Homogenization device

Abstract

The invention presents a device for homogenization and agitation of contents within a suspended cell culture bag. The device comprises a back plate and a compression bar positioned in parallel to the back plate with a variable gap. Attached to the compression bar are at least one push rod, allowing the adjustment of the gap width between a first and second width. When the cell culture bag is positioned between the back plate and compression bar, periodic variation of the gap width causes homogenization and agitation of the bag contents.

Description

[0001] Homogenization Device

[0002] Field of the Invention

[0003] The present invention pertains to the field of machines allowing large scale manufacturing production processes requiring small to medium scale biological steps, such as an electroporation step, with liquid suspensions of vesicles such as cell suspensions. Specifically, the invention focuses on a novel apparatus and method designed for homogenizing stock or feed bags of cell culture for use in such manufacturing processes. The invention is particularly suitable for clinical and industrial applications, and in particular to offer improved efficiency and reliability in electroporation based manufacturing.

[0004] Background

[0005] Recently, there has been increased interest in the processing of vesicles (such as living cells) so as to change and enhance their properties for example by way of electroporation for which electrical pulses are used to reversibly destabilize biological membranes and during the period of destabilization to provide a pathway for the introduction of exogenous material into the vesicles or cells. Typically, such introduced exogenous material can be genetic material to obtain genetically modified cells used for research or therapeutic purposes.

[0006] In industrial and commercial settings, maintaining consistent quality and results is crucial across material batches, even when multiple production runs are performed in a closed system. To achieve this, a constant concentration of cell suspensions feedstock must be ensured throughout the production run.

[0007] The feedstock of cells for such processes is typically supplied in the form of liquid suspensions of cells. Such feedstock is supplied in flexible bags. These bags are suspended with a feed being taken from the bottom of the bag. Subsequently, the suspension of cells is delivered for processing. Often, a cell bag remains hanging for an extended period as the biological processes are typically slow and rely on repeated filling and emptying of a low volume reaction cell. Typically a run requiring several electroporation steps can be of the order of a day.

[0008] One common issue faced with such systems in which feed stock bags of suspended biological material remain hanging for extended periods is the variation in cell concentration flowing from the bag over time. This variation can be caused by factors such as gravitational settling of cells, flocculation whereby cells clump together due to mutual attraction and cell-wall attraction with the walls of the bag.

[0009] US2007140047 discloses a device for blending materials comprising a carrier support arranged to support a closed bag containing material to be blended and a kneading paddle to apply a kneading action to the walls of a supported bag for homogenizing its contents.

[0010] But until now, it has been impossible to achieve the required homogeneity owing to numerous technical challenges which are further compounded by the delicate nature of biological cells. The objective of the current invention is therefore to introduce an apparatus that ensures homogenization within the cell suspension bag as the bag is emptied to supply the manufacturing processes.

[0011] The reader will appreciate how this invention may be applied in any situation where the maintenance of a homogenized state in bags containing liquid or containing suspensions of particles is required.

[0012] Summary of the Invention

[0013] According to a first aspect, a device is disclosed for homogenization and agitation of the contents within a suspended cell culture bag. The device comprises a back plate having a front side and a back side, a compression bar arranged parallel to and spaced from the front side of the back plate by a variable gap, at least one push rod having a front end and a back end with the front end attached to the compression bar and being positioned perpendicular to and passing through the back plate. At the at back end, the at least one push rod is attached to a translation mechanism wherein operation of the translation mechanism causes the at least one push rod to move perpendicular to the back plate such as to cause adjustment of the gap periodically between a first width and a second width. When the bag is positioned between the front side of the back plate and the compression bar and the gap is periodically varied between the first width and the second width the cell culture bag contents is caused to be homogenized and agitated.

[0014] Further, at least one of the front side of the back plate and the compression bar is contoured such that a local gap at a given position on along the compression bar is defined as the gap G minus an offset. The offset may be positive in a central portion of the compression bar such that the local gap is less than the gap in the central portion.

[0015] Yet further, the at least one push rod may be two push rods attached to opposing ends of the compression bar.

