Particle size distribution measuring apparatus with validation and instruction modes

Abstract

This invention provides a particle size distribution measuring apparatus, which has a function of informing an operator of a procedure of validation work of the particle size distribution measuring apparatus. A storage medium which records validation data providing a procedure of validation work for the particle size distribution measuring apparatus and a control unit which has a validation help function which successively reads a validation procedure from the validation data and controls the particle size distribution measuring apparatus according to a measuring procedure without any operation by an operator in the validation procedure while teaching the operator a work procedure requiring an operation by the operator.


Claims

What is claimed is:

1. A particle size distribution measuring apparatus comprising:

a storage medium storing validation data for validating the performance of a particle size distribution measuring apparatus; and

a control unit for providing validation help function which

successively reads a validation procedure from the validation data, and

successively carries out control of the particle size distribution measuring apparatus according to a measuring procedure requiring

no operation by an operator in the validation procedure, and when operator intervention is necessary,

providing step-by-step validation instructions to the operator of a work procedure requiring an operation by the operator, wherein at least one of the operations requires the operator to provide one or more parameters for validating the measuring apparatus.

2. The particle size distribution measuring apparatus according to claim 1, and wherein the control unit includes a warning function of pointing out the operator's mistake in the work procedure to the operator, and teaching a validation work according to a correct work procedure.

3. The particle size distribution measuring apparatus according to claim 1, wherein the control unit includes a speech output section for outputting an instruction to the operator by an audio signal.

4. The particle size distribution measuring apparatus according to claim 1, wherein the control unit has a monitor screen for displaying an instruction to the operator.

5. The particle size distribution measuring apparatus according to claim 1, further including an automatic charger for successively charging a standard sample used for the validation work in the particle size distribution measuring apparatus.

6. The particle size distribution measuring apparatus according to claim 1, wherein the control unit includes a judgment function of comparing an inspection result obtained from the validation work with a performance standard of the particle size distribution measuring apparatus, and making a judgment whether the comparative result is within a predetermined performance standard range.

7. The particle size distribution measuring apparatus of claim 1, wherein the control unit includes a recording function of recording the inspection result obtained by the validation work.

8. A The particle size distribution measuring apparatus of claim 1 wherein providing step-by-step validation instructions to the operator includes,

requesting that the operator provide a predetermined standard sample with which to validate the measuring apparatus.

9. In an improved particle size distribution measuring apparatus having a measuring section for receiving a sample, a source of light for irradiating the sample and sensors for measuring the light after irradiating the sample, the improvement comprising:

a validation unit for determining the accuracy of measurements when a predetermined standard is irradiated in the measuring section; and

an instructing unit for providing step-by-step validation instructions to an operator as the validation unit performs the measurements of the predetermined standard, wherein at least one of the instructions requires the operator to provide one or more parameters for validating the measuring apparatus.

10. The particle size distribution measuring apparatus of claim 9 further including an automatic sample standard unit for inserting the predetermined standard into the measuring section.

11. The particle size distribution measuring apparatus of claim 9 further including a speaker wherein the instructions are generated as audible speech to the operator.

12. A particle size distribution measuring apparatus capable of interactive calibration procedures to guide a user, comprising:

a measuring section for receiving a sample;

a source of light for irradiating the sample;

a sensor unit for measuring the light after irradiation and providing measurement signals;

a measuring unit for storing reference values to enable calibration;

a controller for processing the measurement signals to measure particle sizes;

a display screen;

a user interface unit;

a validation program stored in the memory unit to enable the controller to validate the performance of the particle size distribution measuring apparatus including displaying a series of predetermined screens with indicia to prompt a response from a user on the user interface unit;

means for prompting a user to insert a calibration sample into the measurement section;

means for adjustment of an optical axis between the measuring section and the source of light;

automatic means for measuring light transmittance through the measuring section;

means for comparing the light transmittance measurement with stored reference values to determine when the measurement is one of a value above the stored

reference values and below the stored reference values when the calibration sample is in the measurement section; means for displaying a message to prompt the user to alter the calibration sample when one of a value above the stored reference values and below the stored reference values is measured; and

means for displaying a message that the validation result is within the stored reference values wherein the user is directed through the validation procedures by displayed indicia and by automatic procedures initiated directly by the controller.

13. The particle size distribution measuring apparatus of claim 12 further including a monitoring sensor to determine if the measuring section is open and means to display a message to close the measuring section.

14. The particle size distribution measuring apparatus of claim 12 further including means to display a message to insert dispersion medium in the measuring section.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a particle (grain) size measuring apparatus, which detects a diffraction/scattered light or dynamic light scattering by irradiating a laser beam onto a particle group such as a dispersing powder sample or the like, and measures a particle size distribution of the particle group on the basis of a scattered light intensity signal or the like obtained by the detection and more particularly provides an automatic validation and instruction mode.

2. Description of the Prior Art

Conventionally, a particle size distribution measuring apparatus using a light diffraction phenomenon or scattering phenomenon by a particle has calculated a particle size distribution of a sample particle in the following manner. More specifically, the above apparatus measures intensity distribution of a diffraction light or a scattered light, that is, a relation between a diffraction angle or scattering angle and a light intensity, and then, carries out arithmetic processing based on a Frauunhofer diffraction theory or Mie scattering theory with respect to the measured result. In the above manner, the above apparatus has calculated the particle size distribution of a sample particle. The above particle size distribution measuring apparatus has been used for research and development of raw materials in most mining and industrial fields such as the cement or the ceramic industry, and in a new material field mainly using ceramics.

For example, to give an example of the above apparatus, there is a particle size distribution measuring apparatus disclosed in Japanese Examined Patent publication No. 6-43950. FIG. 16 is a view schematically showing a construction of the particle size distribution measuring apparatus disclosed in this Publication. In FIG. 16, a reference numeral 10 denotes a cell comprising a transparent container for receiving a sample solution 11 dispersing a particle group of the measuring object in a proper dispersion medium, and a reference numeral 12 denotes a laser beam source provided on one side (backward side) of the cell 10. A parallel laser beam 13 emitted from the laser beam source 12 is enlarged by a beam expander (not shown), and then, is irradiated to the cell 10 in an enlarged state.

In FIG. 16, a reference numeral 14 denotes a condenser lens provided on the other side (forward side) of the cell 10, and a ring detector 15 is arranged at a focal position of the condenser lens 14. The ring detector 15 is constructed in a manner that a plurality of photo sensors having mutually different radii and a ring or semi-ring light receiving plane is arrayed concentrically around an optical axis of the condenser lens 14. Further, the ring detector 15 receives light scattered/diffracted at a relatively small angle to the optical axis of the laser beam 13 diffracted or scattered by particles in the cell 10 for each scattering angle, and then, measures their light intensity.

Moreover, a wide angle scattered light photo detector group 16 is provided at the vicinity of the cell 10. The wide angle scattered light photo detector group 16 individually detects light scattered/diffracted at a relatively large angle to the optical axis of the laser beam 13 diffracted/scattered by the particles in the cell 10 for each scattered light. Further, the wide angle scattered light photo detector group 16 is composed of a plurality of photo sensors 17 to 22 provided at an angle different from the condenser lens 14 and the ring detector 15. Thus, the photo detector group 16 can detect a wide angle scattered light exceeding a predetermined angle by the particles in the cell 10 in accordance with each oriented angle. In the photo detector group 16, the photo sensors 17 to 20 detect a forward scattering light, the photo sensor 21 detects a side scattering light, and the photo sensor 22 detects a backscattering light.

A reference numeral 23 denotes a pre-amplifier for amplifying an output of the photo sensors constituting the ring detector 15, and a reference numeral 24 denotes a pre-amplifier for amplifying each output of the forward scattering light photo sensors 17 to 20. Further, a reference numeral 25 denotes a pre-amplifier for amplifying each output of the side scattering light photo sensor 21 and the backscattering light photo sensor 22. A reference numeral 26 denotes a multiplexer for successively capturing each output of the pre-amplifier groups 23 to 25 and transmitting it to an A-D converter 27, and a reference numeral 28 denotes a computer which is used as an arithmetic processor for inputting an output from the A-D converter 27. The computer 28 stores a program for processing each output converted into a digital signal (digital data relative to light intensity) of the ring detector 15 and the photo sensors 13 to 22 on the basis of a Frauunhofer diffraction theory or Mie scattering theory, and obtaining a particle size distribution in a particle group.

In the above particle size distribution measuring apparatus, in a state that the sample solution 11 is received in the cell 10, when the laser beam 13 is irradiated from the laser beam source 12 to the sample cell 10, the laser beam 13 is diffracted or scattered by the particles in the cell 10. Of the diffracted light or scattered light, a light having a relatively small scattering angle is imaged on the ring detector 15 by the condenser lens 14. In this case, the photo sensor arranged on the outer side receives a light having a larger scattering angle; on the other hand, the photo sensor arranged on the inner side receives a light having a smaller scattering angle. Therefore, a light intensity detected by an outer-side photo sensor means a quantity of particles having smaller particle diameter (particle size); on the other hand, a light intensity detected by the inner-side photo sensor means a quantity of sample particles having larger particle diameter. The light intensity detected by each of these photo sensors is converted into an analog electric signal, and further, is inputted to the multiplexer 26 via the pre-amplifier 23.

