Learning Objectives
In this lecture, you will learn•
What is containment in downstream processing?•
Containment in cell separation•
Containment in cell disruptionRecent advances in molecular biology and recombinant DNA (r- DNA) technology have enabled products of animal, plant or microbial origin to be produced in large quantities by culturing bacteria, yeast, plant or mammalian cells. A typical bioprocess will consist of growing cells in a suitable nutrient medium, followed by the recovery and purification of the product:
downstream processing. If the desired product is extra-cellular then
the first stage in processing will be the removal of large solids and cells by centrifugation or filtration. The broth is then fractionated or extracted into major fractions; this can be done using processes such as chromatography, liquid-liquid extraction or
precipitation. The fraction containing the product may then be
purified further, often with more specialized chromatographic techniques. However, the majority of products remain intracellular, enclosed in a soluble or insoluble form within the cell. Some of these products are cytoplasmic, others are associated with cell membranes, cell wall components or the periplasm (where present). In this case, the cells must first be harvested to form a concentrated slurry or paste, then disrupted to release their products into solution for subsequent extraction and purification.
We have seen that the chances of microorganisms coming in contact with the workers are more in case of downstream processing as compared to the fermentation as such.
Downstream processing operations are high energy operations and hence generate fine sized aerosols, which, as we have seen, more potent in terms of causing infections and allergic reactions. Therefore, containment of downstream processing is probably more important than containment of fermentation. Of all process equipment, centrifuges, in addition to fermentors themselves of course, are most likely to release micro-
organisms. It is possible to kill process micro-organisms after the fermentation is complete so there may be no need for containment in further processing steps to eliminate the infectious risk. However, even dead micro-organisms could present an allergenic risk. Most reported health problems have been associated with downstream processing. The greatest demands on biosafety occurred from the time the broth leaves the bioreactor to the final processing steps, as this involves dealing with large amounts of cell debris. Downstream processing frequently involves the use of machinery that rotates at high speeds (centrifuges) or exerts increased pressure (liquid extrusion homogenizers, cross-flow microfiltration and ultrafiltration units). Such energetic processes may generate
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filtration devices available for initial cell separation. However, the choice is restricted in biotechnology due to the limitations imposed by the nature of fermentation broths. The filters used for initial solids recovery (e.g. recovery of biomass from fermentor broths) are of two main types: the rotary vacuum drum, a continuous filter, and the filter press, a batch filter. Generally, filter presses are slow and labour intensive and are usually only used at small scales. They are often found in the older style biotechnology processes such as brewing and distilling. Rotary vacuum drum filters can be used for larger scale continuous operations and they are more often found in the pharmaceutical and food industries. It is easier to contain a rotary vacuum drum filter, e.g. using local exhaust ventilation, than a filter press but it is not possible to operate a rotary vacuum drum in an aseptic manner. Filter presses usually operate at pressures between 5 and 7 bar. Rotary vacuum drum filters operate such that the vacuum pressure is applied internally so the filtrate is drawn through the filter, into the drum and finally into a collecting vessel. Considering the low pressures and low rotational speeds used in such devices, their operation should not present a problem in terms of
containment and aerosol formation. However, when the cake is removed from the filter there is potential for considerable release of biological material.
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What about the membrane filtration?In order to concentrate and purify the product more, it is possible to use membrane filtration. Here, some form of semipermeable membrane is used to separate the components of a liquid stream. In most of the commercially important processes the driving force is pressure, the solvent (usually water) is driven through the membrane while the solute(s) are retained. This type of process includes reverse osmosis, ultrafiltration and microfiltration.
Cross flow membrane filtration has attracted attention in recent years as an alternative to high g force centrifugation. Scaling up from laboratory or pilot scale is relatively easy, as additional modules/units can be added to increase the surface area for filtration; this can, however, be costly. The major disadvantage of these techniques is the detrimental effect of membrane fouling on filtration rates and subsequent product recovery. Generally, membranes are considered to have less potential for the emission of aerosols or breach of containment, compared with centrifuges. Difficulties may be encountered when cleaning membranes in situ. It may only be achieved adequately through the dismantling of the filter units. This process could be hazardous in terms of aerosol production, so adequate precautions should be taken, i.e. the use of secondary containment.
Traditionally, most membranes have been fabricated from plastics such as polysulphones and cellulose acetate. In recent years, inorganic membranes, made from materials such as ceramics and metals, have been introduced and these have found application in cell recovery. The robustness of inorganic membranes are generally higher than plastic membranes, offering higher temperatures (suitable for sterilization) and higher operating pressures. Ceramic membranes, however, are
vulnerable to heat shock and mechanical shock, i.e. they are brittle and can be broken.
A wide range of membrane equipment designs are available for cell recovery and other applications at both pilot and production scale. The inherent containment features vary widely. Plate and frame membrane filters rely on seals on each plate and the clamps on the assembly for containment. Hollow fibre systems are pressure limited and are often fitted with a pressure switch in order to prevent the recirculation pump reaching the bursting pressure of the fibres.