[0016] Optionally, the translation mechanism may comprise an electric motor and may comprise a crank such that operation of the translation mechanism causes the gap to vary periodically sinusoidally.

[0017] Further optionally, the at least one push rod may be removably attached to the compression bar such as to place the compression bar into an open position so as to facilitate insertion of the bag between the front side of the back plate and the compression bar. A sensor may be configured to detect the open position of the compression bar. Yet further still, a compressive force limiter may be included to regulate the level of compression applied during agitation.

[0018] In a second aspect, a method for agitating a liquid suspension of biological material contained within a flexible bag is disclosed. The method comprises: providing a flexible bag containing a liquid suspension of a biological material; providing a back plate; providing a compression bar parallel to and spaced from the back plate by a gap; placing the bag between the back plate and the compression bar; and varying the gap periodically between a first width and a second width such as to compress the bag with a periodic motion and to cause flow of the liquid suspension within the bag.

[0019] Optionally, the periodic motion may be a sinusoidal motion.

[0020] Further optionally, the bag may be suspended and the compression bar may be placed between one third and halfway up the bag.

[0021] In a third aspect, a manufacturing system is disclosed. The manufacturing system comprises the homogenization and agitation device according to the first aspect above, a bag containing a feedstock of a liquid suspension of a biological material, the bag placed between the back plate and the compression bar of the homogenization and agitation device. A manufacturing device for processing the contents of the bag is provided, the manufacturing device being configured to draw a feed from the bag while the homogenization and agitation device is activated to agitate the contents of the bag and to maintain a constant concentration of suspended biological material within the feed.

[0022] Advantageously, the manufacturing device is an electroporation device wherein liquid suspension of a biological material is a liquid suspension of cells and wherein the system further comprises a feedbag of an exogenous material comprising polypeptides, polynucleotides, pharmaceuticals, polymers, carbohydrates and combinations of these. In the accompanying drawings, given by way of non-limiting examples:

[0023] Figure 1 is an oblique view of a homogenization and agitation device.

[0024] Figure 2 is a schematic representation of a device for agitating a bag of cell suspension;

[0025] Figure 3 is the homogenization and agitation device of fig. 1 showing the bag in a compressed state.

[0026] Figures 4a to 4f show various configuration of contouring of the compression bar and back plate of figs 1 to 3.

[0027] Figure 5 is a manufacturing system comprising the homogenization and agitation device of figs 1 to 3.

[0028] Fig 6 is a graph showing cell concentration as a function of time.

[0029] Detailed Description

[0030] Fig 1 shows an oblique view of the device of the present invention designed for homogenization and agitation of the contents within a suspended cell culture bag. A cell culture bag 1 is positioned between a back plate 8 and a compression bar 7 arranged parallel to and spaced from a front side 8a of the back plate 8 by a variable gap G. Contact between the compression bar 7 and the bag is at a region 10 on the compression bar. As shown in fig. 1 , the bag 1 contains a liquid L. In the uses of concern in this application, the liquid L comprises a suspension of vesicles such as living cells. The bag 1 is suspended from hook 15. An outlet 5 at the base of the bag can be connected to and to supply a manufacturing device 100. Fig. 2 shows a schematic view of the device of fig. 1 . The bag 1 occupies a space between the back plate 8 and the compression bar 7 such that as gap G is varies, this causes the bag 1 to be compressed or relaxed and, accordingly, for the liquid L within the bag to be displaced.

[0031] Regarding the back plate 8 this has the front side 8a facing and in contact with the bag 1 and a back side 8b facing towards the inside of the device. Positioned perpendicular to and passing through the back plate 8 is at least one push rod 9a, 9b having a front end and a back end. As shown in the depicted embodiment, the at least one push rod is two push rods 9a and 9b. The front end of each one push rod 9a, 9b is attached to the compression bar 7.

[0032] As depicted in figs 1 to 3, the at least one push rod is two push rods attached to opposing ends of the compression bar. Other arrangement having the push rods attached at other point along the compression bar 7 can also be contemplated. Typically, the compression bar 7 is positioned between a third and half way up the bag 1 . In typical applications, this means that the compression bar is placed a few centimeters from the bottom of the bag. Incorrect positioning of the compression bar 7, such as at the bottom of the bag or too far up the bag results in ineffective mixing.