On the other hand, of the laser beam 13 diffracted light or scattered by the particles, a light, which is not converged by the condenser lens 14 and has a relatively large scattering angle, is detected by the photo sensors 17 to 22, and then, its light intensity is measured. In this case, the forward scattering light photo sensors 17 to 20, the side scattering light photo sensor 21 and the backscattering light photo sensor 22 successively detect a scattering light from a particle having a small particle size. The light intensity detected by each of these photo sensors 17 to 22 is converted into an analog electric signal, and further, is inputted to the multiplexer 26 via the pre-amplifier groups 24 and 25.

The multiplexer 26 captures measurement data from the ring detector 15 and the photo sensors 17 to 22, that is, an analog electrical signal in a predetermined order. The analog electric signal captured by the multiplexer 26 is made into a serial signal, then, is converted by the A-D converter 27 into a digital signal in succession, and thereafter, is inputted to the computer 28.

Subsequently, the computer 28 processes the light intensity data for each scattering angle obtained by each sensor of the ring detector 15 and the photo sensors 17 to 22 on the basis of a Frauunhofer diffraction theory or Mie a scattering theory.

As described above, in the above particle size distribution measuring apparatus, a light intensity distribution of scattering light having a substantially larger particle size is measured by the ring detector 15 and a light intensity distribution of wide-angle scattering light having a mainly smaller particle size is measured by the photo sensors 17 to 22. Further, the output of these ring detector 15 and photo sensors 17 to 22 is processed by the computer 28. Therefore, it is possible to obtain a particle size distribution of particle group over a wide range from a relatively larger particle size to a micro particle size.

By the way, the particle size distribution measuring apparatus as described above requires periodically carrying out a validation work in order to make a decision whether or not its measuring accuracy is correctly made. In particular, in a pharmaceutical company, in the case where the above particle size distribution measuring apparatus is used for a quality control, the validation work must be correctly done at least once per year.

In order to correctly do the validation of the particle size distribution measuring apparatus, an operator must measure a predetermined standard sample according to a determined procedure. For this reason, in the conventional particle size distribution measuring apparatus, the operator prepares a validation manual recording procedures of the above validation work, and then, must carry out the validation work according to the determined procedures.

However, a measuring technique using the above particle size distribution measuring apparatus is complicated, and therefore, it is difficult for an inexperienced operator to memorize and operate all of the working procedures. For this reason, in the case where an inexperienced operator operates the above apparatus, the operator must operate a control unit while referring to a user manual. In fact, when an inexperienced operator performs the above operation while referring to a user manual, such an operator can easily make a mistake in the sequence of work, and may not notice the mistake. Therefore, validation cam be incorrectly carried out; and for this reason, the resulting numerical value has no reliability.

In such a case, the measurement procedure must be performed again. However, in order to again make a measurement, the following steps are required. More specifically, dispersion medium and standard sample in a sample supply apparatus are discharged, and the sample supply apparatus is washed, and thereafter, the dispersion medium and standard sample are charged in the supply apparatus. Excessive time and labor are spent for doing the above work, and in the case where the standard sample is valuable, there is a possibility that many standard samples are wasted.

After the validation work is completed, the operator makes a report based on the measurement result, and must store the report; however, there is the case where the operator forgets this work. For this reason, there is a problem that no record is stored in spite of carrying out the validation work.

In addition, the above scattering type particle size distribution measuring apparatus uses precision optical components, a laser beam source, a motor and the like; for this reason, there is a possibility that an accident happens in the case where a user disassembles the apparatus in error, or makes a handling mistake. In order to prevent such an accident, conventionally, caution procedures have been prescribed in a manual or the like, or a label describing handling caution matters have been stuck onto the apparatus main body. However, the aforesaid accident happens due to a user's careless mistake and an erroneous operation by an inexperienced operator; for this reason, there is a possibility that the number of processes may be required on a maker side or user side.

The present invention has been made in view of the aforesaid problems in the related art. It is, therefore, an object of the present invention to provide a particle size distribution measuring apparatus, which has a function of informing an operator of a procedure of validation work of the particle size distribution measuring apparatus, and thereby, can prevent a generation of mistake in a complicated validation work.

SUMMARY OF THE INVENTION

In order to achieve the above object, the present invention provides a particle size distribution measuring apparatus comprising: a storage medium which records a validation data indicating a procedure of validation work for a particle size distribution measuring apparatus, and a control unit which has a validation help function which successively reads a validation procedure from the validation data and successively carries out a control for the particle size distribution measuring apparatus according to a measuring procedure requiring no operation by an operator in the validation procedure while teaching the operator a work procedure requiring an operation by the operator.

Therefore, when carrying out the validation work, the operator can use the validation help function, and thereby, can operate the particle size distribution measuring apparatus according to the work procedure given from the control unit in a predetermined order without making a mistake. Moreover, the measuring procedure which is controlled by only a control unit is automatically carried out, by which the control unit controls the particle size distribution measuring apparatus according to an operation sequence previously stored as validation data.

The measuring procedure automatically carried out by the control unit includes a procedure for setting a setup value of a measuring object sample such as a refractive index of a standard sample used for the validation work. Therefore, the operator has no need of carrying out troublesome various setup operations for the validation work.

Accordingly, the operator can perform the complicated validation work without referring to a manual, and thereby, it is possible to reduce the responsibility of the operator to the minimum, and to successively carry out the work procedure made by the operator step by step without making a mistake. This serves to more accurately perform the validation work.

In the case where the control unit has a warning function of pointing out the operator's mistake in the work procedure to the operator, and teaching a proper validation work according to a correct work procedure, when the operator makes an erroneous operation, a warning is given from the control unit. Therefore, the validation work is accurately performed, so that a measured result having a high reliability can be obtained.

The following matter is considered as a possible aforesaid mistake in the work procedure; more specifically, the operator charges a standard sample unsuitable for the measurement condition. For example, it is considered that concentration and temperature of the charged sample are outside a range of measurement condition predetermined as a standard sample. In this case, the concentration of the sample is previously measured by a light transmittance, and then, in the case where the measured result is different from the sample concentration condition, the control unit can give an instruction to adjust the concentration of the sample to the operator. Moreover, the control unit may have a warning function which confirms the measurement condition during measurement of particle size distribution, and gives an instruction to the operator to retry the measurement in the case where the measurement condition is beyond a range of a predetermined value.

In the case where the control unit has a speech (voice) output section for outputting an instruction to the operator by a speech signal, an operating instruction is given by machine generated speech; therefore, the operator can readily perform a validation work according to the spoken instruction without giving attention to reading a manual and the like.

In the case where the control unit has a monitor screen for displaying an instruction to the operator, the operator can obtain instructions for validation work via a display screen; therefore, it is possible to readily perform the validation work.

Moreover, in the case where the particle size distribution measuring apparatus of the present invention has an automatic charger for successively charging a standard sample used for the validation work into the above particle size distribution measuring apparatus, it is possible to reduce the work done by the operator to the minimum, and further, to easily handle the above apparatus. In this case, preferably, the control unit has a function of continuously measuring a plurality of standard samples.

The control unit has a judgment function of comparing an inspection result obtained from the validation work with a performance standard of the particle size distribution measuring apparatus, and making a judgment whether or not the comparative result is within the performance standard range. In this case, the operator has no need of collating the comparative result with a standard chart of the particle size distribution measuring apparatus, and making a judgment whether or not an error is within the performance standard range.

In the case where the control unit has a recording function of recording the inspection result obtained by the validation work, the inspection result is automatically stored after the validation work is performed. Even in the case where the operator forgets the output of the inspection result, it is possible to prepare a report using records of the inspection result automatically stored. Therefore, the record is not lost when carrying out the validation work. In this case, the record of the inspection result may be a result data stored in the storage medium, or may be an inspection result report outputted by a printer or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view schematically showing a construction of a particle size distribution measuring apparatus according to one embodiment of the present invention;

FIG. 2 is a view to explain an operation of validation help function of the above embodiment;

FIG. 3 is a view showing a display window on a screen when measuring a standard sample using the validation help function of the particle size distribution measuring apparatus;

FIG. 4 is a view showing another display window on the screen;

FIG. 5 is a view showing another display window on the screen;

FIG. 6 is a view showing another display window on the screen;

FIG. 7 is a view showing another display window on the screen;

FIG. 8 is a view showing another display window on the screen;

FIG. 9 is a view showing another display window on the screen;

FIG. 10 is a view showing another display window on the screen;

FIG. 11 is a view showing another display window on the screen;

FIG. 12 is a view showing another display window on the screen;

FIG. 13 is a view showing another display window on the screen;

FIG. 14 is a view showing a particle size distribution measuring apparatus according to another embodiment;

FIG. 15 is a view to explain an operation of the validation help function of the above embodiment; and

FIG. 16 is a view to explain the measuring principle of a conventional particle size distribution measuring apparatus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventors of carrying out their invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein to specifically provide a particle size distribution measuring apparatus with validation and instruction modes of operation.

One embodiment of the present invention will be described below with reference to the accompanying drawings.

