US3536370A - Controlled environment apparatus - Google Patents

Description

as. T. EVANS ETAL 3,536,370

CONTROLLED ENVlRONMBNT APPARATUS 3 Sheets-Sheet 2 Filed Dec. 18, 1967 IIIIIIIIIIIIII/ ZIKZIJIZIIIL j Inventofs (Ii/muss 66R vnsz Home: Evmv: Roam HHRRw-SmrrH' M OCmwww, Attorney;

FIG. 2

c. G.T. EVANS ETAL 3,536,370

CONTROLLED ENVIRONMENT APPARATUS 3 Sheets-Shea 1 Filed Dec. 18, 1967 III FIG. I [nvmtom [1mm ES GERVHsE mom/came: Eva/vs Foam Hams-8mm: fifiww uflzg 0%, Attorneys Oct; 27, 1970 c. 6.1. EVANS ETAL 3,536,370

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v Inventors Omnes Geravase 7Z1oRlvenT Evnus Roe/N H/IRRIS Smn-H MA fa Mu, Attorneys United States Patent Ofifice 3,536,370 Patented Oct. 27, 1970 US. Cl. 312-1 9 Claims ABSTRACT OF THE DISCLOSURE Apparatus for carrying processes in a controlled environment, and particularly for carrying out microbiological processes involving the production of dangerous pathogenic organisms, comprises an outer casing enclosing a process chamber, a transfer chamber, communicating trap means between said process chamber and said transfer chamber having a sterilising lock, means preventing the ingress to said process chamber of unsterilised air from the atmosphere surrounding said outer casing and means preventing the egress of contaminating matter from said process chamber to the atmosphere surrounding said outer casing; said transfer chamber having sealable door means communicating with said surrounding atmosphere and permitting access for a transfer vessel.

The present invention relates to apparatus for carrying out processes in a controlled environment. It is especially concerned with apparatus wherein a process, particularly a chemical or microbiological process, is carried out, the apparatus being arranged to prohibit as far as possible the ingress or egress of noxious matter or contaminants.

For example, in microbiology a culture of organisms which may be dangerous pathogens may be grown in a process chamber sealed against escape to atmosphere. In other processes the process chamber is sealed so that articles being produced or treated therein may be guarded against contamination from the atmosphere. The process chamber may be a glove box or may contain externally controlled manipulators.

The greatest danger of contamination, either of material or the atmosphere, arises during transfer of substances to or from the chamber, even when some form of lock or trap is employed. Cultures produced in liquor in a sealed fermenter are commonly harvested by pumping or draining from the fermenter into a sealed transfer vessel through a flexible tube introduced into and withdrawn from the sealed chamber through a sterilising lock which usually comprises a liquid trap containing a liquid such as formalin. Hitherto the sealed transfer vessel has been in the outer atmosphere during transfer, with the various attendant disadvantages such as the danger of a fault in the flexible tube or an incomplete seal between the tube and the transfer vessel, the danger of damage to the transfer vessel, tube or seal by accidental knocking or movement of the vessel standing apart from the process chamber, and the danger of implosion or explosion of the transfer vessel when suction or pressure is employed.

According to the present invention, apparatus for carrying out processes in a controlled environment, particularly microbiological processes involving highly dangerous pathogenic organisms, comprises an outer casing enclosing a process chamber, a transfer chamber, communicating trap means between the process chamber and the transfer chamber having a sterilising lock, means preventing the ingress to the process chamber of unsterilised air from the atmosphere surrounding the outer casing and means preventing the egress of contaminating matter from the process chamber to the atmosphere surrounding said outer casing, the transfer chamber having sealable door means communicating with the surrounding atmosphere and permitting access for a transfer vessel and having internal sterilisation means. In operating the apparatus, a sealed flexible tube is connected to the transfer vessel and inserted into one side of the sterilising lock, the sealable door is closed and sealed, the flexible tube is removed from the other side of the lock by use of sealed gloves or a manipulator and connected up within the process chamber to make the transfer connection, after which the transfer is effected with the transfer vessel enclosed within the sealed casing. After transfer, in order to remove the transfer vessel, the flexible tube is discon nected inside the process chamber, sealed, and Withdrawn through the lock. The transfer vessel can then be removed through the reopened door without risk of contamination into or out of the process chamber through the tube, or risk of matter in the lock entering the transfer vessel. An important advantage of the present invention is that relatively large transfer vessels may be used safely which are too large to pass through the sterilising lock.

