Patent Application
20100043883
In a bioreactor process, maintaining a contamination-free environment is key. Whenever a bioprocess system is exposed to the external environment, it faces the risk of contamination by viruses, micro-organisms, and chemicals. Typical bioprocesses involve batch bioreactors where cells are cultured and harvested over a period of time ranging from minutes to days. After a batch is harvested, the reactor vessel is sterilized in preparation for the next batch process. For small volume reactors, the entire reactor system can be placed in an autoclave and completely sterilized. For example, reactors that are about 5 liters or less typically are made of glass and are sterilized in an autoclave. However, large volume reactors, such as those that are about 5 liters or more are typically too large to be placed in an autoclave, and must therefore be sterilized using Clean-in-Place (CIP) and Steam-in-Place (SIP) methods. CIP and SIP are methods used in the pharmaceutical and food industries for the in-line sterilization of processing equipment, including vessels, valves, process lines, and filter assemblies. These methods are used to achieve sterility or a certain level of sanitation required by regulation for a particular process.
In many cases, bioreactor processes do not lend themselves easily to in-situ analysis of the batch. Instead, samples must be physically extracted from the process and examined and manipulated outside the vessel, thereby exposing the entire batch to the external environment and the possibility of contamination. Since loss of a sample run or contamination of the process can have extremely expensive ramifications, it is important to obtain a sample without causing contamination. Furthermore, to minimize waste of the batch material, it is desirable to extract a sample only in the amount necessary for processing and analysis.
Many reactors are equipped with a sampling valve whereby the contents of the reactor may be extracted. Referring to
The described process is susceptible to the introduction of contamination in various ways; the sterilizing and sampling processes are always subject to the possibility of human error, and the routine connecting and disconnecting of the lines brings constant exposure of the system to contamination from the external environment. In some instances, the sample may leak from the sampling valve, unnecessarily wasting portions of the batch and, if the batch material is biohazardous, possibly injuring the operator. In addition, the process places the operator at risk of bum injuries during the steam operation.
What is needed is an improved system and method for acquiring samples from a bioreactor that is safer, more consistent, and less susceptible to contamination.
In one aspect, provided is an automatic sterile sampling system for sampling fluid, including a steam valve; a sampling valve having a steam inlet port in fluid communication with the steam valve, a sample inlet port, and an outlet port, the outlet port having a first inner diameter; a processing system comprising a cleaning fluid source and in fluid communication with a sample transfer channel, the sample transfer channel having a second inner diameter less than the first inner diameter; an isolation valve having an inlet port in fluid communication with the outlet port of the sampling valve, a drain outlet port, and a sample transfer port in fluid communication with the sample transfer channel, the inlet port and sample transfer port having a third inner diameter less than the first inner diameter and larger than the second inner diameter; and a controller. The inner diameters of the valve ports and fluid lines are less than about 8 mm. The system has tapered transitions between the outlet port of the sampling valve and the inlet port of the isolation valve and between the sample transfer port of the isolation valve and the sample transfer channel. The isolation valve drain outlet port protrudes upwardly from the isolation valve and has a longitudinal axis that is angled less than about 45° with respect to vertical.
In another aspect, provided is a method for automatic aseptic sampling from a fluid sample source, including the steps of: providing a steam source, a steam valve connected the steam source, a sampling valve connected to the fluid sample source, an isolation valve, a processing system, a drain valve, a drain, and a controller; and employing the controller to pass cleaning fluid from the processing system through the isolation valve to the drain; pass steam through the steam valve, sampling valve, and isolation valve to the drain for a duration sufficient to sterilize the sampling valve, the isolation valve, and a fluid path therebetween; and pass fluid sample from the fluid sample source through the sampling valve and isolation valve to the processing system.