In FIG. 1, a particle size distribution measuring apparatus 1 of this embodiment has a measuring section 2 for measuring a particle size distribution of a measuring object sample S, and a control unit 3 connected to the measuring section 2. In the measuring section 2, a laser beam is irradiated to the measuring object sample S charged from a charging hole so as to detect a scattered light by particles in the measuring object sample S, and then, the scattered light is analyzed, and thereby, its particle size distribution is measured.

In this embodiment, the control unit 3 is an information processing unit (hereinafter, referred to as a personal computer 3), which is connected to the measuring section 2 via a communication cable C. The control unit 3 has a display 3d (i.e., monitor screen) and a keyboard 3k. However, in the present invention, the construction of the control unit 3 is not limited to this embodiment. For example, the control unit 3 may be a microcomputer provided in the measuring section 2.

The aforesaid personal computer 3 has a CPU 3c, a storage (memory) section 3m such as a RAM, a ROM, a hard disk or the like, and an input-output interface 3i. The storage section 3m previously stores a control program Pc, and controls the measuring section 2 by executing the control program Pc. More specifically, the personal computer 3 executes various arithmetic processings on the basis of a data inputted from the input-output interface 3i, and outputs the processing (output) result via the input-output interface 3i.

Moreover, the input-output interface 3i is connected with the communication cable C so that a signal from sensors (not shown) incorporated into each part of the measuring section 2 can be transmitted to the personal computer main body via the communication cable C.

The storage section 3m stores a validation program P and a validation data D decoded by the validation program P, in addition to the above control program Pc. More specifically, the personal computer 3 has a validation help function by the control program Pc, the validation program P and the validation data D.

In this embodiment, regardless of the validation work, the validation program P is executable by reading a control data (including the above validation data D) recorded by a simple language for controlling the measuring section 2, and thereby, the validation program P has a general-purpose characteristic.

Further, the validation program P automatically and successively carries out a procedure executable in the particle size distribution measuring apparatus 1, to provide the procedures of validation work recorded as the validation data D. Meanwhile, regarding a procedure requiring an operation by the operator, the validation program P is programmed so as to give a work instruction relative to the procedure to the operator. As a result, the operator merely carries out a necessary and minimum operation, so that the validation work can be simplified.

The work instruction to the operator is outputted by a message on display screen 3d and a voice announcement. More specifically, in the case of carrying out a validation work, the operator has no need of reading a manual describing a working procedure for the validation, and can perform the validation work according to a message display on the display screen 3d or the announced instruction; therefore, the operator is able to concentrate on the validation work.

The validation data D includes at least one of an announcement by recorded speech and a message displayed on the display screen. For example, in the case of carrying out an announcement by audio speech, the personal computer 3 has a converter 3t for converting digital data stored as validation data D into a speech (audio) signal, and a speaker 3s which is a speech output section.

The validation program P is programmed in a manner that a work situation of the operator is detected and grasped by a sensor incorporated into each part of the measuring section 2. In the case where the operator makes a mistake in the work procedure, the validation program P is programmed in a manner of giving a warning to the operator, and giving an instruction for carrying out the validation work according to a correct work procedure to the same.

The following matter is considered as an example of a possible mistake in the work procedure; more specifically, the measuring object sample S charged into the measuring section 2 by the operator is beyond a range of concentration usable as a standard sample. In this case, it is possible to use a sensor for measuring a light transmittance of the measuring object sample S. Moreover, in the case where the inexperienced operator makes a mistake of handling the measuring section 2 by his careless or erroneous operation, a warning can be given to the operator. More specifically, when the operator opens a cover of the apparatus, which should not be opened during measurement work, the following announcement or message is made or displayed; for example, "Please close the cover. Please do not look at laser beam source in the apparatus."

FIG. 2 is a flowchart showing a procedure of the validation work in this embodiment, and shows measuring and operating procedures. FIG. 3 to FIG. 13 are views showing examples of window screens displayed on the display 3d pursuant to the progress of the validation work.

In FIG. 2, step S_t1, shows one example of an operation previously executed before the validation data D is read and the validation program P is executed. Namely, the operator presets validation standards.

FIG. 3 is a view showing a window displayed on the display 3d of the personal computer 3 in the above step S_t1.

In FIG. 3, W₁, is a main window, and W₂ is a display condition setup window. When seeing the display condition setup window W₂, in this embodiment, it is possible to input standard values relative to three kinds of diameters (median diameter, 10% diameter, 90% diameter) with respect to one standard sample. W₃ is a validation setup window for setting validation judgment factors such that each standard diameter has a set size (µm) or a permissible error in the size of diameter is applicable by what percents in plus and minus as an allowable range.

More specifically, in this embodiment, three kinds of standard diameters are set with respect to one standard sample, and then, in each diameter, a judgment is made whether or not the measured value is within a permissible error range; therefore, it is possible to perform a more detailed validation work. In the present invention, the number of diameters set as the validation standard diameter is not limited to three. For example, only a standard value and an allowable range relative to median diameter may be set.

The judgment standard of validation work set in the above manner is reflected in the validation data D. Namely, the operator can freely change the judgment standard of validation work. Likewise, the operator can freely change a kind of standard sample used in the validation work and physical information such as a refractive index of the standard sample or the like.

In the following embodiment, a PSL (Polystyrene latex) sphere having a diameter of 1 µm is used as the standard sample. Moreover, various data such as a kind and allowable range of the standard sample may be predetermined as the judgment standard of validation work by a maker of the particle size distribution measuring apparatus.

Next, when the validation program P starts up, the validation program P reads the validation data D, and then, successively executes various processings based on the validation data D.

In Step S_t2 shown in FIG. 2, the personal computer 3 displays a measuring window W₄ on the display screen, and further, displays a message window W₅ thereon. Moreover, in a message window W₅, a message M₁ "Please put dispersion medium in cell" is displayed, and thereafter, the same announcement as the message M₁ is converted into speech, and outputted from the speaker.

Subsequently, the operator sets a cell, into which distilled water is put as a dispersion medium, in the measuring section 2, and thereafter, operates an OK button B displayed on the message window W₅ (step S_t3). In this case, a method for operating the OK button B is as follows. For example, in the case of a keyboard, the OK button B is selected by a cursor key, and thereafter, a return key is pressed. Moreover, in the case of a mouse, the mouse is clicked in a state of bringing the cursor key to the OK button B. In order to simplify the description, the operation as described above is referred simply to "press the OK button".

In this embodiment, the personal computer 3 senses the completion of work when the operator presses the OK button B; however, the present invention is not limited to this embodiment. More specifically, the completion of work may be sensed by a sensor attached to each part of the measuring section 2.

Next, the personal computer 3 reads the following procedure from the validation data D, and then, executes the procedure. The next procedure of validation work is blank measurement, and is a measuring procedure executable in the particle size distribution measuring apparatus 1 without requiring any work by the operator. Therefore, after waiting for the operator's instruction, the personal computer 3 automatically controls each part of the measuring section 2, and then, executes the blank measurement.

FIG. 5 shows a message window W_6. In this message window W₆, a message M₂ "execute blank measurement" is displayed, and the personal computer 3 is waiting for the operator to press the OK button B while making announcement of the above message M₂ (step S_t4).

Subsequently, when the OK button B is pressed, the personal computer makes an adjustment of an optical axis according to the measuring procedure shown by the validation data D, and thereafter, executes a blank measurement. In this case, the personal computer 3 is controlled by the validation program P, and each part required for the blank measurement is automatically adjusted; therefore, the operator has no part in any of the operations.

When the blank measurement is completed, the personal computer 3 again reads the validation data D, and then, executes the next validation work. The next validation work is a work procedure on which the operator puts the standard sample S in the cell. For this reason, the personal computer 3 displays a message window W₇ as shown in FIG. 6. In the message window W₇, a message M₃ for instructing the work procedure to the operator is displayed, and a speech of the same content as the message M₃ is announced (step S_t5).

Subsequently, the operator mixes a PSL sphere in the cell according to the above message M₃, and then, inputs the completion of work by pressing the OK button B (step S_t6).

Then, the personal computer 3 reads the validation data D so as to execute the next validation work. More specifically, as shown in step S_t7, the personal computer 3 executes an adjustment of the optical axis, and thereafter, measures a light transmittance in order to confirm whether or not a measuring object sample put in by the operator has a transmittance within a range predetermined as a standard sample S for validation.

Namely, a decision is made whether or not the light transmittance of the measuring object sample S is higher than the upper limit of a predetermined transmittance (step S_t8).

At that time, in the case where the transmittance is too high, as shown in FIG. 7, a message window W₈ is displayed, and a message M₄ "concentration is too thin. Please add sample thereto" is displayed on the window screen while an announcement by speech is being outputted. In FIG. 8 and FIG. 9 described later, a reference numeral 4 denotes a bar graph showing a light transmittance measured in the above step S_t8, and a reference numeral 4a denotes a predetermined range of the light transmittance. When the operator presses the OK button B, the validation work is returned to step S_t6, and then, measurement for the light transmittance can be retried (step S_t9).

