This invention relates to sparging apparatus for a reaction vessel for carrying out a cell culture, to a cell culture and mixing vessel having such sparging apparatus and to a method of sparging a culture medium contained in a cell culture and mixing vessel.

Background to the Invention

Many academic and industrial processes utilise cell cultures to generate cells or in the production of biomaterials or compounds of interest.

Sparging involves introducing one or more gasses from a submerged position in a liquid medium for these cells to be cultured. The gas enters the liquid in the form of bubbles and rises from the submerged position to the liquid surface. The gas is typically used to aerate an aerobic culture (in which case the gas is, or includes, oxygen) and/or in the mixing of the contents of the vessel.

However, the sparging process can have a detrimental effect on the efficacy of a bioreactor system that utilises the sparged culture. This is because the bubbles introduced by the sparging process can in some circumstances kill the cells being cultured. It is believed that cell damage or death can occur in cells in the immediate wake of a bubble as a result of the bubble breaking the surface of the liquid. As the bubble approaches the liquid surface, a thin film of liquid separates the bubble from that surface and rises from the normal surface level of the liquid with the bubble. When the film breaks liquid rapidly recedes back into the bulk of fluid, and the larger bubbles results in a fluid jet being projected upwards from the liquid surface. This results in a reaction which produces a submerged jet in the opposite direction into the liquid body, resulting in a build-up of pressure which is believed to cause the cell damage.

It is thus believed that the size of the bubbles within the vessel is closely related to cell mortality. Conventional sparging apparatus comprises a porous or sintered pipe or sheet through which the gas is supplied to the liquid in the vessel. The sizes of the pores/apertures through which the gas is supplied have a bearing on the bubble size, but it has been found that selection of aperture size alone does not ensure that bubbles of the desired size or range of sizes are reliably generated by the sparging apparatus.

Summary of the Invention

According to a first aspect of the invention, there is provided sparging apparatus for a reaction vessel for carrying out a cell culture, the sparging apparatus comprising conduit means for conveying gas into the vessel and two groups of outlet apertures through which, in use, gas travels from the conduit means into liquid in the vessel to form bubbles in the liquid, each group of apertures being situated in a respective zone on the conduit means, the zones being separated from each other so as to prevent or inhibit the coalescence of bubbles emitted through one group of apertures with those emitted through the other group of apertures.

Preferably, the conduit means and the zones are elongate, the zones extending along the conduit means.

The applicant has realised that coalescence of bubbles gives rise to a seemingly random variation in bubble sizes produced by conventional sparging equipment, so that conventional selection of design parameters such as aperture size, shape and spacing for a sparge tube does not in itself lead to a design which reliably and consistently causes bubbles of the desired sizes to pass through the liquid. The invention avoids or mitigates the size variation caused by coalescence of some of the bubbles.

The arrangement of apertures into two separate spaced groups enables sparging gas to be introduced into liquid in the vessel whilst avoiding or inhibiting the creation of bubbles of a size which can cause cell damage.

Preferably, the conduit means is arranged to extend non-vertically, preferably horizontally, into the vessel and each aperture is axially spaced from the other apertures of its group so as to prevent or inhibit the coalescence of bubbles from the apertures of the same group.

Preferably, the apertures in each group are arranged in a respective linear array.

It will be appreciated that, in this case, the zones comprise the axes defined by the arrays.

Having each group of apertures linearly arranged, in a respective single row, means that the bubbles from any aperture are unlikely to coalesce with those from any other aperture in the same group, if the conduit means is generally horizontally disposed in the vessel.

Preferably, said zones are substantially parallel to each other and are laterally spaced from each so as to achieve said separation.

Preferably each of the holes is not less than 3mm from any neighbouring one or more of the other holes.

The conduit means may conveniently comprise a common conduit in which both groups of apertures are formed. Preferably, the portion of the common conduit between the two groups of apertures is unapertured.

This enables the sparger to be formed from a single component.

Preferably, the common conduit comprises a pipe.