In microbiological production plant (including a culture vessel, stirring means therefor, a medium vessel, and a harvester) the process chamber containing the culture vessel is advantageously in the upper part of the casing with the transfer chamber, for receiving the transfer vessel below so that liquid can be drained into the transfer vessel by gravity.

The transfer chamber may be kept substantially sterile by being flushed by air entering through a filter and leaving it through another filter. When contamination of the atmosphere is at risk then preferably suction means is employed at exit of air from the apparatus; when the sealable door is opened the slightly low pressure inside will cause air to be drawn into the transfer chamber and filtered before it can re-enter the atmosphere rather than air which might possibly have been contaminated from the culture, being blown out through the door. In plant for handling substances which might be contaminated from the outer atmosphere the air is preferably forced in so that a slight pressure will be maintained within the transfer chamber and air will be blown out when the sealable door is open. Means are provided for sterilising the transfer chamber and the process chamber. The means may include ultraviolet or irradiation means, and an evaporator of sterilising vapour.

Sealed process chambers for growing or handling of dangerous cultures have hitherto been more or less permanent fixtures and have been constructed of metal plates bolted together, possibly with windows of glass or transparent plastics material such as polymethylmethacrylate, and the joints between the plates and between the fixing bolts and the plates have had to be effectively sealed and the seals maintained. For reduction of weight and increased safety it is an important feature of the present invention that the sealed process chamber or an apparatus incomporating the chamber is of jointless construction in reinforced plastics material such as, for example, glass fibre reinforced plastics material. The casing, in so far as it cannot be moulded, may be built up of plates of plastics material fused or welded together to avoid any joints requiring seals except for access panels where needed. A complete casing may be built up by welding together moulded parts. Where the process is such that it is necessary or permissible that the material of the sealed process chamber should be exposed to light, at least the Walls of this chamber or the walls of the complete casing may be of translucent or transparent material; otherwise, for viewing the interior of the chamber a transparent window portion can be welded or sealed into the wall and if it be desirable to reduce to a minimum the amount of light getting into the chamber the window welded in can be small and normally covered externally by a blind. The construction according to the invention allows any feature such as this to be integrally included without requiring sealable joints. The light weight resulting from this construction makes possible the design of a mobile apparatus which can be moved about from one laboratory to another as required.

The apparatus of the invention may be provided with means whereby liquids or objects can be transfered from outside the apparatus into the process chamber. The means may include a lock or trap such as a sterilising liquid or vapour lock which can be part of the transfer lock or trap. When a dry lock is used an air pressure difference should be employed between the process chamber and the outside, the difference being such as to prevent the fiow of contaminated air in one direction or the other as required. Further sterilising means may be provided in the dry lock, if desired.

According to a feature of the invention the process chamber and/or the transfer chamber are each provided with a well suitable for retaining all liquid accidentally split during use of the apparatus.

The apparatus of the invention may be used, for example, in microbiological processes such as the continuous or batch cluture of pathogens and tissue cells. In the culture of organisms three main vessels are employed, these are a medium vessel for containing substance required as an environment for growth of organisms, a culture vessel wherein the organisms are grown, and a harvest vessel to which the organisms are temporarily transferred after growth. These vessels would be mounted in the process chamber of the invention, and may be mounted on a common mounting removable through an access panel for the purposes of, for example, sterilisation in an autoclave. The transparent window in such cases is advantageously provided with glove ports by which the stages of the process can be manipulated and samples taken for such purposes as dilutions and plate counts, which can also be carried out in the safety and aseptic atmosphere of the process chamber. Moreover an incubator can be incorporated within the process chamber to enable the plate counts and routine sterility tests to be made within the confines of the apparatus.

Mobile apparatus in accordance with the invention will now be described, by way of example, with reference to the accompanying drawings, of which:

FIG. 1 is a front elevation of the apparatus with the access door open and the transfer vessel in cross section.