On the other hand, in the case where the light transmittance 4 of the measuring object sample S is not higher than the upper limit of the predetermined transmittance range 4a, conversely, a decision is made whether or not the light transmittance of the measuring object sample S is lower than the lower limit of predetermined transmittance range 4a (step S_t10).

In the case where the light transmittance 4 is too low, as shown in FIG. 8, a message window W₉ is displayed, and a message M₅ "concentration is too strong. Please make sample thin" is displayed on the window screen while an announcement by speech is also being outputted. When the operator presses the OK button B, the validation work is returned to step S_t6, and then, measurement for the light transmittance can be retried (step S_t11).

In the above manner, in the case where the light transmittance 4 is set within the predetermined range 4a, as shown in FIG. 9, a message window W₁₀ is displayed, and a message M₆ "Start measurement" is displayed on the window screen while an announcement by speech is being outputted. Then, when the operator presses the OK button B, a measurement of the particle size distribution is started (step S_t12).

In this case, the personal computer 3 automatically executes the following various settings for the particle size distribution measuring apparatus according to the measurement procedure recorded as the validation data D. The various settings include an input of refractive index of the standard sample S and solvent required for measuring a particle size distribution, etc. Namely, the operator has no need of doing troublesome various setting steps for the validation work. Moreover, it is possible to prevent a mistake which could be made in the case where the operator does the above various settings manually, and therefore, even an inexperienced operator can confidently perform the validation work.

Subsequently, when the particle size distribution measurement is completed, the personal computer 3 displays the measured result on windows Wa to Wc according to the measurement procedure recorded as the validation data D, and thereafter, makes a decision whether or not the measured result is suitable. Namely, the personal computer 3 makes a decision whether or not the measured result of the particle size distribution measuring apparatus 1 is within an allowable error range.

Then, the personal computer 3 displays a message window W₁₁, as shown in FIG. 10 in the case where the above measured result is within an allowable range, and displays a message M₇ "Validation OK" on the window screen while outputting an announcement by speech. On the other hand, in the case where the above measured result is outside the allowable range, as shown in FIG. 11, a message window W₁₂ is displayed, and a message M₈ "VALIDATION FAULT" is displayed on the window screen while an announcement by speech is being outputted (step S_t13).

In each case, when the operator presses the OK button B shown in FIG. 10 and FIG. 11, the personal computer 3 displays a message window W₁₃ as shown in FIG. 12, and then, displays a message M₉ "PRINT?" on the window screen while outputting the same announcement by speech (step S_t14).

When the operator presses a "YES" button By, the personal computer 3 prints inspection results such as measured result, validation standards, measuring person's name, date and time, kind of measuring apparatus, operational parameters or the like via a printer (not shown) (step S_t15). Namely, the operator can determine whether the result should be printed when performing the validation work; therefore, the operator never forgets to record the validation work.

On the other hand, in the window W₁₃ shown in FIG. 12, when the operator presses a "NO" button Bn, the personal computer 3 does not perform any procedure, and then, executes the next procedure (step St_t16).

FIG. 13 shows an example of the next step S_t16. In this embodiment, the personal computer 3 displays a window W₁₄ for recording inspection results such as measured result, validation standards, measuring person's name, date and time, the kind of measuring apparatus, and operational parameters in a storage section 3m as digital data. The window W₁₄ has an input bar N, an input bar F, a list L, a save button Bs for executing a save, and a cancel button Bc for canceling a save. More specifically, the input bar N is used for setting a file name for adding a file bundling a saving data, and the input bar F is used for setting a folder saving the file, and further, the list L is used for displaying a file name list of the file already stored in the folder.

Therefore, the operator can input a folder and a file name by using the above input bars F and N (step S_t16). The operator presses the save button Bs, and thereby, it is possible to readily save the inspection result (step S_t17).

On the other hand, when the operator presses the cancel button Be, the saving of the inspection result is cancelled, and the validation work ends.

The aforesaid embodiment is merely one embodiment of the present invention, and therefore, the present invention is not limited to this embodiment. More specifically, in the above embodiment, respective contents of the messages M₁ to M₉ displayed on each of the windows W₅ to W₁₃ may be announced by speech (voice), and thereby, the operator can extremely readily obtain validation help from the personal computer 3. The present invention is not limited to this modification and the message may be made by either of display on the display screen 3d or announced by speech.

Likewise, this embodiment has disclosed the case of printing the inspection result record on a paper and the case of storing it in a file as a digital data. Either of two disclosures may be carried out. Moreover, the inspection result record may be separately carried out by the operator.

In the above embodiment, as shown in the window W₂ of FIG. 3, three parameter values (median diameter, 10% diameter, 90% diameter) are set with respect to one standard sample S as validation standards. The present invention is not limited to this embodiment. For example, a geometrical mean particle diameter or the like may be set.

Further, the standard sample S to be measured is not limited to one kind.

FIG. 14 is a view showing a particle size distribution measuring apparatus according to another embodiment of the present invention. A particle size distribution measuring apparatus 1' will be described below with reference to FIG. 14. In the particle size distribution measuring apparatus 1', like reference numerals are used to designate the same member as FIG. 1, and therefore, the details are omitted.

In this embodiment, a reference numeral 2' denotes a measuring section for measuring a particle size distribution of measuring an object sample. The measuring section 2' is different from the measuring section 2 detailed described in FIG. 1 in that it 2' has an automatic sample charger 5 for automatically charging a plurality of standard samples S₁ to S_n according to the control by the personal computer 3.

FIG. 15 is a flowchart showing a procedure of validation work performed according to the control of the particle size distribution measuring apparatus 1'. In FIG. 15, the same steps S_t1 to S_t17 as FIG. 2 have almost the same description as FIG. 2, and therefore, the details are omitted for simplification of description.

In each procedure shown in FIG. 15, the procedures which are different from the description of FIG. 2 is as follows. More specifically, in step S_t3, standard samples S₁ to S_n are set with respect to the automatic sample charger 5, and there is no need of making a message displayed in step S_t5 and charging a sample by the operator in step S_t6.

Moroever, in this embodiment, the following step S_t18 for making a confirmation by the operator is added. More specifically, when measurement of one sample is completed, a message "next, measure another sample" is displayed on the screen, and then, announce by speech is made. In this case, when the operator presses the OK button, the personal computer 3 controls the automatic sample charger 5 so as to charge the next sample, and then, the procedures from step S_t4 is repeatedly made.

In this embodiment, when the procedure jumps from step St₁₈ to step S_t4, a blank measurement is made when the standard samples S₁ to S_n are changed in succession, and thereby, a measurement accuracy is improved. The present invention is not limited to this embodiment. For example, the procedure jumps from step S_t18 to step S_t7, and thereby, measurement may be made at a high speed.

As is evident from the above description, by using the particle size distribution measuring apparatus of the present invention, when the operator performs a validation work, the operator can receive a suitable validation help. More specifically, by validation help function, it is possible to operate the particle size distribution measuring apparatus according to a work procedure given from a control unit in a predetermined order without making a mistake. Moreover, a measurement procedure controlled by only control unit is automatically made according to a sequence of the measurement procedure previously stored as a validation data. Therefore, the operator can perform the complicated validation work without referring to a manual, so that a load acting on the operator can be reduced to the minimum. In addition, the work procedure is executed by one step without making a mistake, so that the validation work can be accurately performed.

Method and apparatus for aseptic growth or processing of biomass

Abstract

A method and apparatus for aseptic biological production or processing of cells, tissues and/or microorganisms is provided. The apparatus includes a support housing having an interior chamber, a disposable liner lining the interior chamber and a head plate attached to the liner forming a sealed chamber with the liner. After use the liner can be disposed and the apparatus can be reused with a new liner. In this way, the apparatus simplifies cleaning and ensuring validation required by pharmaceutical and food industry standards.


Claims

What is claimed is:

1. A bioreactor for culturing or processing a biomass, comprising:

a. a liner support;

b. a sterilized plastic liner having an opening, wherein the liner is mounted on the support and forms a reservoir for receiving a biomass dispersion;

c. a closure releasably sealingly engageable with the liner to close the liner opening, wherein the closure is separable from the liner, and the closure comprises;

a port in fluid communication with the reservoir; and

d. a connector comprising a support ring for releasably connecting the closure with the liner;

wherein the liner is sandwiched between the support ring and the closure, and the support ring and liner support are configured such that the liner and the closure can be removed from the liner support while maintaining a fluid-tight seal between the closure and the liner.

2. The bioreactor of claim 1 comprising an aerator for aerating the biomass dispersion in the reservoir.

3. The bioreactor of claim 1 wherein the closure comprises a second port in fluid communication with the reservoir.

4. The bioreactor of claim 1 wherein the closure comprises a third port in fluid communication with the reservoir.

5. The bioreactor of claim 1 comprising a circulator for circulating the biomass dispersion.

6. The bioreactor of claim 1 comprises a second reservoir in fluid communication with the reservoir.

7. The bioreactor of claim 6 wherein the biomass dispersion comprises culture medium and the bioreactor comprises a conduit for circulating culture fluid from the reservoir to the second reservoir.

