JP6731172B2 - Calibration method of dissolved oxygen sensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a dissolved oxygen sensor, and more particularly to calibration thereof.

A single-use bag is used for culturing animal cells used for antibody drug production in a culture solution. A dissolved oxygen sensor is used to measure the dissolved oxygen concentration in the culture solution in the single-use bag.

FIG. 4 is a diagram illustrating the principle of the optical dissolved oxygen sensor. When the dissolved oxygen sensor unit or the dissolved oxygen sensor chip is irradiated with excitation light modulated in a sinusoidal wave, phosphorescence having a phase angle corresponding to the oxygen concentration is obtained.

In Equation 1, A is a constant, and the phase angle in nitrogen (oxygen concentration 0%) or zero oxygen water is Pn, in air (oxygen concentration 20.9%) or in atmospheric saturated water (dissolved oxygen concentration 8 at 25° C.). 11 is an example of an approximate expression for obtaining the dissolved oxygen concentration DO from the phase angle P in a gas having a certain oxygen concentration when the phase angle at 0.11 mg/L) is Pa.

DO=A*(P-Pa)/(Pn-Pa) (1)

Here, the measurement of the parameters Pa and Pn of Equation 1 is called calibration. In reality, the phase angle and the oxygen concentration do not have a linear relationship, so it is necessary to approximate them with a polynomial or the like, but linear approximation is assumed. Further, the phase angle measured in air and the phase angle measured in liquid are treated as equivalent, and the oxygen concentration in air and the dissolved oxygen concentration in liquid are not distinguished.

FIG. 5 is a configuration explanatory view showing an example of a probe type dissolved oxygen sensor which has been conventionally used. In FIG. 5, a dissolved oxygen sensor probe (hereinafter also simply referred to as a sensor probe) 1 is autoclaved by a high-pressure steam sterilizer (not shown).

A connector 3 is integrated with the outside of the side of the single-use bag 2 so as to communicate with the inside thereof, and the inside of the single-use bag 2 has a stirrer 5 rotatable at one end through a shaft 4 protruding outside. Is provided.

The single-use bag 2 in which the connector 3 is integrated and the agitator 5 is provided inside can be purchased from a manufacturer that has been sterilized in advance by γ-ray irradiation, for example.

The user purchases a single-use bag 2 that is integrated with the connector 3 and provided with an agitator 5 inside and is sterilized by γ-ray irradiation from a manufacturer, so that a desired animal cell or the like suitable for a purpose can be obtained. The use bag 2 can be cultured in an environment in which a predetermined appropriate culture liquid 6 is injected.

FIG. 6 is an explanatory diagram of the configuration of the single-use bag. As shown in FIG. 6A, a disk-shaped dissolved oxygen sensor chip 7 (hereinafter also simply referred to as a sensor chip) is attached to the inner wall of a single-use bag 2 made of a transparent material such as polyethylene. ing.

For example, as shown in FIG. 6B, the sensor chip 7 generates and outputs phosphorescence by irradiating the optical system signal processing section 8 with excitation light, and the optical system signal processing section 8 generates and outputs phosphorescence. The detected phosphorescence is received and detected and converted into an electric signal.

FIG. 7 is a configuration explanatory view showing a conventional example of a calibration device for a sensor probe, and parts common to FIG. 5 are given the same reference numerals. In FIG. 7, a calibration nitrogen gas cylinder 9 is connected via a valve 10 to a pipe 11 whose end is opened in the culture solution, and an air cylinder 12 is also connected via a valve 13 to a common pipe 11. Has been done.

FIG. 8 is a structural explanatory view showing an example of the calibration device of the sensor chip 7, and the same parts as those in FIG. 5 are designated by the same reference numerals. Also in FIG. 8, the calibration nitrogen gas cylinder 9 is connected via a valve 10 to a pipe 11 whose end is opened in the culture solution, and the calibration air cylinder 12 is also shared via a valve 13. It is connected to the pipe 11.

FIG. 9 is a flowchart illustrating the flow of the procedure of sterilization and calibration of the sensor probe. First, a single-use bag sterilized by γ-ray irradiation is purchased from the manufacturer (step S1).

Next, the sensor probe is autoclave sterilized by the high-pressure steam sterilizer as described above (step S2).

The sensor probe is inserted into the single-use bag 2 so as to communicate with the inside through the aseptic connector (step S3).