FIG. 2 is a front elevation of the apparatus with the front face cut away and the transfer vessel and instruments removed, and

FIG. 3 is a view on IIIIII in FIG. 1, with the transfer vessel removed and the access door is closed.

As shown in the figures, the apparatus comprises a casing 1 constructed of glass fibre reinforced plastics material. The interior of the casing 1 is divided into distinct chambers, a process chamber 2 having an accident well 3, a transfer chamber 4 having an accident well 5, and an instrument chamber 6. The process chamber 2 occupies the upper half of the interior of casing 1 and the accident well 3 a rearward portion of the lower half. The transfer chamber 4 occupies the lower portion of the interior of the casing 1 in front of the accident well 3, and the instrument chamber 6 occupies the remaining side half of the lower half of the interior of the casing 1. A Well 7, of similar material to that of the casing 1, divides the instrument chamber 6 from the accident Well 3 and the transfer chamber 4, and a wall 8, also of the said material, divides the accident well 3 from the transfer chamber 4. A process chamber floor 9 divides the process chamber 2 from the transfer chamber 4 and the instrument chamber 6, the rear part of the floor 9 above the instrument chamber 6 being sloped to provide a draining board into the accident well 3. The accident well 3 may be divided from the process chamber by a grill which also extends above the sloping part of the floor 9. A window 10 is sealed into the floor 9 of the process chamber to permit viewing of the interior of the transfer chamber. The transfer chamber 4 and the instrument chamber 6 are thus sealed from each other and from the process chamber 2. and the accident well 3. The casing 1 has a window 12 by which one can see into the process cham ber 2, and an access door 13, which also has a glove port 14 (shown in dotted outline only), in the front of the transfer chamber 4. The casing 1 may have a further sealable access panel for the process chamber 2, or if desired this access can be obtained by bolting the window 12 in position together with a sealing cushion. The window 12 is fitted with glove ports 15 in convenient positions. The access door 13 is hinged to the casing 1 and has an outer flange 16 surrounding, but spaced from, a flange 17 on the casing 1, the space between them accommodating an air pressurisable tube or a rubber seal (not shown) for sealing the access door 13 to the casing 1. A screw seal access hatch 18 is provided in the wall 7 at a height which will permit a person to be able to reach to any part of the bottom of the accident well 3 and yet which is above the level that all the liquids being used in the process chamber 2 would reach if they were drained into it. The glove port 14 into the transfer chamber 4 is sited so that one can open the hatch 18 through it with the access door 13 closed. The hinging of the door 13 is such that the door can open about An instrument and control panel 19 is let into the casing 1 in the forward face of the instrument chamber 6, in such a manner that no instruments or controls project beyond the front of the casing 1. The instruments may be covered with a transparent lockable door, if required, to forestall tampering and accidental alteration of control settings.

A transfer and exit liquid lock 20 is fitted in the side of the casing 1, with its exit well 21 protruding from the casing 1 and having a hinged lid 22. An internal wall 23 of the lock juts into the transfer chamber 4 and is open directly into the process chamber 2 where it is fitted with a sliding lid 24'. The sliding lid 24 extends the working surface in the process chamber 2 and can be used to restrict liquid loss by evaporation. A transfer well 25 is let into the internal well 23 from the transfer chamber. The arrangement is such that with sufficient liquid in the lock 20, the liquid seals the process chamber 2 and the transfer chamber 4 from each other and from the exterior of the casing 1. The look 20 may be of a translucent plastics material whereby the level of the liquid can readily be seen. The lock may also be fitted with an automatic feed and constant level device (not shown). The transfer well 25 is provided with a removable sealing cap 26 to prevent loss of liquid by spillage. An inlet lock 27 is mounted in the casing 1 to communicate between the exterior and the process chamber 2. The lock 27 is a straight tube of bore sufiicient for the passage of small containers or bottles. It has a screw-compressible seal cap at each end and is mounted so that it slopes downwards into the process chamber 2 at an angle of between about 30 and 60 to the horizontal. The ultraviolet strip lights are fitted in a recess 28 along the bore of the inlet lock 27 and the inside surfaces of the inlet lock are coated with aluminised paint to retain and reflect ultraviolet radiation within the inlet lock. Conveniently the inlet lock is composed of a translucent material and one or more gaps are left in the aluminised paint coating so that visual confirmation may be obtained that the lights in 28 are functioning.