8. A bioreactor for culturing or processing a biomass, comprising:

a. a sterilized plastic liner having an opening;

b. a support having a top opening adjacent the opening of the liner, wherein the support supports the liner so that the liner forms a first reservoir for receiving a biomass;

c. a second reservoir for receiving fluid;

d. a first fluid line connecting the first reservoir to the second reservoir for providing a flow of fluid from the second reservoir to the first reservoir through the top opening in the support; and

e. a second fluid line connecting the first reservoir to the second reservoir for providing a flow of fluid from the first reservoir to the second reservoir through the top opening in the support.

9. The bioreactor of claim 8 comprising a circulator for circulating the fluid in the second reservoir.

10. The bioreactor of claim 8 comprising an innoculation tube.

11. The bioreactor of claim 8 comprising an aerator for aerating the biomass with an aerating fluid.

12. The bioreactor of claim 11 comprising a vent for venting the aerating fluid from the bioreactor.

13. The bioreactor of claim 8 comprising a closure releasably sealingly engageable with the liner to close the liner opening.

14. The bioreactor of claim 13 comprising a connector for releasably connecting the closure with the liner.

15. The bioreactor of claim 13 wherein the first and second fluid lines extend through the closure.

16. A bioreactor for culturing or processing a biomass, comprising:

a. a first reservoir for receiving fluid;

b. a support having an internal configuration;

c. a sterilized plastic liner conforming to the internal configuration of the support to provide a second reservoir configured to grow a biomass;

d. an aerator in the second reservoir for aerating the biomass in the second reservoir;

e. a first fluid line connecting the first reservoir to the second reservoir for providing a flow of fluid from the second reservoir to the first reservoir; and

f. a second fluid line connecting the first reservoir to the second reservoir for providing a flow of fluid from the first reservoir to the second reservoir.

17. The bioreactor of claim 16 comprising a circulator for circulating the fluid in the first reservoir.

18. The bioreactor of claim 16 comprising an innoculation tube.

19. The bioreactor of claim 16 comprising a vent for venting the bioreactor.

20. The bioreactor of claim 16 comprising a closure releasably sealingly engageable with the liner to close the liner opening.

21. The bioreactor of claim 20 comprising a connector for releasably connecting the closure with the liner.

22. The bioreactor of claim 20 wherein the first and second fluid lines extend through the closure.

Description

FIELD OF THE INVENTION

The present invention relates to the field of biologic cell production. More specifically, the present invention relates to the aseptic production and processing of cells, tissues and/or microorganisms in a bioreactor.

BACKGROUND

The production of chemicals in bioreactor systems is expensive. The differential between production costs and product market value is the dominant driving force for drug discovery and development of potential bioprocesses. The importance of production costs is reflected in the economic observation that the volumetric productivity of a wide range of biologically produced products is about the same at $0.17 per liter per day. The expense of biological production is a motivation for pursuing chemical synthesis when possible; however, the complexity of synthesis of many natural products often makes this route equally costly. In the absence of chemical synthesis, metabolites derived from microorganisms must be produced in aseptic bioreactors. In contrast, plant-derived chemicals can be harvested from intact plants. Therefore, agronomic production or collection from natural environments is a formidable competitor to growth of plant tissues in bioreactor systems. Many rationales are given for pursuing plant tissue culture as a potential production system. The most compelling are those situations where intact plants are poor competitors. Some plants either grow very slowly, or are not amenable to agronomic production. In addition, environmental degradation is limiting the attractiveness of natural harvest, particularly from endangered environments such as the rain forest where the biochemical diversity is the greatest.

Although the bioreactor described herein is not limited to use for plant tissue culture, the economic constraints and stringent asepsis requirements presented by this production system provide an excellent context to demonstrate the effectiveness of the bioreactor. There have been many efforts to commercialize plant metabolites from cell culture; however, few have achieved commercial success. Low productivity is usually cited as the reason for failure despite the fact that production rates and tissue concentrations are very often substantially higher than the intact plant. In fact, tremendous productivities have been achieved by plant tissue culture. There are at least eight different systems where the metabolite levels are greater than 10% of the cell dry weight, and several of these productivities have been achieved with cultures that display relatively high growth rates. One example is anthocyanin pigments production by P.C.C. Technology, Japan where the cell content was greater than 17% and effective specific growth rates were 0.22 day^-1 at a scale of 500 L. Rosmarinic acid production was successfully scaled up by A Nattermann & Cie GmbH in a 30 L stirred tank. The titer of rosmarinic acid reached 5.5 g/L with volumetric productivity of nearly 1 g/L/day, and tissue content as high as 21% of dry weight. The failure of these processes is more a failure to compete economically with whole plant material rather than a failure of the cultures to be biochemically productive. There have also been significant advances in strategies to improve cellular productivity by cell line selection, genetic engineering, elicitation and root culture or enhance reactor productivity by operational strategies such as high density culture, integrated product recovery and immobilization. However, there is a limit to the improvements that can be achieved by these strategies, and for compounds where there is a low-cost alternative from intact plant material, it simply does not make sense to attempt production in bioreactor systems.

Despite the limitations, the potential of plant-tissue culture derived chemicals has resulted in a tremendous investment from both industry and academia in developing this technology. It has been demonstrated that large-scale production is technically feasible. The first commercial process was the production of shikonin. Since the market for this dye is limited, production has been on hold to focus efforts on taxol as a more profitable target. Similarly, the efforts of EscaGenetics on the production of vanillin were way-layed in favor of taxol development. Ginseng has been produced commercially by Nitto Denko (Japan) for 10 years at a scale of 25,000 liters. There are other reports of industrial scale cultivation of plant cells including tobacco at 15,500 L and three different plant species by DIVERSA (Hamburg, Germany) up to 75,000 L. Taxus sp. is being grown at industrial scale by both Phyton, Inc. and Sam Yang. These examples show that technical problems of scale-up can be overcome.

The preceding indicates that the technology for production of chemicals by plant tissue culture is available provided the secondary metabolite has a sufficiently high value. The required product price to consider plant tissue culture production has been estimated to be in the range of $1000 to $5000 per Kg. The issue arises as to whether this technology can be extended to lower value/higher volume biochemical production. To achieve this objective, it is useful to understand what contributes to production costs.

Based on experience with the commercial development of shikonin, the Mitsui group estimated that 64% of the production costs for cultured plant cells was due to fixed costs (depreciation, interest and capital expenditures). A similar number can be calculated from the recent analysis presented by Goldstein based on general plant tissue culture characteristics. Using Goldstein's 2000 kg product per year basis (which is implicitly 22 tons of cell mass based on assumed productivity), the fixed costs (calculated as capital charges) were 55.4% of the manufacturing costs. The estimate of Yoshioka and Fujita is likely to be more generally applicable since it uses a cycle time of roughly 14 days as compared to the 5-day reactor cycle time assumed by Goldstein. Clearly capital investment is an important target for cost reduction. This is not surprising since equipment and support facilities associated with aseptic bioprocessing are extremely expensive because vessels are constructed of stainless steel and pressure rated for autoclave sterilization. Accordingly, eliminating the need for expensive autoclave construction could substantially reduce production costs by reducing the initial capital investment.

SUMMARY OF THE INVENTION

In light of the foregoing, the present invention provides a method and apparatus for producing cells, tissues and/or microorganisms. The method includes providing a disposable liner forming a reservoir having an opening. A closure is attached to the liner to close the opening. The liner and attached closure are sterilized. A biomass dispersion is then introduced into the reservoir.

The present invention further provides a bioreactor for culturing cells, tissues and microorganisms. The bioreactor includes a support, and a liner mounted on the support and forming a reservoir for receiving a biomass dispersion. A closure sealingly engages the liner to close the liner opening. The closure sealingly engages the liner and is separable from the liner. The closure includes an inlet port in fluid communication with the reservoir.

DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the preferred embodiments, will be better understood when read in conjunction with the accompanying drawing, in which:

FIG. 1 is a side sectional view of an aseptic bioreactor in accordance with the present invention;

FIG. 2a is a graph of the growth of the cell cultures for Examples 1 and 2;

FIG. 2b is a graph of the growth of the cell culture for Example 3; and

FIG. 3 is a side sectional view of second embodiment of an aseptic bioreactor in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings in general, and to FIG. 1 specifically, an aseptic bioreactor 10 is illustrated. The bioreactor is lined with a disposable liner 30. The bioreactor is used for biological production of cells, tissues and/or microorganisms in a culture medium. After use, the cells and culture medium are removed from the bioreactor 10 and the liner 30 is disposed. The bioreactor can then be used again with a fresh liner.

The bioreactor 10 can be used to produce any of a number of microorganisms and/or cells, including, but not limited to bacteria, fungi, animal cells, nematodes and plant cells. In addition to the production of biomass, the bioreactor is operable to process biomass grown in alternative bioreactors or tissues produced by conventional non-aseptic methods, such as: animal husbandry, field growth plants or general biomass byproducts. Specifically, the bioreactor is operable in connection with biotransformation of biochemicals utilizing enzymatic capabilities of tissue--such as transgenic plant tissue grown in the field to express a heterologous enzymatic activity. The bioreactor is also operable in connection with expressing proteins from post-harvest activated promoters in which the biomass is grown in the field. In such circumstances, the tissue is not growing within the vessel, however, the viable tissue is carrying out the desired chemistry within a process vessel that can be of the type described herein.