Tubular membrane systems appear to offer the best
containment features because they are usually constructed with hard piping and require fewer seals to the outside environment. The collection shrouds on the low pressure side would provide a convenient shield should the membranes or the filter seals fail. Metal membranes can be constructed using welding and this negates the need for seals.
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The centrifugation operations would also require strict containment. Won’t they?Most certainly. The separation of biomass from growth media is a difficult operation, as cells have almost the same density as their surrounding medium, are small, are able to form stable colloids and are cohesive. Sedimentation of cell debris presents an even more difficult problem for biotechnologists and the choice of separation technique is limited. Solid bowl and tubular bowl centrifuges are relatively inexpensive and have in the past been chosen for use in the biotechnology industry. They are useful for small batches, but are labour intensive because the solids have to be dug out by hand. The scroll decanter centrifuge has limited use in the biotechnology industry because of the low g forces generated. It should, however, be better contained than the traditional solid or tubular bowl type of centrifuge. In reality, only the higher g force devices such as disc stack and tubular bowl centrifuges are used at large scale. Decanter and solid bowl centrifuges are however used for separating bigger particles, such as yeast or flocculated bacteria.
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Could we study such an example of commercially available centrifuge?Why not? Let me tell you about the Centritech Cell Separator. This is relatively recent development. The Centritech Cell Separator is designed for aseptic separation of mammalian cells in a completely closed system without any rotating seals. It contains a spinning disposable bladder which lies within the rotor that spins at speeds up to 1200 rpm. The centrifugal force created within the bladder separates the culture into cell concentrate and fluid. A system of tubing and pumps enables the cell culture to enter the bladder directly from the
fermentation vessel. The tubing is connected to the rotating bowl in a way that allows one end of the tube to rotate while the other end is standing still. Thus the separation system is totally enclosed. Further primary containment is provided by a sealed lid on the rotor chamber and an external hood which acts as built on secondary containment. The separation insert is delivered as a pre-sterilized disposable plastic bladder. The novel design of the Centritech Cell Separator, with no openings to atmosphere and no rotating seal, means that it is unlikely to
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produce biological aerosols during normal operation. The containment of this device can be tested by simulating rupture of the bladder. Micro-organisms are detected outside the primary containment of the sealed lid; however, none are detected outside the secondary containment. If the interior becomes heavily contaminated, decontamination may be difficult.
The Centritech Cell Separator has a very low separating capacity (100 1/ hour) and therefore cannot compete with disc-stack separators.
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What are disc-stack separators?Disc-stack centrifuges predominate at production scale in biotechnology. They consist of a solid bowl containing a series of hollow truncated cones (‘discs’) stacked one upon another. Feed suspension enters the centrifuge through a central feed pipe, passes out of the edge of the bowl then upwards and inwards through the stack of discs. Solids settle onto the lower surface of each cone and clarified liquid moves inward and upwards to reach an annular overflow channel, emerging at the neck of the bowl around the feed pipe. The sedimented solids slide off the disc and collect in the space between the stack of discs and the bowl wall.
The different types of disc-stack centrifuge are distinguished by the method in which they discharge solids from the space between the discs and the wall. In solid-bowl or solids-retaining disc-stack centrifuges, the machine has to be stopped for solids to be removed manually. In nozzle discharge disc-stack centrifuges solids are discharged continuously. Opening bowl, solids-ejecting or intermittent discharge disc-stack centrifuges discharge solids either at preset time intervals or discharge is automatically triggered by the load on the bowl.
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What are nozzle- discharge disc – stack centrifuges? Solids discharge from nozzle discharge disk-stack centrifuges is normally continuous. Two different types exist. In the Alfa Laval BTUX 510, the solids are collected in conical storage spaces, with concentrate tubes located around the largest diameter of the bowl in the apex of the cones. Solids pass through the concentrate tubes and the vortex nozzles into the paring tube chamber. The concentrate is skimmed off by the paring tube and discharged under pressure. The clarified liquid phase is displaced towards the centre through the disc-stack. The centrate is then discharged under pressure via a paring disc pump at the top of the frame hood. In the BTUX 510, the unique vortex nozzles automatically compensate for variations in feed flow rate or feed solids concentration to ensure a constant concentration of the discharged solids phase. (See diagram)In the second type of nozzle-discharge disc-stack centrifuge the solids are collected in a triangular storage space with nozzles located around the largest diameter of the bowl. The size and number of nozzles can be optimized for each application, so that too dilute slurry is not discharged, but it is sufficiently fluid to flow through the nozzles.
A further development of the nozzle-discharge disc-stack centrifuge incorporates an additional annular valve at the periphery of the bowl. This centrifuge therefore has the same
type of solids discharge as a solids ejecting disc-stack centrifuge. This hybrid is also equipped with extra nozzles around the bottom of the bowl. As well as giving the centrifuge a CIP facility, the additional feature means that blocked discs can be cleared by initiating a full desludging.
Another type of disc stack centrifuges are opening bowl disc stack centrifuges.