[0033] The back end of each push rod is attached to a translation mechanism 20. Upon operation of the translation mechanism, the at least one push rod is caused by the translation mechanism to move perpendicular to the back plate such as to cause adjustment of the gap G between the back plate 8 and the compression bar 7. When the bag 1 is positioned between the front side of the back plate 8 and the compression bar, cycling of the gap G is periodic between a first width and a second width causing the bag contents L to be agitated. Where the bag contents L is a suspension of vesicle such as living cells, operation of the translation mechanism will cause homogenization of the suspension within the bag 1 . Figs. 3 shows the device from the same view point as fig. 1 with the bag 1 , positioned between the compression bar 7 and the front side 8a of the back plate 8. As described above, the compression bar 7 and the back plate 8 are spaced by the gap, G. As shown in fig 1 , the gap G is of a first width which, as illustrated, is wider than the width of the bag such that the compression bar 7 and the back plate 8 do not cause compression of the bag. Fig 3 shows the situation where the gap G is of a second width. As illustrated, the second width is narrower than the natural width of the bag 1 such that the compression bar 7 exerts a pressure on the bag 1 . Alternatively, the first width may be as illustrated in fig. 3 with the second width being yet smaller than the first width.

[0034] Operationally, when the gap G is varied between the first width and the second width, the bag 1 is compressed and fluid is displaced within the bag 1 . Such displacement of fluid as the gap G is narrowed from the first width to the second width results in flows within the bag. These flows are depicted on fig. 3 as arrows f. Periodical cycling of the gap G between the first width and the second width causes the contents of the bag to be mixed and so remain homogenized.

[0035] Where the contents of the bag 1 is a suspension of cells or other biological material, such mixing ensures that the concentration of cells within the bag is maintained uniform and prevents cells from settling within the bag, sticking to the walls of flocculating. A uniform concentration within the bag ensures a nonchanging concentration of cells entering the manufacturing device 100 as the bag is emptied. This is important for ensuring uniform and reproducible outcomes.

[0036] In the arrangement shown in fig. 2, the compression bar is parallel to with all points on the compression bar being equally spaced from the front side of the back plate along its length. Optionally, the compression bar 7 and / or the front side of the back plate 8 is contoured such the distance, that is to say the local gap Gx, between the front side of the back plate and the compression bar is equal to the gap G minus a local offset. Contouring in this manner is used to achieve optimal flow patterns within the liquid L during operation.

[0037] Fig. 4a to 4f shows various possible arrangements. As shown in Fig. 4a the compression bar may have a step shape. This is the arrangement depicted in fig. 1 and fig. 3. That is to say, the offset is positive in a central portion of the compression bar such that the local gap Gx is less than the gap in the central portion.

[0038] The compression bar shown in fig. 4b has a more rounded profile than that shown in fig. 4a. The compression bar shown in fig. 4c has a double step shape resulting in two narrowed portions. The compression bar shown in fig. 4d is parallel to the back plate along its entire length.

[0039] Alternatively, as depicted in fig. 4e, it may be the front of the back plate as opposed to the compression bar which is contoured. The front of the back plate may have a protruding step causing a local narrowing of the gap between the back plate and the compression bar 7. Further, alternatively, as depicted in fig. 4f, both the front of the back plate and the compression bar are contoured. Although as depicted, the contour of the back plate 8 and of the compression bar match one another, other arrangement, such as a combination of figs 4b and 4f, are also to be contemplated.

[0040] Turning now to the translation mechanism 20 as depicted in fig. 2. As depicted in fig. 2, the translation mechanism consists of a crank 12 turned by a motor 13. Conveniently, the motor is an electric motor powered through a controller 14. A connecting rod connects the crank 12 to a cross member 11 positioned between and attached to the push rods 9a, 9b.

[0041] When the motor 13 is energized, movement of the crank, through the connecting rod, the cross member 11 and the push rods 9a, 9b causes the gap G between the back plate 8 and the compression bar 7 to open and close sinusoidally with respect to time. Where the bag 1 is placed between the compression bar 7 and the back plate 8, fluid within the bag 1 is displaced.