In the following description, the bioreactor is described in connection with the production of plant cells. However, the design and/or operation of the bioreactor can be modified to accommodate production of other types of cells. For example, the production of plant cells progresses slowly so that respiratory heat removal is not problematic to maintain the proper temperature within the bioreactor. However, when producing microorganisms that exhibit rapid progression, such as bacteria, respiratory heat removal is generally necessary to maintain the proper temperature within the bioreactor. Therefore, to accommodate such an application, the bioreactor can be configured to include a heat exchange coil for heat removal. Accordingly, although the following description exemplifies use of the bioreactor in connection with producing plant cells, the bioreactor is not limited to such use.

The bioreactor 10 includes a hollow support housing 20 lined by the disposable liner 30. A head plate 40 is attached to the liner to form a sealed reservoir within the liner 30. The head plate 40 and liner 30 are then sterilized to form an aseptic environment within the reservoir. Culture medium is introduced into the container 20 through an inoculation tube 46 that is in fluid communication with the reservoir. The culture medium is then inoculated through the inoculation tube 46. An aerator 50 extends through the head plate 40 to aerate the biomass dispersion during the production cycle. During the production cycle, the reservoir is sealed to prevent contamination from the outside environment. After the production cycle is complete, the biomass dispersion is removed from the reservoir. After the biomass dispersion is removed, the liner 30 is disposed. Since the liner 30 prevents the biomass dispersion from coming into contact with the support housing 20, the support housing need not be sterilized before reusing the bioreactor for another production cycle. Instead, the reusable head plate 40 is attached to a new liner, and the two are sterilized. In this way, the sterilization of the bioreactor is simplified, and validation of the "clean in place" procedures is significantly simplified.

STRUCTURE OF THE APPARATUS

The structure of the bioreactor 10 will now be described in greater detail. The support housing 20 is an elongated hollow generally cylindrical container. The upper end of the support housing is generally open and the lower end or bottom of the support housing is closed. In the present instance, the support housing 20 includes a circumferential flange 24 projecting outwardly from the side walls 22 of the housing. Baffles 26 are disposed in the interior of the support housing 20 adjacent the bottom on the support housing. As shown in FIG. 1, the baffles 26 form an asymmetric or offset V-shape. As discussed in more detail below, the V-shape improves circulation of the bioreactor.

The support housing 20 can be configured in almost any shape. If an "air-lift" circulation system is utilized as described below, preferably, the aspect ratio of the container is such that the height is 2 to 5 times the width of the container. However, the aspect ratio may be higher or lower. For instance, in certain situations it may be desirable to use a support housing that is shallow and wide. For such a shallow support housing, the fluid pressure of the aeration fluid can be reduced because the pressure at the bottom of a shallow reservoir is less than that of a deeper reservoir, and the aeration fluid must overcome this fluid pressure to aerate the reservoir.

The support housing 20 may be formed of glass thereby exposing the biomass dispersion 15 to light, and allowing visual inspection of the biomass dispersion. However, the support housing can also be formed of any number of materials including metal or plastic. In addition, the support housing can be opaque or semi-opaque.

As can be seen from the foregoing, the configuration of the support housing 20 can be varied considerably. Generally, a structure that mechanically supports the weight of the cell suspension 15 can be utilized. Specifically, a structure that supports the vertical force of the weight of the biomass dispersion 15 and the lateral or horizontal force of the fluid pressure of the biomass dispersion can be utilized. For instance, the support housing 20 could be in the form of an open framework or mesh rather that the solid walls of the housing shown in FIG. 1.

The liner 30 is preferably formed of plastic. The plastic may be transparent or translucent to allow light into the reservoir and to permit visual inspection of the bioproduction/bioprocessing if desired. The type and thickness of plastic will depend upon several variables, including the size of the support housing 20 and the type of sterilization process that will be utilized to sterilized the liner 30 and the head plate 40. For instance, a 2 mil thickness autoclavable polypropylene bag can be utilized if autoclaving is used. For other sterilization processes, such as gas-phase or plasma-phase sterilization, the liner 30 may be formed of a 6 mil thickness polyethylene bag.

As shown in FIG. 1, the area in the support housing 20 above the baffles 26 and between the side walls 22 forms an internal chamber. The liner 30 lines this internal chamber to prevent the cell suspension 15 from coming into contact with the interior of the support housing 20. The upper edge of the liner 30 overlays the flange 24 at the top of the support housing 20. The upper edge of the liner 30 may be sandwiched directly between the head plate 40 and the flange 24. However, in the present instance, the upper edge of the liner 30 is disposed between the head plate and a support ring 28 that rests on the flange 24. The support ring 28 is a flat ring that is approximately as wide as the flange 26 of the support housing. By sandwiching the upper edge of the liner 30 between the head plate 40 and the support ring 28, the head plate and liner can be removed from the support housing without detaching the liner from the head plate.

The head plate 40 is a substantially round plate having a diameter that is greater than the open end of the support housing 20. The head plate can be made from a variety of materials including metal and plastic, For instance, the head plate 40 may be formed of polycarbonate. The lower surface of the head plate 40 confronts the upper surface of the support ring 28. Preferably, a sealing groove 42 extends around the periphery of the lower surface of the head plate, spaced inwardly from the outer edge of the plate. A seal 44 in the form of an O-ring is disposed in the sealing groove 42. The head plate 40 can be attached to the sealing ring 28 in any of a number of ways. Preferably the head plate is releasably attached to the sealing ring 28 so that the liner 30 can be detached from the head plate, allowing the head plate to be reused. In the present instance, the head plate 40 is clamped to the sealing ring 28 with the upper edge of the liner 30 disposed between the head plate and the sealing ring. In this way, a fluid-tight seal is provided between the head plate and the opening in the liner.

The bioreactor 10 includes an inoculation tube 46 and a sampling tube 48 that project into the sealed reservoir formed by the liner 30 and the head plate 40. The inoculation tube 46 and the sampling tube 48 are in fluid communication with the sealed reservoir and provide access to the interior of the bioreactor from exterior of the bioreactor. A seal is formed between the exterior of the inoculum and sampling tubes 46, 48 and the head plate to provide a fluid-tight seal. The inoculation tube 46 is of sufficient diameter for introducing the culture medium into the sealed reservoir through the inoculation tube, along with the inoculum. Depending on the application, the inoculation tube can be configured in any one of a number of designs that facilitate introducing inoculum and resealing the inoculation tube to prevent contamination. The sampling tube is of sufficient diameter for withdrawing media samples.

The bioreactor 10 further includes an aerator 50. In the present instance, the aerator is a sparger. A filter is provided for filtering the gas used by the aerator to prevent contaminants from entering the reservoir. As shown in FIG. 1, the aerator projects through the head plate 40 and into the bottom of the reservoir. For submerged cultures, the aerator 50 is preferably aligned with the vertex of the V-shaped bottom of the reservoir formed by the baffles 26 in the bottom of the support housing 20. In the present instance, the aerator 50 includes two spargers attached in a T-configuration that is parallel to the crease in the V-shaped baffle 26.

In the present instance, the aerator 50 also operates to circulate the biomass dispersion within the bioreactor 10. This is referred to as an "air-lift reactor." The gas bubbling through the biomass dispersion 15 from the aerator 50 causes variations in the density of the culture medium. This operates to circulate the culture medium in the reservoir. For this reason, it is desirable to have the bottom end of the aerator 50 adjacent the lowest point of the reservoir.

The bottom of the reservoir may be flat. However, to improve circulation it is desirable to eliminate the lower corners that form dead spaces. For this reason, the baffle 26 is V-shaped. Offsetting the vertex of the V-shaped baffle 26 as shown in FIG. 1 further improves the circulation in the reservoir. In addition, in a large bioreactor it may be desirable to include a plurality of aerators to improve aeration and circulation.

As described above, in the present instance the aerator 50 operates as an aerator and a circulator. In certain instances it may be desirable to further include a separate circulator. For instance, an impeller can be included to improve the circulation in the reservoir. Such an impeller can be journalled in a bearing mounted in the head plate so that it extends downwardly into the bottom of the reservoir.

As another example, a magnetic impeller may be provided to improve the circulation in the reservoir. A magnetic impeller would not require the mechanical connection between the impeller blade and the drive mechanism. The magnetic impeller utilizes magnetic force to drive the impeller blade. Since there is no mechanical connection between the magnetic impeller blade and the impeller drive, there is no need to provide an additional seal as is required around the drive shaft of a standard impeller. In addition, since magnetic impeller blades are can be relatively inexpensive, the magnetic impeller blade could be disposed along with the liner 30 after a production cycle is completed. This further simplifies cleaning the apparatus and preparing for a subsequent production cycle.

The embodiment illustrated in FIG. 1 shows a gas-dispersed liquid culture. However, the process can also be implemented as a liquid-dispersed bioreactor in which medium is dispersed over the top of the biomass. For example, referring to FIG. 3, an alternative bioreactor embodiment 110 is illustrated. In the alternative embodiment, the biomass is in a primary reservoir and fluid is circulated into the primary reservoir from a secondary reservoir 180 that is separate from the primary reservoir.