Then, a proofreading gas consisting of sterilized air is blown into the culture solution to bring it into a saturated state (step S4).

Then, the phase angle Pa in the saturated state of air is measured (step S5).

Then, a calibration gas consisting of sterilized nitrogen is blown into the culture solution to remove oxygen (step S6).

The phase angle Pn in the zero oxygen state is measured (step S7).

By measuring the respective parameters along these flows, it is possible to obtain an approximate expression such as the following expression for obtaining the dissolved oxygen concentration DO (step S8).
DO=A*(P-Pn50)/(Pn-Pa)

However, if the sensor probe is not sterilized by γ-ray irradiation, such an approximate expression is not obtained.

FIG. 10 is a flowchart illustrating the flow of the procedure for sterilizing and calibrating the sensor chip. First, the sensor chip is attached to the single-use bag at the manufacturer of the single-use bag (step S1).

Further, the manufacturer sterilizes the single-use bag to which the sensor chip is attached by irradiating with γ-rays (step S2).

The user purchases a single-use bag from a manufacturer, to which a sensor chip is attached and which is sterilized by γ-ray irradiation.

Subsequently, the user blows sterilized air as a calibration gas into the culture solution to bring it into a saturated state (step S3).

Then, the phase angle Pa in the saturated state of air is measured (step S4).

Then, a calibration gas consisting of sterilized nitrogen is blown into the culture solution to remove oxygen (step S5).

The phase angle Pn in the zero oxygen state is measured (step S6).

By measuring the respective parameters along these flows, the approximate expression as described above can be obtained.

FIG. 11 is a diagram showing an example of the phase angle variation characteristic of the sensor chip due to γ-ray irradiation. As is clear from FIG. 11, the phase angle Pn in zero oxygen water decreases from more than 60 degrees to less than 60 degrees as the γ-ray irradiation dose increases, and the phase angle Pa in saturated air in the atmosphere decreases from about 15 degrees to 5 degrees. It has fallen back and forth.

As described above, the fact that the measured values of the optical sensor chip fluctuate before and after the sterilization treatment by γ-ray irradiation is the reason why the calibration is performed after the sterilization.

Patent Document 1 describes a dissolved oxygen sensor that can be automatically cleaned and calibrated.

JP-A-6-242057

In the case of the probe type dissolved oxygen sensor shown in FIG. 5, it was possible to insert the sensor probe 1 into the single use bag 2 after sterilization. Therefore, it was possible to calibrate the sensor probe 1 alone before insertion. However, in the case of the sensor chip type shown in FIG. 6, since the sensor chip is attached to the inner wall of the single-use bag 2, it is difficult to sterilize the sensor chip and the single-use bag 2 separately. Therefore, in the conventional technique, the calibration value of the sensor chip fluctuates before and after the γ-ray sterilization, so that the effect of the sterilization on the calibration value is removed by performing the calibration before the use after the sterilization.

However, when the procedure shown in FIG. 9 or FIG. 10 is performed, the time required for saturating the culture medium with air and the time required for saturating it with nitrogen are both several hours each. is necessary.

Performing these procedures has the advantage that reliable calibration values can be obtained, but since it is necessary to keep the culture solution filled in a single-use bag for several hours for calibration, the manufacturing equipment is It will be used only for the saturation treatment of the culture solution, which is problematic from the viewpoint of effective utilization of the manufacturing apparatus.

The present invention is to solve such a problem, and its purpose is to sterilize in a state in which a sensor chip of dissolved oxygen is attached to a single-use bag and sterilized in an integrated manner, or after sterilization that has been conventionally performed or It is to realize the effective use of manufacturing equipment by eliminating the need for a calibration procedure before culture.

In order to achieve such a subject, the invention according to claim 1 of the present invention,
Dissolved oxygen sensor chip that is sterilized in an integrated state by attaching a dissolved oxygen sensor chip that emits phosphorescence with a phase angle corresponding to oxygen concentration when irradiated with excitation light modulated in a sinusoidal shape on the inner wall of a single-use bag In the calibration method,
Obtain the degradation coefficient of the dissolved oxygen sensor chip from the change in phase angle before and after the sterilization treatment of another dissolved oxygen sensor chip of the same type as the affixed dissolved oxygen sensor chip,
It is characterized in that the measured value of the adhered dissolved oxygen sensor chip before sterilization is corrected using the deterioration coefficient.