Preferably, the pipe is of circular cross-section, the two zones being angularly spaced from each other around the pipe, and preferably extending along the pipe parallel to the pipe axis. This angular separation not only gives rise to lateral spacing between the zones but also facilitates an arrangement in which bubbles are emitted through the apertures in one zone in a direction away from those emitted by the apertures in the other zone.

Preferably, the zones are on opposite sides of the bottom dead centre of the pipe.

Preferably, the apertures of at least one group, preferably of both groups, are angularly spaced from the bottom dead centre of the pipe by less than 90°.

Preferably the two groups of apertures are angularly spaced from each other by 20°.

Preferably, the apertures of one or both groups are angularly spaced by 10° from the bottom dead centre of the pipe.

It has been found that, if the angular spacing between the groups of apertures is too small, bubbles tend to coalesce under the pipe. On the other hand, too large an angular spacing can result in a tendency for bubbles to coalesce above the pipe. The applicants believe that the 20° spacing (10° either side of bottom dead centre) represents an optimum position , in many situations, for avoiding coalescence, at least for gas flow rates of the order of 1 standard litre per minute.

Preferably, each aperture has a diameter or equivalent circular diameter, of 0.4mm.

According to a second aspect of the invention, there is provided a cell culture and mixing vessel having sparging apparatus in accordance with the first aspect of the invention.

The invention also lies in a method of sparging a culture medium contained in a cell culture and mixing vessel, the method comprising introducing gas through apertures in gas conduit means extending into the vessel, the apertures being arranged in two groups separated from each other in such a way as to avoid or inhibit the coalescence of bubbles emitted through one group of apertures with those generated at the other group of apertures. Preferably, the method includes inhibiting or preventing the coalescence of bubbles produced by apertures of the same group by ensuring that said apertures are non- vertically spaced from each other.

Preferably, the method is performed using sparging apparatus in accordance with the first aspect of the invention.

Preferably, the size of each aperture and the rate of flow of gas for each aperture are such that the bubbles emitted through the apertures have an aspect ratio predominantly in the range of 1-4.

Preferably, the pipe includes deflection means for deflecting gas travelling along the interior of the pipe, preferably so that the bulk of the gas does not follow a path parallel to the pipe axis.

The deflection means prevents or inhibits variations in gas pressure, along the length of the pipe. Such variations could cause corresponding variations in the sizes of bubbles emitted through apertures at different positions along the pipe.

The deflection means may comprise a series of baffles within the pipe or one or more elongate pieces of cellular material, such as an open celled foam. Alternatively, the member can take the form of a sintered plastics dampener.

Brief Description of the Drawings

The invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a cross-sectional view of a sparging tube which constitutes sparging apparatus in accordance with the invention;

Figure 2 is a side elevation of part of the sparging tube shown in Figure 1; Figure 3 is a view of part of the underside of the sparging tube;

Figure 4 represents the change in velocity and pressure of gas along the length of the tube, when the tube is not fitted with any type of internal deflection means;

Figure 5 graphically shows relative flow rates of gas passing through the apertures in the sparging tube, as a percentage of total gas flow and percentage drop in flow between adjacent apertures.

Figure 6 is a graph which shows the characteristics of various bubbles which are observed to be emitted from apertures of different possible sizes and relative positions, and conveying gas at various different rates through those apertures into a liquid;

Figure 6A is a similar graph for the other arrangements of apertures.

Figure 7 is a sectional side view of a cell culture vessel, also in accordance with the invention, incorporating the sparging apparatus shown in Figures 1 and 2;

Figure 8 is a rear view of the vessel shown in Figure 7; and

Figure 9 is a simplified isometric view of the vessel.

Detailed Description

With reference to Figures 1 and 2, an embodiment of sparging apparatus in accordance with the invention comprises a sparging tube formed from a length of pipe 1 of circular cross-section in the lower portion of which a number of radial apertures, such as apertures 4 and 6, are formed. The pipe may be of a plastics material, or may be of stainless steel. The apertures may be formed by a process of drilling or laser cutting.