Specifically, the bioreactor 110 includes a reservoir lined by a disposable liner 130 that lines a support 120. A head plate 140 seals the liner as in the first embodiment and forms a primary reservoir for growing or processing a biomass. Preferably, a bed of material 123, such as industrial process packing is introduced into the primary reservoir. A biomass is introduced into the primary reservoir and culture medium is circulated through the reservoir.

A secondary reservoir 180 is also provided. Preferably, the secondary reservoir is also formed by a disposable liner that lines a support and is sealed by a head plate. The secondary reservoir contains a quantity of fluid, which may be culture medium or biomass dispersion as is discussed further below.

A feed line 163 and return line 164 are provided for recirculating fluid between the primary and secondary reservoirs. The feed line and return line are in fluid communication with the primary and secondary reservoirs. The return line 164 extends into the bottom of the primary reservoir to recycle fluid after it flows through the primary reservoir.

In this way, a "trickle bed" can be utilized to grow or process biomass. Specifically, biomass is introduced into the primary reservoir. Culture medium is circulated through the primary reservoir from the secondary reservoir. If a bed of material 123 is provided, the culture medium flows into the primary reservoir over the bed of material 123. The culture medium trickles through the bed of material and is then recycled through the return line 164. As the fluid flows through the primary reservoir over the bed of material 123, the fluid promotes the growth or processing of the biomass in the bed of material.

Depending on the biomass, the circulated fluid can be either culture medium alone or biomass dispersion. Specifically, if the biomass dispersion is a suspension, then the suspension is circulated from the secondary reservoir, into the primary reservoir, over the bed of material, and then recycled through the return line 164. In other applications, the biomass dispersion may not be a suspension. In such applications, substantially all of the biomass remains in the primary reservoir and culture medium is circulated over the biomass.

The re-circulating system has been described in connection with a bed of industrial process packing through which the culture medium or biomass dispersion circulates to promote growth or processing of biomass in the bed. However, the bedding material need not be industrial process packing. The bedding material can be other granular material or matrix material.

In addition, the recirculating system is operable in certain applications without a bed of material. In such applications, the biomass is introduced into the primary reservoir, and culture medium is circulated over biomass.

METHOD OF OPERATION

The bioreactor 10 operates as follows. The head plate 40 and liner 30 are attached and sterilized to form an aseptic container. The head plate and liner can be sterilized in any of a number of ways. For instance, autoclaving can be used if the liner 30 and head plate are formed of materials the withstand the temperature and pressure of the autoclaving process. Alternatively, the head plate and liner can be sterilized by gas-phase sterilization using ethylene oxide gas in accordance with hospital guidelines for ethylene oxide sterilization of medical devices. In addition, other sterilization processes such as a vapor phase oxidant, plasma or radiation sterilization.

Furthermore, although the operation of the device contemplates the ability to be sterilized in accordance with the stringent guidelines of the pharmaceutical industry, the term sterilization as used herein is not limited to pharmaceutical or medical sterility. The term sterilization is meant to include cleaning to less stringent standards such as the standards for sterility for the food industry. In addition, as used in this description, the term sterilization is also meant to include marginal sterility, meaning reduction or suppression of non-productive contaminating organisms to sufficiently low numbers so that their presence does not prevent or significantly impede the desired biomass growth or processing. Accordingly, as can be seen from the foregoing, operation of the bioreactor is not limited to a particular sterilization process.

If sterilization is to be used, the head plate 40 and attached liner 30 can be inserted into the autoclave without the support housing because the support housing need not be sterilized since it does not come into contact with the biomass dispersion. Accordingly, a liner and head plate can be autoclaved in a collapsed state, so that a smaller autoclave vessel can be utilized to service a plurality of large-scale production bioreactor tanks. If gas-phase sterilization is to be used, the head plate is attached to the liner, a gas mixture of ethylene oxide and carbon dioxide is introduced into the sealed reservoir formed by the head plate and liner. Preferably, prior to introducing a gas into the reservoir, steam is introduced into the reservoir to provide a damp environment and assure hydration of contaminant spores. After gas-phase sterilization, the toxic ethylene oxide is removed by aeration using a slow flow of air. Surface sterilant gases such as hydrogen peroxide may require little or no aeration.

During gas-phase sterilization, the ethylene oxide gas can permeate the liner 30 and accumulate between the liner and the inner wall of the support housing 20. This accumulated ethylene oxide may remain in the bioreactor after standard aeration procedures. This residual ethylene oxide can permeate through the liner 30 into the reservoir during the production cycle to adversely affect cell production. Accordingly, it is desirable to minimize the gap between the liner and the inner wall of the support housing. In addition, it is desirable to aerate the inside and outside of the liner and head plate assembly to reduce the amount of residual ethylene oxide. This can be accomplished by removing the sealed head plate and attached liner assembly from the support housing and aerating the support housing in addition to aerating the reservoir. It should be noted that potential toxicity of residual ethylene oxide release will become less significant for larger scale reactors since the film surface area to tank volume rapidly declines with scale-up. In addition, the use of non-permeating surface sterilant gas could greatly reduce aeration considerations for gas or plasma phase sterilization.

After the liner and head plate assembly are sterilized, the liner is inserted into the support housing. Alternatively, depending on the type of sterilization used, the liner and head plate assembly can be inserted into the support and then sterilized. Culture medium is then introduced into the sealed medium through the inoculation tube 46. If desired, a growth regulator is also introduced into the reservoir through the inoculation tube. This combination is then inoculated with a cell culture. For clarity, the resultant mixture of cell culture, growth regulator, if any, and culture medium are referred herein to as a biomass dispersion. The biomass dispersion is aerated by the aerator 50 during the production cycle.

The biomass growth is monitored during the production cycle so that the composition of the aeration fluid and/or the flow rate of the aeration fluid can be adjusted to optimize cell growth. Depending on the cell or microorganism being produced, the characteristics monitored during the production cycle may vary. For instance, utilizing dissolved oxygen as a control parameter may be desirable for rapid-production cultures such as bacteria. Conversely, using dissolved oxygen as a control parameter for slow-production cultures, such as plant tissue cultures, can be problematic. Similarly, off-gas analysis can be used to monitor respiration. However, off-gas analysis is generally more amenable to monitoring rapid-production cultures then slow-production cultures. Nonetheless, other measurements indicative of biomass growth can be utilized to effectively monitor biomass growth of slow-production cultures. For instance, incertain culture media, the refractive index of the culture medium is an indicator of sugar levels, and the electrical conductivity of the culture medium is an indicator of inorganic nutrients. These characteristics can be measured and used to correlate biomass accumulation based on nutrient consumption. The examples detailed below describe plant tissue production, utilizing refractive index, electrical conductivity and medium osmolality as control parameters. These details of such production control are set forth in greater detail in Ramakrishnan, D.; Luyk, D.; Curtis, W. R., "Monitoring biomass in root culture systems", Biotechnology and Bioengineering, 62(6): 711-721, 1999, which is hereby incorporated herein by reference as is fully set forth herein.

None of these measurements mentioned above require introduction of a measuring instrument into the reservoir. However, if the bioreactor is utilized to produce other types of cells and/or organisms, the bioreactor may include one or more measuring devices that extend into the reservoir, preferably through the head plate in a fluid-tight relation, to prevent introduction of contaminant cells and/or microorganisms. For instance, a pH meter may be attached to the head plate, extending downwardly into the reservoir to monitor the pH of the culture medium.

In response to the measured characteristic, the operating environment may be modified to optimize biomass production or processing. For instance the gas composition of the aeration gas can be modified. Alternatively, the flow rate of the aeration gas can be varied. In addition, the biomass dispersion may be supplemented with sugar and/or inorganics to encourage continued growth.

EXAMPLE 1

Two 9 L (6.5 L working volume, w.v.) bioreactors were provided: one steam autoclave sterilizable and the other ethylene oxide (EtOX) sterilizable. For the autoclavable configuration, the plastic liner was a 2 mil polypropylene autoclavable bag (25 cm width by 46 cm height). The top edge of the bag was clamped to a 12.7 mm thickness polycarbonate head plate which contained a 17 mm internal diameter (ID) stainless steel inoculation tube, a 1.4 mm ID tube for withdrawing medium samples, a 4.5 mm ID gas outlet tube, and an additional 4.5 mm ID tube which extended to the bottom of the bag with a 0.2 micron sintered metal mobile phase sparger attached for sparging of gas. A bead of hot melt glue stick was applied between the bag and head plate, which melted upon autoclaving to provide an air-tight aseptic seal. The reactor was steam sterilized at 121° C. for 30 minutes in a collapsed state, and expanded after autoclaving inside a 15.2 cm diameter non-sterile glass vessel.