An air ventilation system is provided by which the instrument chamber 6, the transfer chamber 4, and the process chamber 2 can be continuously flushed with air, at a pressure lower than that of the exterior. A coarse filter 29 is fitted across a hole in the casing 1 where it forms part of the Wall of the instrument chamber 6. Fine filters 30 and 31 are fitted, the first low in the wall 8 between the instrument chamber 6 and the transfer chamber 4, and the second high in the wall 7 between the transfer chamber 4 and the accident well 3. Two or more fine filters 32 are fitted on either side of the process chamber 2, and these communicate through the casing 1 with sets of pump fans 33- (only one set of filters and fans are shown). A sterilising heater may be fitted to sterilise exhaust air from the process chamber after filtration. While the filter 29 may be made of gauze, a suitable material for the fine filters 30', 31 and 32 is a glass fibre filtration medium which can successfully retain microbial organisms, viruses or radioactive dust. The transfer chamber 4 is made with as small a volume as possible so that when in operation a maximum airflow can be obtained therein to substantially obivate the possibility of any contamination. The access door 13 is also as small as possible so that if opened during operation the inrush of air through it will further minimise possibility of loss of contaminating substances. A small evaporator 34 for sterilising liquid is also fitted near the bottom of the transfer chamber 4 just above the level of access door 13, i.e., above the accident well 5. The evaporator 34 is arranged to be fed from a pot 35 outside the chamber 4, via a tap 36 and a U-tube 37 which will form a liquid lock. A small window 38 may be sealed into the casing at the side of the transfer door to allow viewing of the evaporator liquid level. Ultraviolet lamps 39 and 40 may be fitted in the roofs of the process chamber 2 and the transfer chamber 4 respectively. Shields 41 mounted on the dividing walls 7 and 8 protectthe filters 30 and 31 from liquid drops.

On the floor of the transfer chamber 4 is a stand 42 having a perforated deck which is level with or just above the bottom of the opening for the access door 13. The stand 42 is for supporting the transfer vessel, shown at 43 in FIG. 1, made of glass. The transfer vessel 43 is, for safety purposes, housed in a sealable metal container 44 having a sealing lid 45 and is preferably offered to the access opening on a trolley of similar height to the deck of the stand 42. The transfer vessel 43 has a flanged neck surrounded by a chemically resistant rubber collar 46 which seats a metal clamping collar 47. A bung 48 carrying two transfer tubes, 49 and 50, is held tightly into the neck of the vessel 43 by an arrangement of clamping bolts 51 acting between a clamp 52 and the clamping collar 47. To the two tubes 49 and '50 are fitted flexible transfer tubes 53 and 54 of sufficient length to pass down through the transfer Well 25, up through the internal well 23 and well into the process chamber 2. Alternatively the tube 50 may be fitted with an air filter for simply balancing pressure.

The casing 1 is mounted on a trolley 55 fitted with foot operated brakes and the overall height of the apparatus shown is less than 7 ft. The breadth is less than 4 ft. and the depth less than 3 ft. The apparatus is thus mobile and can thus easily be pushed from room to room as required.

The particular apparatus shown is adapted for use in the production of pathogenic organisms, by a microbiological fermentation process. Inside the process chamber 2, there are a culture vessel 56 with its associated stirring motor assembly 57, a medium vessel 58 below and to one side of the culture vessel 56, and a harvesting vessel 59 below and to the other side of the culture vessel 56. Thus the culture vessel may be drained by gravity.

The vessels 56, '58 and 59 may be constructed conveniently from glass pipeline lengths; for example the vessels 58 and 59 from 12" lengths of 12" diam. pipeline and the culture vessel 56 from. 6" length of 6" diam. pipeline closed at the ends by gasketted top and base plates. The base plates of vessels 8 and 59 can be bolted to lengths of metal angle 60 bearing wheels which run on guides set in the floor of the process chamber and the top plates support the base plate of the culture vessel 56, the stirrer motor and the various necessary pumps and air filters. This arrangement results in a compact, easily operated apparatus which can be removed from the process chamber through the sealable access panel 61 for sterilising in an autoclave without the need to break any liquid or air lines. Wiring f0 rthe various measurement and control instruments is conveniently carried in a conduit channel 62 from the instrument chamber 6 to hermetically sealed plugs 63 in the process chamber 2. The only external supply as needed to the apparatus in this case are electrical power, and compressed air which are both taken directly into the instrument chamber 6.