The invention according to claim 2 is the method for calibrating the dissolved oxygen sensor according to claim 1,
The deterioration coefficient is obtained for each same manufacturing lot.

The invention according to claim 3 is the method for calibrating a dissolved oxygen sensor according to claim 1,
The sterilization process is performed by irradiation with electromagnetic waves.

The invention according to claim 4 is the method for calibrating a dissolved oxygen sensor according to claim 3,
The electromagnetic waves are gamma rays.

As a result, the calibration procedure after sterilization or before culture, which is required in the conventional dissolved oxygen sensor, can be eliminated, and the production efficiency of the same manufacturing apparatus can be improved.

It is a structure explanatory view showing one example of the present invention. It is explanatory drawing which shows the structural example of the concrete power supply part based on this invention. FIG. 3 is an operation explanatory diagram illustrating a flow of a transmission/reception sequence operation of FIG. 2. It is a principle explanatory view of an optical type dissolved oxygen sensor. It is a structure explanatory view showing an example of the probe type dissolved oxygen sensor used conventionally. It is a structure explanatory view of a single-use bag. It is a structure explanatory view showing the conventional example of the calibration device of a sensor probe. It is a structure explanatory view showing an example of a calibration device of a sensor chip. It is a flow chart explaining the flow of the procedure of sterilization and calibration of a sensor probe. It is a flow chart explaining the flow of the procedure of sterilization and calibration of a sensor chip. It is a figure of a phase angle variation characteristic example of a sensor chip by γ-ray irradiation.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a configuration explanatory view showing an embodiment of the present invention, and the portions common to FIG. 8 are designated by the same reference numerals. The difference between FIG. 1 and FIG. 8 is that the optical system signal processing unit 8 is provided with a deterioration coefficient storage unit 81.

FIG. 2 is a flow chart for explaining the flow of procedures for sterilization and calibration of the sensor chip 7 shown in FIG. Note that the γ-ray irradiation in the following procedure will be described as being performed by the manufacturer of the single-use bag equipped with the γ-ray irradiation equipment, but the user of the single-use bag uses the same γ-ray irradiation equipment as the manufacturer. If provided, the user may also irradiate γ-rays.

The manufacturer attaches the sensor chip to the single-use bag (step S1).

Then, the single-use bag to which the sensor chip is attached is filled with air as a calibration gas (step S2).

In this state, the phase angle PaO in air is measured (step S3).

Next, the single-use bag is filled with nitrogen as a calibration gas (step S4).

The phase angle PnO in the zero oxygen state is measured (step S5).

After that, the entire single-use bag is irradiated with γ-rays (step S6).

A calibration calculation is performed based on a predetermined calculation formula (step S7).

The dissolved oxygen concentration DO is obtained from the measured value P of the phase angle by the above-mentioned formula 1. In reality, since it is not a linear relationship, it is necessary to approximate it with a polynomial or the like, but here, it is assumed to be linear approximation. The phase angle measured in gas and the phase angle measured in liquid are treated as equivalent here and are not distinguished.

Similarly to the above, A is a constant (A=8.11 mg/L) representing the saturated dissolved oxygen concentration when the temperature is 25°C.

Then, the phase angle in nitrogen (zero oxygen water) before γ-ray irradiation is Pn0, the phase angle in air (atmosphere saturated water) is Pa0, and the phase angle in nitrogen (zero oxygen water) after 50 kGy irradiation. Is Pn50, the phase angle in air (atmospheric saturated water) is Pa50, the deterioration coefficient in air (atmospheric saturated water) is Ba, and the deterioration coefficient in nitrogen (zero oxygen water) is Bn.

Obtain the deterioration coefficient.
After measuring the phase angles Pn0 and Pa0 before γ-ray irradiation with a separately prepared sensor for measuring deterioration factors, the phase angles Pn50 and Pa50 after γ-ray irradiation are measured, and the deterioration factors Bn and Ba are calculated in advance. .. Here, the sensor for measuring the deterioration coefficient is assumed to be γ-ray sterilized at the same time when the single-use bag is γ-ray sterilized.