The apertures are circular, and are arranged in a pair of linear arrays (i.e. rows) which run parallel to the axis of the tube 1. Figure 2 shows that the aperture 6 is one of a number of apertures, others of which are referenced 7-10, which is in one of these rows. Each row has 26 apertures, so that the total number of apertures (through which gas connects at the pipe 1) is 52. The apertures in each row are spaced apart by no less than 3mm along the length of the row. The spacing between neighbouring apertures in this example is 10mm.

The radial axes passing from the centre of the pipe 1 through the central axes of the apertures in each group lie in a plane extending along the line of the pipe 1. In Figure 1, reference numeral 12 denotes the plane (viewed end-on) associated with the group of apertures which include the aperture 6, whilst reference numeral 14 denotes the corresponding plane for the second row of radial apertures, which include the aperture 4. Figure 1 also shows a vertical axis 16 which lies on a plane that extends through the centre of the pipe 1 and through the bottom dead centre position 18 on the pipe 1. It will be appreciated that the bottom dead centre position defines an axis which runs along the bottom of the pipe 1 between the two rows of apertures. Each of the planes 14 and 12 makes an angle of 10° with the axis/plane 16. Thus each group of apertures is separated, angularly, by 20° from the other group, and, because the apertures are radial, the holes in the two groups are angled away from each other by 20°.

It will be appreciated that Figures 1 and 3 are not drawn to scale, and exaggerate the angular separation between the two rows of apertures. Thus the rows are depicted as being angularly separated from each other by 40 ⁰ and from the plane by 20 °, twice the actual angular separations.

In other embodiments of the invention, the apertures can be orientated non- symmetrically about the bottom dead centre of the sparge tube and can be located between 10° and 30° on either side of bottom dead centre. If the apertures are non- symmetrically arranged, the angular difference between the angle made by one set of apertures with bottom dead centre and that made by the other set of apertures with bottom dead centre will be between 0° and 10°. In other words, the angle made by the angular mid-point between the two sets of apertures with bottom dead centre will be between 0° and 10°. The pipe 1 contains an elongate parallelepidal block 20 of foam or sintered polyethylene material. The block 20 is frictionally held in place by its contact with the inner walls of the pipe, and acts as a diffuser or baffle which defects air flowing along the pipe 1.

The function of the block 20 can be understood from Figure 4 in which a plot 19 of the air pressure within the tube 1 versus the length of the tube is superimposed on an image of part of the tube 1. Thus the y axis of the plot 19 represents pressure, whilst the x axis indicates position along the tube 1. As can be seen from Figure 4, the plot 19 is a straight line which proceeds from a minimum at the inlet end of the tube to a maximum value at the distal end of the tube 21. The pressure in the tube has an inverse relation to the velocity of gas along the tube so that, whilst the shaded area under the plot 19 can represent pressure, the height of the portion above the line 19 and beneath the top of the tube 1 as shown in Figure 4 is indicative of velocity. Thus the velocity of gas flowing along the tube 1 decreases from a maximum at the inlet 23 to a minimum at the distal end 21.

In the absence of the block 20 from the pipe as depicted in Figure 4, the air velocity progressively decreases along the length of the pipe from the inlet end to the opposite end of the inlet. This is consistent with the fact that the exiting of gas from all of the apertures contributes to the velocity of gas along the pipe at the inlet end, but that progressively fewer apertures make this contribution as the distance along the pipe from the inlet increases, until at the remote end from the inlet it is only the final pair of apertures, one from each group, through which the gas exits. This reduction in the number of downstream apertures with distance from the inlet end will have the opposite effect on pressure.