EXAMPLE 2

The second bioreactor is designed to eliminate the need for autoclave sterilization. The second bioreactor utilizes sterilization through exposure to ethylene oxide (EtOX)-carbon dioxide gas mixture. The ability to sterilize the reactor without high temperature eliminated the need for autoclavable materials. For this reason, the plastic liner was constructed from 6 mil thickness polyethylene, and Norprene tubing was used in all connections to minimize degradation and diffusion of EtOX during the sterilization cycle. The liner was constructed to fit inside the same 15.2 cm ID (internal diameter) glass column, with the seams sealed with a hot glue gun and a hand iron wrapped in paper towel. The head plate was constructed from 20-gauge (0.95 mm) 304 stainless steel sheet metal where the ports analogous to those described for the polycarbonate head plate were silver-soldered in place. The head plate and liner were held in place by bolting through the collar of the Corning conical glass connections. A bead of silicone glue was applied between the head plate and plastic to facilitate the seal. Gas-phase sterilization was accomplished using a commercially available mixture of ethylene oxide: 10% EtOX in carbon dioxide to avoid the danger of flammability. Prior to gas introduction, steam under ambient conditions was briefly introduced from the release valve of a commercial pressure cooker. This pre-sterilization steam treatment provided a damp environment and assured hydration of contaminant spores. Exposure to ethylene oxide was accomplished in a fume hood where roughly four reactor volumes of the EtOX gas mixture were introduced three times over a period of two days. Gas was introduced through the air sterilization filter, and the gas outlet was rotated using pinch clamps through the medium sample, gas outlet, and inoculation ports. After gas-phase sterilization, the toxic EtOX was removed by a slow flow of 200 mL/min of sterile air for a period of roughly two days based on hospital guidelines for ethylene oxide sterilization of medical devices.

EXAMPLE 3

A 40 L bioreactor with a working volume (w.v.) of 28.5 L was constructed from 6 mil plastic to fit inside a 59.7 cm height by 28.3 cm diameter glass tank. The head plate sealed against a compression ring so that the liner and head plate could be independently removed from the tank to facilitate aeration from both sides of the liner and eliminate pockets of ethylene oxide between the liner and the support tank. Circulation was facilitated by off-center placement of the sparger, and contouring of the tank bottom with a baffle. The baffle was `V`-shaped with the crease off-center to align with the sparger. The sparger consisted of two 0.2 micron sintered metal mobile phase spargers attached in a T-configuration so that they could be placed parallel to the crease in the V-shaped baffle. The reactor was fitted with a 28.6 mm ID inoculation port and 9.5 mm ID sample port similar to the other reactor configurations. The 34 cm ID head plate and compression ring were constructed of 12.7 mm thickness polycarbonate with an o-ring groove machined in the upper plate 25.4 mm from the edge to provide for a seal against the plastic liner.

EXAMPLE 4

A 150 L (100 L w.v.) bioreactor was constructed from 6 mil polyethylene film to fit inside a 85.7 cm height by 45.1 cm diameter stainless steel process tank. The baffling geometry, head plate compression ring, and sparging arrangement were analogous to Example 3. The polycarbonate head plate included a 19.1 mm ID inoculum port, and two 12.7 mm ID compression fittings for the sparge tube inlet and sample port. A separate 6.4 mm ID compression fitting was used for gas outlet. A thin silicone film on the 5.7 cm width aluminum compression flange 28 was utilized to accomplish the seal between the head plate and plastic liner.

The root culture used in EXAMPLE 4 was culture of Hyoscyamus muticus, line HM90T, established by Agrobacterium rhizogenous transformation in 1990 and grown on `Gamborg B5` medium. The cell suspension line used for the four EXAMPLES was established from the same root culture by de-differentiating the root culture through the addition of 0.2 mg L^-1 of the growth regulator 2,4 dichlorophenoxyacetic acid (2,4 D). The root line and cell lines have been maintained for more than 3 years through serial bi-weekly subculturing on their respective medium. Autoclaved B5 medium was introduced aseptically for Examples 1 through 3, and Example 4 utilized filter sterilization through a cartridge filter sterilization unit. The initial media volumes were 6.5 L (example 1), 6.7 L (example 2), 28.5 L (Example 3) and 100 L (Example 4, for both cell and root culture run).

The operational strategy for the bioreactors relied upon the refractive index and conductivity (as well as visual observation through the glass reactor) for making operational changes. The initial gas flow rate to the prototype reactors was minimized (0.05 volumes of gas per volume of medium per minute, VVM) corresponding to 0.33 liters per minute. Air was used for the first day to avoid potential problems of oxidative stress and the low flow rates reduced foam fractionation and loss of cells on the reactor vessel walls at the surface of the medium. When the conductivity started to decline--indicating culture growth--the gas composition was changed to 30-40% oxygen in air by oxygen supplementation. This oxygen enrichment permitted low gas flow rates and minimized volatile stripping. Low gas flows also minimized wall growth. The gas flow rate was incrementally increased to 0.25 VVM for the 9 L autoclaved bag and 0.20 VVM for the 9 L EtOX sterilized bag as the culture nutrients declined. The gassing program for the 40 L reactor was initiated at 0.05 VVM, and incrementally increased to 0.25 VVM as the culture nutrients declined. CO₂ and O₂ supplementation for the 40 L reactor were initiated at day 2 and 10, respectively. The glass support tank permitted visual observation, which confirmed that the gas flow should be increased more rapidly in the air-lift reactors than the gas-flow used in a typical stirred tank to avoid cell sedimentation.

The gassing strategy in the 150 L bioreactor paralleled that of the smaller reactors. This pilot-scale run was undertaken to verify the utility of nutrient feed based on the measurements of media conductivity, refractive index and osmotic pressure. When refractive index and conductivity indicated 90% nutrient consumption (day 9 after inoculation), a sucrose feed corresponding to an additional 20 g sucrose per liter was added to the bioreactor. The root culture was operated in simple batch mode without media supplementation.

RESULTS

Biomass accumulation for the two smaller vessels is plotted in FIG. 2a. Growth ceased in the EtOX sterilized reactor in Example 2 due to sugar depletion as indicated by a zero refractive index for the final cell concentration data points. The autoclaved reactor in Example 1 also reached a zero RI; however, a supplementation of sugar was added at day 11, which permitted continued growth. This indicated that substantially higher cell concentrations are possible. The autoclaved bag reactor was terminated when a low-level contaminant was observed at day 13 as the appearance of slow growing colonies on LB media plates.

Growth in the ethylene oxide sterilized reactor in Example 2 was clearly attenuated--presumed to be due to toxicity of residual ethylene oxide. Based on recommendations for hospital use of EtOX, it was anticipated that the two days aeration period should have been adequate for removal of residual EtOX. However, it was later realized that the sand below the plastic bag could have acted as a reservoir that would diffuse small amounts of residual EtOX for a long period of time. After 15 days of slow growth, the rapid consumption of nutrients indicated a rapid growth period, which was visually evident through the glass reactor as well, suggesting a recovery from the ethylene oxide toxicity.

As discussed previously, residual sterilant gas removal predicted that the potential for residual toxicity can be minimized for a well fit reactor liner. For the Third Example, the contouring of the support housing was accomplished by baffles which resulted in large reservoir volumes between the liner and the tank wall. The design of the 40 L bioreactor permitted removal of the liner and head-plate from the tank to provide aeration on both sides. As shown in FIG. 2b, this successfully eliminated the long lag observed in the smaller prototype reactor in Examples 1 and 2, and the specific growth rate of 0.26 day^-1 is approximately the same as the results for stirred tank bioreactors. The bioreactor of the Third Example produced 2.9 Kg FW (199 g DW) of cells in 13 days.

The 150 L bioreactor with sugar supplementation produced over 53.8 Kg fresh weight (1.5 Kg DW) of plant cell suspension biomass during the 33 day culture period. This represents one of the highest biomass productivities achieved in plant cell culture at the pilot scale. This run demonstrated the ease of scaleability of the bioreactor and utility of the monitoring technique. By the end of the run, more than half of the water within the bioreactor was inside the biomass so that more simplified methods of attempting to monitor bioreactor performance based on media concentrations were not accurate. The same reactor inoculated with homogenized root tissue produced 24.1 g FW Kg FW (881 g DW) or root tissue for a 31 day culture period. The growth of root cultures demonstrates the versatility of the bioreactor in growing an organized tissue.

The method and apparatus of the present invention provide a simple, low cost bioreactor for bench and pilot scale discovery and developmental studies. A plastic lined reactor has very attractive characteristics besides the reduction in costs. Such a design could potentially eliminate much of the costs associated with validation of clean in place (CIP) procedures since the plastic liner portion of the reactor is disposable. Validation of sterilization only requires verifying sterilization of the reusable head plate, and a clean plastic liner. Accordingly, the method and apparatus of the present invention should be capable of meeting the very stringent cGMP (good manufacturing practices) guidelines currently imposed on the pharmaceutical industry.

Costs for gas sterilization could be improved by sterilization in a collapsed state to minimize gas use. Similarly, the bag and head-plate assembly could be autoclaved in a collapsed state--thereby utilizing one smaller autoclave-type vessel to service many larger-scale production tanks.

It will be recognized by those skilled in the art that changes or modifications may be made to the above-described embodiments without departing from the broad inventive concept of the present invention. For instance, the bioreactor may be utilized for anaerobic fermentation. For such production, the bioreactor need not include means for aerating or circulating the cell suspension in the reservoir. Accordingly, it should be understood that the present invention is not limited to the particular embodiments described herein but is intended to include all changes and modifications that are within the scope and spirit of the invention as set forth in the claims.