Thus provided are a system and a method that delivers safer, more consistent sampling, while reducing the risk of contamination during extraction of a sample from a vessel. Waste of the sample can also be minimized.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
a is a drawing of the isolation valve and drain valve functioning in cooperation during a first part of a sanitizing operation;
b is a drawing of the isolation valve and drain valve functioning in cooperation during a second part of a sanitizing operation;
a is a drawing of the automated system during a first part of a sanitizing operation;
b is a drawing of the automated system during a second part of a sanitizing operation;
a is a drawing of the isolation valve and drain valve of an improved system functioning in cooperation during a first part of a sanitizing operation;
b is a drawing of the isolation valve and drain valve functioning in cooperation during a second part of a sanitizing operation;
The present invention provides improvements for an automated system and method for extracting a sample from a batch reactor while maintaining sterility of the key components through which the sample is extracted. The improvement relates to the system and method described in U.S. Ser. No. 61/133,209, entitled, “System and Method for Automated Sterile Sampling of Fluid From a Vessel,” of George E. Barringer, Jr., (Attorney Docket No. 3551.1013-000) which application is filed concurrently herewith, and which application is incorporated herein by reference in its entirety. Particularly, the improvement is directed to the automatic sterile sampling of heterogeneous fluid from a bioreactor vessel, for example, a mammalian cell culture in suspension. This invention is not limited to sampling from a bioreactor, but rather can be applied to the aseptic sampling of any vessel containing a fluid.
A description of example embodiments of the invention follows. The embodiments provide an automated system and method for extracting a sample from a batch reactor while maintaining sterility of the key components through which the sample is extracted. The invention is not limited to sampling from a bioreactor, but rather can be applied to the aseptic sampling of any vessel containing a fluid. The system employs a series of pneumatically actuated valves to control the flow of steam, fluid sample, cleaning fluid, and optionally air through the system at specified times and includes a connection whereby a fluid sample is routed from the bioreactor vessel to a downstream processing system. As used herein, the term “valve” refers to a single valve or system of valving that achieves a particular flow configuration.
Referring to
Returning to
Steam valve 13 controls the flow of steam through a steam channel 2. Steam valve 13 is typically a diaphragm valve, such as GEMÜ® Type 650/015/D80415A0-1537, which is a ½ inch two-port pneumatically actuated sanitary valve. When steam valve 13 is open, steam is allowed to pass through steam channel 2 to sampling valve 3.
Sampling valve 3 is typically a three-port plunger valve specifically adapted for sterile sampling of a liquid sample from a container, such as the Keofitt® W15™ sampling valve, or the valves described in U.S. Patent Application Publication No. 2007/0074761 incorporated herein by reference in its entirety. An example of a suitable Keofitt® sampling valve is shown in
Isolation valve 17 is typically a three-port diaphragm valve. An example of a suitable isolation valve is a GEMÜ® Type 650 TC TFE 15RaEP Con1, which is a ⅜ inch three-port pneumatically actuated sanitary valve. A first port of isolation valve 17 is connected to the steam/fluid channel 4, while a second port of isolation valve 17 is connected to drain channel 8 and a third port of the isolation valve 17 is connected to sample transfer channel 6.
Sample transfer channel 6 establishes fluid communication between isolation valve 17 and processing system 11. As used herein, “fluid communication” refers to a relationship between two components by which fluid can be permitted to flow from one component to the other. Processing system 11 can include cleaning, processing, and analytical instrumentation, as well as controller 27, which will be described further below. An example of a suitable processing system is described in U.S. Patent Application Publication No. 2004/0259266, incorporated herein by reference in its entirety. Processing system 11 further includes a cleaning fluid source 40, a sterile water source 30, and an internal valve 29, which opens and closes fluid communication to isolation valve 17.
In one embodiment, the isolation valve 17 essentially operates in the manner shown in
As shown in
The drain valve is typically similar to the isolation valve, but has two ports instead of three. An example of a suitable drain valve is a GEMÜ® Type 650 TC TFE 15RaEP Con1 having two ⅜ inch ports, which is also a pneumatically actuated sanitary valve. In the alternative, isolation valve can perform the above functions without the assistance of drain valve 19, so long as isolation valve is a true three-way valve, rather than a three-port valve with two ports always coupled together.