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What are they?Opening-bowl, or solids-ejecting disc-stack centrifuges are very common in large and pilot scale biotechnology plants. They have been the most widely researched in terms of sterile or contained operation and are similar in design to solid bowl and nozzle-discharge disc-stack centrifuges, but here peripheral ports in the solids collection area are held closed by water or air pressure to retain sedimented cells during separation. At a predetermined time interval, the feed-stream ceases and the ports open to allow the solids to eject (termed ‘desludging’). They are often the only type of centrifuge capable of continuous separation of cells and cell debris because the frequency of solids discharge can be set to maximize the sedimented solids concentration.
Most devices have cyclone receivers to contain the discharge of sludge. However, a considerable shock wave is generated by the centrifuge and the air which is then displaced from the cyclone may contain aerosols of cells or debris unless suitable vent filters are fitted. Lawrence and Barry report shock waves during discharge from an Alfa-Laval AX 213 Separator, thought to be sufficient to allow aerosol to escape from cartridge housing air vents. Walker et at. describe modifications to a Westfalia CSA 19-47476 centrifuge. The vent filter was blocking due to massive aerosol formation during desludging, so it was removed and attached to the main frame drain, thus increasing the distance between the solids receiver and the filter. This alleviated the vent filter blockage problem.
There are several such types of centrifuges. But we will see about just one more.
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Which is that?That is solid bowl centrifuges. Solid bowl centrifuges have the feed stream entering from the bottom of the bowl and moving upwards. Solids are sedimented in the bowl and centrate flows out over a weir. Single chamber, triple bowl and multichamber devices are available, each with a larger surface area and hence greater efficiency. Sedimented solids can be removed
intermittently manually or automatically using a plough with the bowl rotating slowly. Normal operational speeds lie between 450 and 3500 rpm, developing centrifugal forces in the range of 500 to 1200 g force.
A novel design, the Alfa Laval-Sharples SP-725 Superhelix, is shown in the figure. This is a vertical solid bowl centrifuge. The product stream is fed through a stationary feed nozzle at the bottom of the bowl and gently accelerated to bowl speed in the conical feed zone. Under the action of centrifugal force, the solid phase moves to the bowl wall where the helical conveyor forces it downwards to the beach. Here, the solid phase is further concentrated. Solids are finally discharged into the solids chute at the bottom of the centrifuge, and to prevent escape of
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material the method of solid collection must be contained. The manufacturers state that they can supply suitable equipment for solids handling. The centrate is discharged by a centripetal pump at the top of the bowl. The automatic solids discharge of the Alfa Laval-Sharples SP-725 represents an improvement in solid bowl centrifuge design.
The Btux 510 Nozzle Discharge Disc Stack Centrifuge
The SP-725 Superhelix
Exercise: Find out what are the different types of centrifuges, their manufacturing companies and their brand names. Feel free to use internet and make a list in the space provided below:
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After we have separated the cells we need to break themup. How do we do that?
We can do that by a number of cell disruption techniques. Let us see more about cell disruption first.
Cell disruption
Disruption may involve physical, mechanical or chemical steps to allow intracellular products (usually proteins) to be extracted from cells. Alternatively, it may consist of merely removing certain components from the cell wall or membrane, to permit product leakage. There are many methods of disrupting cells. The suitability of each method depends on the scale of production, the protein to be isolated, the individual cell suspension, and the disruption techniques available. The performance of each technique is dependent on cell type, culture conditions, pretreatment, and the device used.
Physical or mechanical methods of cell disruption are the most widely researched in terms of containment. The underlying principle is either by breakage of the cell wall by mechanical
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contact, the application of liquid or hydrodynamic shear forces, or the application of solid shear forces. Cell disruption by non- physical methods generally involves simple operations which may be carried out in large tanks or vessels, which mayor may not require agitation.
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What are the various physical methods of cell disruption?Again, there are several methods. We will see some of them.
Agitation with abrasives
Micro-organisms in dry or frozen solid form can be disrupted by conventional ball or vibratory mills used in the chemical process industries. Whilst the method of dry milling may be efficient, it raises a number of problems, including caking of fine powders (at around 1 mm most bacteria are smaller than powders that are generally milled in the chemical process industries), erosion of the mill surfaces including liners and balls (which leads to contamination of the disruptate), and the generation of heat energy which can denature the desired product. In the biotechnology industry, it is much more common to employ wet milling where disruption is caused by a mixture of hydrodynamic shear forces and mechanical crushing. Bead mills are generally operated at near ambient pressure. When disrupting very thick cell pastes, there may be a slight build up of pressure in the vessel, but it is unlikely to exceed 0.2 bar, so bead mills are unlikely to cause aerosols to be released during operation. In the event of seal failure or a leak, however, even this low pressure is likely to lead to aerosol formation. Design of a typical continuous bead mill is given below:
Liquid Extrusion
This method has been widely studied and relies on the principle that forcing a cell suspension at high pressure through a narrow orifice will provide a rapid pressure drop. This is a very powerful means of disrupting cells. It is a relatively simple matter to design equipment to subject the cell suspension to shear forces before releasing the pressure. By varying the pressure applied, cells may be completely or only partly disrupted (the latter usually being sufficient for the release of periplasmic enzymes).
The earliest devices to employ this principle were the French Press and the Chaikoff Press. Both these devices are relatively crude and simple which can only disrupt small batches of cell suspensions. The next stage in the development was the