[0042] Because of the sinusoidal variation of the gap, no abrupt or damaging forces are imposed on the contents of the bag 1 .

[0043] The controller 14 may include circuitry which measures the current drawn by the motor. Such measurements may be used to limit the force which the mechanism is able to impart on the bag 1 . In the event of a blockage, the motor is designed to stop immediately or reverse its direction. This safety mechanism is activated when an overcurrent situation occurs, indicating a current exceeding a user-adjustable threshold in mA. The overcurrent is occasionally detected when the motor encounters a resistive torque.

[0044] Further, sensors may be included to detect the position of the compression bar 7.

[0045] Installing and removal of the bag 1 from between the back plate 8 and the compression bar may be facilitated by arranging the compression bar to be detached from at least one of the pushrods into an open state. In the open state, the bag 1 can be easily inserted. The compression bar can then be returned to the closed state and secured to the pushrods. Sensors may be installed on the compression bar to detect the open produce an appropriate error signal if operation is attempted in the open state.

[0046] To ensure safety and visibility during operation, a fixed protective cover is installed, made from transparent PMMA material. This cover shields the internal components while allowing visual inspection and monitoring of the system's performance.

[0047] In a representative device, components are constructed from stainless steel material, providing durability and strength. The maximum gap G distance between the compression bar and the back plate is typically set at 175mm with a compression speed operating at 22 compression cycles per minute.

[0048] There will now be described a method for agitating a liquid suspension of biological material contained within a flexible bag 1 . This method comprises the steps of: a) providing a flexible bag 1 containing a liquid suspension of a biological material; b) providing a back plate 8; c) providing a compression bar 7 parallel to and space from the back plate 8 by a gap G; d) placing the bag between the back plate and the compression bar; e) varying the gap periodically between a first width and a second width such as to compress the bag with a periodic motion and to cause flow of the liquid suspension within the bag.

[0049] The periodic motion described above may be a sinusoidal motion such as produced by an electric motor powering a crank. For best results, the bag is suspended and the compression bar 7 is place between one third and halfway up the bag.

[0050] The device described above can beneficially form part of a system for manufacturing or processing biological material. Fig 5 shows an example of such a system comprising the homogenization and agitation device of described above. A bag 1 contains a feedstock of a liquid suspension of a biological material. This bag 1 is placed between the back plate and the compression bar of the homogenization and agitation device as described above. The outlet 5 of the bag is fed into a manufacturing device 100 for processing the contents of the bag in a processing chamber 3. The processed product is passed to a contain 4 for further processing and use. The manufacturing device is configured to draw a feed from the bag while the homogenization and agitation device is activated to agitate the contents of the bag and to maintain a constant concentration of suspended biological material within the feed. As such, the feed from the bag 1 to the manufacturing device 100 remains homogenous throughout. As set out in the introduction above, this is vital is consistent results are to be obtained.

[0051] An example of such a manufacturing system is an electroporation machine. Here, the liquid suspension of a biological material is a liquid suspension of cells. In this case, an additional bag 2 is included containing a feed of an exogenous material comprising polypeptides, polynucleotides, pharmaceuticals, polymers, carbohydrates and combinations of these. The additional bag feeds the manufacturing device 100 through a line 6. In such a case, the processing chamber 3 is an electroporation chamber.

[0052] Example: Efficient homogenization of the cells using the mixer of the present invention

[0053] A cell suspension was prepared from a mix of different cell cultures. The mix was centrifuged 5 min, 1500 rpm, the cell pellet was washed with Cytoporation Medium T (Harvard Apparatus) and centrifuged 5 min at 1500 rpm. The washed cell pellet was then resuspended in 185 ml of Cytoporation Medium T to obtain a final cell concentration of 1 .5 million cells / ml, and the cell suspension was placed in a 250 ml culture bag.

[0054] The bag was then placed in the homogenization system described above and power applied to the translation mechanism to start the mixing. 10 ml samples were withdrawn from the bag using a pump at 0, 5, 45, 90, 120 and 150 minutes after the start of the mixing. After 150 minutes, the translation mechanism was turned off thus the mixing was stopped. Further 10 ml samples were withdrawn from the bag at 15, 45 and 90 min after the stop.