As shown in
The steam valve 13, sampling valve 3, isolation valve 17, drain valve 19, and internal valve 29 are controlled in sequence to perform various system operations, which will be described in detail below. Each of the valves is pneumatically actuated by one of two control valves in parallel: a solenoid control valve and a manual control valve. For example,
Before a new sample can be extracted from the reactor vessel, parts of the sampling system are sterilized, while others are sanitized. As used herein, the term “sterile” refers to a system or components of a system that are absolutely free of unknown living organisms or bioactive DNA. As thus defined, sterility has been proven by experiment to be achieved only by high temperature steam or radiation. As used herein, the term “sanitized” refers to a system or components of a system that are free of unknown organisms in measurable levels.
In the embodiment shown in
Internal valve 29 opens to permit fluid to flow. For example, when internal valve 29 is open, cleaning fluid can flow from cleaning fluid source 40 through sample transfer channel 6 to isolation valve 17. As shown in
The second part of the sanitizing operation is shown in
The system then undergoes a sterilizing operation, as shown in
In one embodiment, sample transfer channel 6 can be at least partially sterilized. As shown in
Once the sterilizing operation has completed, steam valve 13 closes and the system is sufficiently free of contamination. However, the system components generally remain hot from the sterilizing operation. To reduce the temperature of the components, the system can undergo an optional cooling operation, as shown in
Immediately prior to the sampling operation, drain valve 19 closes so that fluid samples cannot flow to drain 10. As shown in
Immediately prior to the sampling operation, drain valve 19 closes so that fluid samples cannot flow to drain 10. As shown in
In some embodiments, where even the smallest amount of the batch material is highly valuable, the dead volume of the sample transfer channel 6 is sized as small as possible to avoid drawing more fluid sample than is needed for analysis. Typically, the sample transfer channel 6 has an inner diameter between about 1 mm and about 2 mm, and a dead volume of less than about 60 ml. Thus provided is a safer, more consistent sterile sampling system that minimizes sample waste and performs sampling operations automatically.
To ensure that the system extracts a quality sample for analysis, i.e., a sample that is representative of the batch, the fluid lines of the system are primed. In other words, during the sampling operation, the system extracts more fluid than necessary to perform an analysis. For example, a total of 30 ml of the batch is extracted in order to obtain a 10 ml aliquot; the first 20 ml is primer to flush the fluid lines of residual fluid and the final 10 ml is the actual sample to be analyzed. This practice is typical for previously known manual systems as well as the presently described system, and it prevents the analysis sample from being diluted by residual fluid as it flows through the system. In contrast to previously known manual systems, the present automated system is capable of consistently and accurately providing the exact amount of fluid sample required to prime the fluid lines, thus minimizing waste of the sample.
The quality of an automatically extracted sample is evaluated by comparing output values of the sample to known output values of the batch. A sample is of acceptable quality only when the output values of the samples equal the output values of the batch. A system that dispenses acceptable samples is considered “primed.” For the purpose of comparison, the output values for manually extracted sample are considered to represent the actual output values of the batch. That is, known output variables, such as cell count and glucose concentration, are determined from samples manually extracted from the batch and are used as a base line for comparison against the automatically extracted samples. Therefore, the system is considered “primed” when the output values of the samples of the automated system match the output values of the samples extracted manually. It is understood that the manually extracted sample used for comparison is provided by a manual system that has already been primed.
When the above described automated system is applied to batch processes of homogeneous fluids, the amount of sample required to prime the system is comparable to that required when the sample is extracted manually. However, the above system requires priming with significantly larger quantities of fluid sample when the sample to be analyzed is a heterogeneous fluid, such as a mammalian cell culture.
For example,
However, the system could not sufficiently be primed by the heterogeneous fluid to provide a representative sample for VCC. The triangles represent the VCC in the fluid for each aliquot, while dotted line 42 indicates the known VCC of the batch (as measured from a batch sample extracted by a primed manual system). Like the first aliquots tested for glucose, the first four aliquots of the tested for VCC are substantially dilute, and a steady state concentration is reached shortly thereafter. However, unlike the steady state reached by the homogeneous fluid for glucose concentration, the steady state level achieved by the heterogeneous fluid for VCC is not equal to the value observed in the batch. Thus, even after 200 ml of priming, the system could not provide an aliquot that was truly representative of the batch. Because the experiment was terminated after the 20th aliquot, the actual volume of batch fluid required to achieve a representative sample was not determined. In any case, the cost of wasting such relatively large quantities of fluid for the purposes of priming the system for an accurate sample is prohibitive in most, if not all applications. Several features can be added to the above described system to increase sample quality and ensure automated delivery of representative batch samples.