This pressure gradient has a direct effect on the size of bubbles created at the apertures at different lengths along the pipe 1. The pressure variation is avoided, or at least mitigated by the block 20 as a result of the turbulence it causes in the flow of gas along the pipe 1. Figure 5 shows how, in the absence of the block, the distance of any given aperture in any given row from the inlet, more particularly the number of other apertures in the group upstream of that aperture, has an effect on the relative amount of gas that passes through that aperture. There are two curves, the curve 28 which shows a percentage of the total gas flowing out of the group of apertures that flows out of each given aperture in the group and the curve 30 which shows the percentage drop between apertures of the same group. The block helps to present this variation. Additionally or alternatively the size of the apertures may vary in order to compensate for the variation in flow caused by any pressure gradient in the gas flowing along the tube.

With reference to Figure 6, the size of bubbles produced by the sparging apparatus, and their shape, can be influenced by such parameters as the aperture diameter, the flow rate of gas through the apertures, aperture orientation and pipe diameter. The aspect ratio, plotted on the ordinate axis is the ratio of the maximum to the minimum bubble diameter as the bubble rises through the liquid. The equivalent circular diameter is the diameter of a pure circle that would have an area equivalent to the area of the bubble. The aspect ratio and equivalent circular diameter are calculated from both simulations and physical tests.

In those tests, bubbles are tracked in Multiphysics Computational Fluid Dynamics (CFD) simulations and using physical tests in which high speed video camera footage of rising bubbles is obtained, and the necessary data determined using established image and tracking analysis algorithms as the bubbles rise. Initially, physical tests were conducted on simplified models in order to help to calibrate the CFD simulation model. That model was then used for simulation of bubbles generated by arrangements having various different parameters and combinations of parameters. Once a preferred set of parameter values has been identified from the simulations, a final physical test may be performed in order to verify the design results and outcomes of the CFD simulations or the preferred set.

The simulations and tests determine the area of bubbles as they rise, and this data is used to obtain equivalent circular diameter (ECD) and aspect ratio (AR) values by means of the following equations. ECD = (4Α/π) , where A is measured area (cross-sectional) of bubble as it rises.

While the AR, is the ratio of the maximum bubble diameter to the minimum bubble diameter as the bubble rises the animation, or:

AR = dmax/dmin, where dmax and d _m in are the maximum and minimum diameters, respectively.

In the described example, the tube (i.e. the pipe 1) has a diameter of 12.5mm, the apertures under consideration each subtends an angle of 10° with bottom dead centre of the pipe and is 0.4mm in diameter. In addition, those apertures are supplied with gas at a rate of .02 litres per minute through each aperture (so that for 50 apertures the flow rate is 1 litre per minute).

It will be appreciated that the plot is for a very simplified arrangement in which there are only two apertures, one in each group, however it is believed that similar results should be achieved if the analysis were conducted on a pipe having two groups of apertures, each consisting of a row of 25 apertures (i.e. ports), or more.

The selected parameters give bubbles having a relatively narrow range of aspect ratios (approximately between 1 and 4) and equivalent circular diameters (in the range of just under 4mm to just over 8mm), this amounts to far less variation than the other possibilities shown in Figure 6.

For the sake of clarity, only a relatively small number of plots for each set of parameters is shown ⁰ in Figure 6. However, if more points were plotted on the graph, then the points for the preferred set of parameters, e.g. the point 24 (i.e. all of the roughly diamond-shaped points) would in the main show an ECD value of greater than or equal to 7mm and less than/or equal to 20mm and aspect ratio of close to 1 (e.g. greater than or equal to 0.75). Preferably, at least 90% of the points for the selector parameter values satisfy both of these criteria. Figure 6A is a similar graph of various plots which represent observed aspect ratio and equivalent circular diameter values for bubbles emitted through various arrangements of ports. In the Figure, the "optimized parameter set" corresponds to the preferred set of parameters shown in Figure 6, i.e. tube outer diameter of 12.5mm, having apertures of 0.4mm in diameter which subtends an angle of 10° with bottom dead centre and which each is supplied with gas at a rate of 0.02 litres per minute.

The plots of data set 1 in Figure 6 A are for bubbles observed for an arrangement having a tube with an outer diameter of 11.89 mm and an inner diameter of 7.86 mm. The tube has 3 ports longitudinally spaced 38.22mm apart and each having a diameter of 0.50mm. The combined flow rate of gas through the 3 ports was 0.5 litres per minute and each port was angularly spaced by 15° from bottom dead centre.

