed (m s ( f i x e d at 8.33 x 1 0^ m s

B = barrel internal diameter (m) (fixed at 9.54 x 1 0 ^ m) d = internal bore of capillary (m) (fixed at 5.75 x 1 0 * m)

From this, a shear rate of 1.70 x 10"* s'^ was calculated. The mean residence time was calculated according to the following equation:-

0 = d^L VB^

(Thomas, 1977) where : -

0 = mean residence time (s)

d = internal bore of capillary (m) (fixed at 5.75 x 1 0 ^ m)

L = capillary length (m) (set at either 0.075 m or 0.15 m) V = crosshead speed (m s'^) (fixed at 8.33 x 10 ^ m s ') B = barrel internal diameter (m) (fixed at 9.54 x 10'^ m) From this, the residence time was calculated as either 0.03 3 seconds (for 0.075 m capillary) or 0.065 seconds (for 0.15 m c apillary).

Problems with this method arose due to the use of a large bore capillary; unless the bottom of the capillary was blocked off during sample loading, the sample flowed out before shear could be applied.

6.13.2 Comparison of sienna values.

In order to compare centrifuges of different scales, the following relationship is used:-

Û1 = Ü2

where : -

Qg = flowrate through centrifuge 2 (m^ s'^) = sigma value for centrifuge 1 (m^) Eg = sigma value for centrifuge 2 (m^)

The sigma value is calculated from several parameters of a centrifuge and denotes the area of a gravitational settling tank with the same separation performance as the centrifuge. Different centrifuge types require different equations to calculate this value, and it is not recommended that scale-up is carried out between different types of centrifuge. In this case however, no disc-stack centrifuge small enough for use on a bench scale was available, so there was no option but to attempt to compare different types of centrifuge.

For a disc-stack centrifuge, the equation for calculating the sigma value is as follows:-

E = 27Tù)^(s- 1) (rg^-r^^) 3 g tan.n

(Ambler, 1952) where:-

E = area of equivalent settling in a gravitational field(m^) 0) = angular velocity (rads s'^) = 27r(rps) = TT(rv>m)

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