In the preparation of the apparatus for a process such as a fermentation process the vessels 56, 58 and 59 and the culture medium are installed, and the access panel 61, window 12 and door 13 sealed. A sterilising liquid such as Formalin is placed in the look 20, and the fans 33 are switched on. Because of the filters 30, 31 and 32 a pressure of /2-2 inches of water below atmospheric is thereby maintained in the process chamber 2. Control of the process is effected through the instrument and control panel 19 and the glove ports 15. Instruments and sampling bottles etc. may be introduced into the process chamber 2 by removing the outer cap of the lock 27, inserting the instruments or sampling bottle etc. and replacing the outer cap. The ultraviolet lights 28 may then be switched on to sterilise the objects before they enter the process chamber. With the operators left and right hands in the left and centre gloves 15 respectively, the inner cap of the lock 27 is removed from the object to be transferred into the process chamber 2. With the air pressure difference as described the air flow through the lock 27 during loading inhibits the escape of bacteria even when neither cap is in place. The ultraviolet lights should be switched on when either cap has been removed and subsequently replaced.

Replenishment of the medium vessel 58 is preferably carried out from a transfer vessel 43, and in order to do this when the process is operating, the access door 13 is opened.

A transfer vessel 43 containing sterile culture medium and housed in a sealable container 44 is loaded into the transfer chamber 4 with the two tubes 49 and 50 projecting through a threaded hole 65 in the lid into which a filter 66, conveniently attached loosely to the lid, may be screwed. Tube 54 connects with the transfer vessel 43 via a filter 67 and tube 53 is equipped with a suitable fitting for connection to the medium vessel 58. The two tubes are passed (after removal of the cap 26 which prevents over-spill of the sterilising liquid from the transfer well during passage of articles through the sterilising lock 20) through the transfer well 25 into the liquid sterilising lock 23 and the door 13 is closed and sealed. The tube 53 is retrieved from the sterilising lock 20 using one of the gloves 15 and is connected to the medium reservoir 58. The tube 54 is retrieved from the sterilising lock 20 and connected outside the apparatus to a source of low pressure compressed air. Medium is thus forced from the transfer Vessel 43 into the medium reservoir 58. After completion of the transfer the tube 53 is clipped off in the process chamber, tube 54 is disconnected from the compressed air supply and, by use of the glove 14 in the transfer chamber door, is drawn through the lock 25 together with tube 54--after a suitable period in the sterilising liquid-and both are passed through the hole 65 in the sealable lid of transfer vessel container 44. .When all the tubing has been passed into the container, the filter 66 is screwed into the threaded hole 65 and the transfer well 25 is covered with the cap 26. The sealable door 13 can then be opened and the sealed container removed for sterilising in an autoclave for safety.

Any container or instrument to be removed from the process chamber 2 should be dropped into the well 23. The operator can retrieve it from the well 21. The slope of the lock 27 renders it awkward to remove containers and instruments therethrough by unskilled operators. It

7 is easier to remove the containers or instruments through the lock 20 and thereby sterilise them.

When liquid such as harvested culture or unwanted medium is to be removed from the process chamber 2, a transfer vessel in its container, similar to that previously described but with a filter in place of tube 54 and with wider bore tubing for 53 which alone projects through the threaded hole 65, is placed in the transfer chamber. The tube 53 is passed through the transfer well 25 into the well 23 where it is lifted into the process chamber and connected to the harvesting vessel 59. Grown culture then flows from the harvesting vessel 59 into the transfer vessel by gravity until empty. The tube 53 is then disconnected, sealed and the ends left soaking in the sterilising lock 23 for a suitable time after which the tube 53 is withdrawn into the transfer vessel container as previously described. The subsequent procedure is as described for the medium transfer.