It was unexpectedly found that orienting the isolation valve so that the drain outlet port points in an upwardly direction reduces the level of dilution in samples extracted by the system, thereby improving sample quality. In particular, it was found that a quality sample can be obtained when the isolation valve drain outlet port protrudes upwardly from the isolation valve and has a longitudinal axis that is angled at less than about 45° with from vertical. As used herein, the term “vertical” refers to a line parallel to a gravitational force vector. As used herein, “upward” refers to a direction that is at least partially opposite the gravitational force vector. As used herein, “protrude” means to project outwardly from a particular object in space.
The system for automated sterile sampling of a vessel described above can therefore be improved by reorienting the system components as shown in
Surprisingly, using the above described configuration yielded samples having output variables, such as lactate concentration, ammonium concentration, and VCC, that more closely matched those of the samples extracted manually. Lactate and ammonium concentrations are indicators of metabolic activity of cells. Higher concentrations of lactate and ammonium in a fluid sample suggest that more cells exist in the fluid sample. Thus, lactate concentration, ammonium concentration, and VCC are each directly or indirectly indicators of the quantity of cells in the fluid sample, and therefore important output variables of a heterogeneous fluid (cells in suspension).
Without wishing to be bound to a theory, it is believed that orienting the isolation valve so that the drain outlet port protrudes upwardly from the isolation valve and has a longitudinal axis that is angled less than about 45° with from vertical prevents steam condensate from pooling in drain channel 9. If the drain outlet port is pointed downwardly, as in
As described above, the manual sampling valve 15, isolation valve 17, and drain valve 19 of the sampling system each has a ⅜ inch inner diameter port. This sizing had been considered appropriate for both homogeneous and heterogeneous fluid samples. However, it was found that the dimensions of the valve ports and fluid lines substantially impacted sample quality for heterogeneous fluids. More specifically, it was discovered that sample quality is improved when the inner diameters of the valves ports and fluid lines were decreased. This was determined after the original manual sampling valve 15, isolation valve 17, and drain valve 19 of the sampling system were each replaced with a corresponding smaller valve having ¼ inch inner diameter ports. It is therefore preferable that the valve ports and fluid lines downstream from the sampling valve have an inner diameter that is less than 8 mm. This number was determined to be appropriate because the ⅜ inch (9.5 mm) diameter valves did not work as well as the ¼ inch (6.3) diameter valves. A system having lines of an inner diameter of 8 mm could not be tested because no valve sizes between ¼ inch and ⅜ inch exist for the valves specified in detail above.
In addition to valve resizing, transitions between substantial changes in fluid path size were added. As previously described, ports of the sampling valve 3 have an inner diameter of over 9 mm, while the steam/sample transfer channel connected to it was changed (by virtue of the valve resizing described in the preceding paragraph) to a ¼ inch (0.63 mm) inner diameter. Thus, junction between the sampling valve outlet port 37 and the steam/sample transfer channel had a substantial decrease in inner diameter. The connection was modified to have a tapered transition 39 between the sampling valve outlet port and the steam/sample transfer channel, as shown in
These modifications in combination yielded a surprisingly substantial reduction of variance in cell count between automatically extracted samples and manually extracted samples.
Again without wishing to be bound by a theory, it is believed that the concentration of a heterogeneous fluid remains dilute in the above described system because the heavy phases (such as cells) are permitted to settle out when the linear velocity of the fluid in the system is too low. Decreasing the inner diameter of the valve ports and fluid lines increases the linear velocity of the fluid, thereby maintaining sufficient turbulence in the fluid to keep the cells in suspension while still maintaining the same flow rate. Furthermore, tapered transitions are believed to eliminate dead zones where cells can become trapped.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/133,171, filed on Jun. 25, 2008. The entire teachings of the above application are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61133171 | Jun 2008 | US |