The phase angle for the approximate expression after γ-ray irradiation is obtained.
Using the deterioration factors Ba and Bn obtained above,
Pn50=Pn0*Bn
Pa50=Pa0*Ba
To get

Expression 2 is an example of an approximate expression for obtaining the dissolved oxygen concentration DO from the phase angle P measured in a liquid using a sensor probe or a sensor chip sterilized with γ-rays. As in the case of Equation 1 above, since the phase angle and the oxygen concentration are not in a linear relationship in practice, it is necessary to approximate them with a polynomial, but here, linear approximation is assumed.

DO=A*(P-Pa50)/(Pn50-Pa50)} (2)

FIG. 3 is a table showing the calibration values of the phase angle in the phase angle variation characteristic example diagram of FIG. The dissolved oxygen concentration DO when the phase angle P measured after γ-ray irradiation is 26 degrees is calculated by substituting the calibration value of FIG.

DO=8.11×(26-4)/(58-4)=3.30 mg/L

According to the present invention, in the procedure of sterilization and calibration of the sensor probe shown in FIG. 9, it is not necessary to measure the parameters of the approximate expression, so that steps S3 to S6, which require time for measurement, are unnecessary and work is performed. The time can be greatly shortened, and the manufacturing apparatus can be effectively used as compared with the conventional one.

As a sensor for measuring the deterioration coefficient to obtain the deterioration coefficient, by using a sensor chip that was created from the same manufacturing lot as the sensor chip actually used in the culture tank, the effect of the difference in the sensor lot on the deterioration coefficient Can be reduced.

In addition, the uncertainty of the measured value in the culture solution can be improved by correcting with the calibration formula using the calibration value and deterioration coefficient measured in air using another sensor chip and the calibration value and deterioration coefficient measured in the liquid. Can be reduced.

In addition, when the standard deviation is small when the fluctuation of the phase angle due to the γ-ray irradiation dose shown in FIG. 11 is measured by performing γ-ray irradiation a plurality of times with a plurality of sensor chips and the representative value is small, single-use It is also possible to omit the measurement of the deterioration coefficient of the sensor chip for γ-ray sterilization of the bag at the same time as the γ-ray sterilization and to perform the measurement before and after the sterilization, and use the representative value to obtain the deterioration coefficient.

When deterioration due to other factors appears as a change in the phase angle, the deterioration coefficient may be obtained using the measured values before and after the deterioration factor and correction may be performed.

In addition, a stable correction coefficient can be obtained by using a sensor that is saturated by strong γ-ray irradiation or a large number of times of irradiation and the characteristic does not change any more.

Furthermore, the sterilization method is not limited to the irradiation of γ-rays described in the embodiments, and β-rays, which is a similar method using electromagnetic waves, may be used. If the deterioration coefficient can be obtained, a sterilization method by heating such as autoclave sterilization or a sterilization method by a chemical action using ethylene oxide gas may be used.

As described above, according to the present invention, the calibration procedure after sterilization or before culture, which is required in the conventional dissolved oxygen sensor, becomes unnecessary, so that the effective use time of the manufacturing apparatus is significantly expanded as compared with the conventional method. A dissolved oxygen sensor that can be realized can be realized, and the production efficiency of the same manufacturing apparatus can be improved.

2 Single-use bag 4 Shaft 5 Stirrer 6 Culture solution 7 Sensor chip 8 Optical system signal processor 81 Degradation factor storage 9 Calibration gas (nitrogen)
10, 13 Valve 11 Pipe 12 Calibration gas (air)

Claims (4)

Dissolved oxygen sensor chip that is sterilized in an integrated state by attaching a dissolved oxygen sensor chip that emits phosphorescence with a phase angle corresponding to oxygen concentration when irradiated with excitation light modulated in a sinusoidal shape on the inner wall of a single-use bag In the calibration method,
Obtain the degradation coefficient of the dissolved oxygen sensor chip from the change in phase angle before and after the sterilization treatment of another dissolved oxygen sensor chip of the same type as the affixed dissolved oxygen sensor chip,
A method for calibrating a dissolved oxygen sensor, characterized in that the measured value of the attached dissolved oxygen sensor chip before sterilization is corrected using the deterioration coefficient. The method for calibrating a dissolved oxygen sensor according to claim 1, wherein the deterioration coefficient is obtained for each same manufacturing lot. The method for calibrating a dissolved oxygen sensor according to claim 1, wherein the sterilization process is performed by irradiation with electromagnetic waves. The method for calibrating a dissolved oxygen sensor according to claim 3, wherein the electromagnetic waves are γ rays.