Data sets 2 and 3 are the plots for an arrangement having a tube of 11.93mm outer diameter and 7.91mm in a diameter and a single port of 1.5mm diameter. In data set 2 the flow rate of gas through the single port is 0.07 litres per minute and the port makes an angle of 45° with bottom dead centre, whilst the corresponding values for data set 3 are 0.05 litres per minute and 15°. The data for data set 1 were obtained for a fluid having a temperature of 37°C and a fluid head (at the pipe) of 264mm. The corresponding values for data sets 2 and 3 were 17°C and 213mm. In Figure 6A, the shaded zone 25 represents where the majority of the plots for the optimized parameter set lie, and it can be seen that most of these plots show an aspect ratio value of at least 0.75 and an ECD value greater than 7mm and less than 20mm.

As can be seen from Figures 7-9, the sparging apparatus in accordance with the invention is, in use, situated towards the bottom of a vessel 32, the pipe 1 being horizontal and extending across substantially the whole width of the vessel. In addition, the pipe 1 is orientated in the way shown in Figure 1, so that 18 is the bottom dead centre.

The vessel 32 has a back wall 34, a front wall 36, two side walls 38, a top wall 40 and a bottom wall 42 constituting a base of the vessel. Front wall 36 has a lower portion 44 inclined at about 20° to vertical. The front wall has an upper portion including a lower part 46 inclined at about 60° to vertical and an upper part 48 which is substantially vertical.

The back wall 34 includes a lower part 50 that rises vertically and an upper part 52 that is inwardly inclined at about 32°, constituting an abutment that comprises means for redirecting rising gas.

The vessel contains culture medium 54 which has a top surface 56, the volume of the vessel filled with medium constituting the working volume of the vessel. A headspace 57 is provided above the medium surface.

The vessel has an overall height of 55cm and a length at the base of 20cm. The upper chamber portion has a maximum width of about 22cn. The vessel has an aspect ratio of about 2.

The inlet end of the pipe of the sparging apparatus is connected to an associated inlet tube 58 (Figure 8), leads to the exterior of the vessel, and is provided with an associated filter assembly 60.

Additional inlet ports 62,64 are provided for addition of liquid reagents and/or gases. A pressure regulation valve 66, set to operate at 2 psi gauge, is provided on the top of the vessel.

A temperature regulation plate 68 is provided slightly spaced from the back wall lower 50.

A sensor 70 is provided on the lower part 46 of the upper portion of the front wall, which can be used to monitor operating parameters.

The walls of the vessel are made from two layers of co-extruded nylon and polyethylene with the nylon on the outside, and is free from materials derived from animal sources. The vessel is supported by an outer frame (not shown) or may be a support made of steel, glass or other appropriate materials. The vessel is intended to be disposed of after use and a similar fresh vessel can be used for any subsequent cell culture fermentations or mixing operations. The vessel is initially supplied in sterile pre-validated condition, sealed in packaging.

In use, sterile gas 72 is introduced continuously to the pipe 1 via inlet tube 58 from a supply (not show) at a rate of e.g. 1 litre per minute. Gas bubbles with a size range indicated in Figur3 6 for the chosen parameters are produced and the liquid medium above the pope 1 rises vertically generally in the direction of arrows 74 (Figure 7). The rising liquid medium and gas bubbles are redirected generally in a substantially horizontal direction as represented by arrows 76.

This flow at the culture surface 56 increases the rate of mixing and gas transport from the headspace 57 into the medium 54, Thereafter some of the medium circulates within the upper chamber portion about a horizontal axis as indicated by arrows 76 and 78. Some of the medium flows downwards into the lower chamber portion in a downcomer as indicated by arrows 80, eventually rising again generally in the direction of arrows 74. Thus a tubular circulation pattern is established within the upper chamber portion parallel to the back wall 34.