If there should be an accident in the process chamber 2 such that live pathogens are certain to have escaped in quantity into the process chamber and accident well 3, the ultraviolet light 39 or the evaporator 34 are switched on. The port 18 may be opened using the glove 14, with the door 13 sealed, to allow Formalin vapour loaded air into the accident Well 3 and process chamber 2 to sterlise them. Liquid in the accident well 3 may be drawn off into a transfer vessel 43 placed in the transfer chamber 4.

Should there be an accident in the transfer chamber 4 such that liquid is split, this will be retained in the accident well 5, the volume below the level of the access door 13. For sterilisation in this circumstance, the ultraviolet light 40 or the evaporator 34 is switched on.

In the electrical circuitry of the apparatus, provision is made to ensure that as well as the usual information regarding the conditions of the culture, for example pH, temperature, aeration rate and stirrer speed, information about the factors that would affect the safety of the apparatus are also presented at the instrument panel. Such factors include failure of the ventilating fans, failure to maintain the pressure differential between the process chamber and the external atmosphere (as indicated by a pressure differential switch co-operating with the filter 64) increased pressure in the culture vessel and power failure. In the event of any such failure the information is presented on the instrument panel, an alarm sounds and the culture apparatus (stirrer, air to the culture, medium feed, temperature control, and pH control) automatically stops and remains off until the cause of the stoppage has been ascertained and corrected by the operator in charge of the apparatus. Unless the fault is complete failure of the fans or of the mains supply, the fans will remain on and are supplied through a separate key operated switch to ensure that they cannot be turned off by unauthorised persons. These provisions are particularly advantageous when growing dangerous pathogenic organisms.

The above apparatus has been particularly described by way of example only, and other alternatives will no doubt occur to those involved in laboratory art or in the art of handling sterile or dangerous substances. For example, mobile apparatus of dimensions similar to those of the apparatus described may have air pumped into it through filters, rather than out, when contamination of the process chamber is to be prevented, or both pump means may be incorporated. The transfer chamber may be sited above the process chamber, a feature which would be particularly advantageous when one is more concerned with feeding in liquids when sterility is required inside. Apparatus in accordance with the invention may be employed for biological processes such as the growing of plants, animal or plant tissue cells, micro organisms, viruses, the production of vaccines, and the observation of animals, in a controlled environment.

We claim:

1. Apparatus for carrying out processes in a controlled environment which comprises an outer casing enclosing:

a process chamber,

a transfer chamber,

trap means for containing liquid providing communication between said process and transfer chambers and including a sterilizing lock,

means preventing the ingress to said process chamber of unsterilized air from the atmosphere surrounding said outer casing, and means preventing the egress of contaminating matter from said process chamber to the atmosphere surrounding said outer casing, said transfer chamber having scalable door means communicating with said surrounding atmosphere and permitting access for a transfer 'vessel,

and internal sterilization means.

2. Apparatus according to claim 1 and in which said transfer chamber has sterile-air flushing means restricting the escape of contaminating matter from said transfer chamber including when said sealable door is open.

3. Apparatus according to claim 1 and in which said transfer chamber has sterile air flushing means restricting the ingress of unsterile air into said transfer chamber including when said scalable door is open.

4. Apparatus according to claim 1 wherein said trap means also communicates with the atmosphere surrounding said casing by way of a sterilizing lock.

5. Apparatus according to claim 1 and in which said process chamber and said transfer chamber are provided with remote handling means.

6. Apparatus according to claim 1 and wherein said outer casing has wheeled support means conferring mobility on the apparatus.

7. Apparatus according to claim 1 and having an accident well disposed below said process chamber to receive spillage from an accident occuring in said process chamber.

8. Apparatus according to claim 1 and having an accident well disposed below said transfer chamber to receive spillage from an accident occuring in said transfer chamber.

9. Apparatus according to claim 1 and in which said transfer chamber is adapted to accommodate a transfer vessel of size considerably larger than the maximum size vessel transmissible through said communicating trap means.

References Cited STATES PATENTS JOSEPH SCOVRONEK, Primary Examiner B. S. RICHMAN, Assistant Examiner U.S. Cl. X.R.

2l61; 23259; l28-l; 220-45

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