[0055] To measure cell concentration, for each sample collected, cells were centrifuged 5 min, 1500 rpm, and resuspended in 10 ml medium. Cell concentration was measured using Nucleocounter, according to manufacturer protocol.

[0056] Fig. 6 show the concentration of cells as function of time during the first 150 minutes that the bag was agitated using the device of the invention and then during 90 minutes that the device was switched off. Apparent is that the mixing system allowed to keep cell concentration constant at approximatively 1 .3 million cells / ml during 150 minutes. When the mixing is turned off the cell concentration decreased from 1 .3 million cells / ml to 0.8 million cells / ml. Although the mechanism by which the cell concentration was caused to decrease upon turning off the mixing is the subject of academic speculation, these results demonstrate an efficient mixing when using the mixer of the present invention.

[0057] In the above description, the agitation device is given for the bag 1 of cell suspension. It will be appreciated that similar devices can also be attached the bag of exogenous material. Further, it will be appreciated that such a device will find application across the life sciences away from this specific example of an electroporation device to any application where a cell suspension requires agitation. Applications for non-living suspensions are also envisaged.

Claims

CLAIMS1 . A device designed for homogenization and agitation of the contents within a suspended cell culture bag (1 ), comprising: a back plate (8) having a front side (8a) and a back side, a compression bar (7) arranged parallel to and spaced from the front side of the back plate by a variable gap (G) at least one push rod (9a, 9b) having a front end and a back end; the front end of the at least one push rod attached to the compression bar (7), the at least one push rod being positioned perpendicular to and passing through the back plate (8); the at back end of the at least one push rod attached to a translation mechanism (20); wherein operation of the translation mechanism causes the at least one push rod to move perpendicular to the back plate such as to cause adjustment of the gap (G) periodically between a first width and a second width; wherein when the bag (1 ) is positioned between the front side of the back plate (8) and the compression bar (7) and the gap (G) is periodically varied between the first width and the second width the cell culture bag contents is caused to be homogenized and agitated.

2. The device of claim 1 wherein at least one of the front side of the back plate and the compression bar is contoured such a local gap (Gx) at a given position on along the compression bar is defined as the gap G minus an offset.

3. The device of claim 2 wherein the offset is positive in a central portion of the compression bar such that the local gap (Gx) is less than the gap (G) in the central portion.

4. The device of any of claim 1 to claim 3, wherein the at least one push rod is two push rods attached to opposing ends of the compression bar (7).

5. The device of any one of claims 1 to claim 4, wherein the translation mechanism comprises an electric motor (13).

6. The device of any one of claims 1 to 5, wherein the translation mechanism comprises a crank (12) wherein operation of the translation mechanism causes the gap (G) to vary periodically sinusoidally.

7. The device of claim 1 or claim 6 wherein at least one of the at least one push rod is removably attached to the compression bar such as to place the compression bar into an open position so as to facilitate insertion of the bag (1 ) between the front side of the back plate (8) and the compression bar (7).

8. The device of any one of claims 7, further comprising a sensor to detect the open position of the compression bar.

9. The device of any one of claims 1 to 8, further comprising a compressive force limiter to regulate the level of compression applied during agitation.

10. A manufacturing system comprising: a homogenization and agitation device of claim 1 to 9, a bag containing a feedstock of a liquid suspension of a biological material;the bag (1 ) placed between a back plate (8) and a compression bar (7) of the homogenization and agitation device; a manufacturing device (100) for processing the contents of the bag (1 ); the manufacturing device being configured to draw a feed from the bag while the homogenization and agitation device is activated to agitate the contents of the bag and to maintain a constant concentration of suspended biological material within the feed.11 . The system of claim 10 wherein the they manufacturing device is an electroporation device wherein liquid suspension of a biological material is a liquid suspension of cells.

12. The system of claim 10 or claim 11 wherein the system further comprises a feedbag (2) of an exogenous material comprising polypeptides, polynucleotides, pharmaceuticals, polymers, carbohydrates and combinations of these.

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