JPH0269655A - On-line automatic analyzing instrument for bioreactor - Google Patents

Abstract

PURPOSE:To attain continuation and automation of a series of pretreating processes by assembling a filter which filters pH-adjusted samples in a turntable. CONSTITUTION:The sample of the product of a bioreactor 8 is sucked into and held in a sample loop 7 through a low-pressure six-way changeover valve 6 while the gas produced from the sample is exhausted by means of an exhaust pump 22 by opening a solenoid valve 5. A fixed quantity of a buffer liquid for dilution having a desired pH value in a tank 1 is fed to a dilution tank 9 by pushing out the sample into a loop by means of a pump 3 through a changeover solenoid valve 4 and the valve 6 after the buffer liquid is passed through a filter 2 and mixed with the sample by means of a stirrer. Thus the sample is diluted and adjusted in pH. The sample liquid thus adjusted is fed to a filter 32 by means of a pump 14 through a solenoid valve 23 and small holes 36 and 33 through pipelines and filtered by the filter under a pressure. The liquid filtered by the filter 32 is fed to a sample loop 17 through small holes 31 and 27 and a hexagonal valve 16 and further fed to a column 18. A solvent for gradient in a reservoir tank 20 is sent to the column 18 by means of an HPLC pump 19.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention is an online analysis device for products produced from a bioreactor, and more specifically, an online analysis device using high performance liquid chromatography.

[Prior Art 1] A bioreactor is defined in a narrow sense as an actor that utilizes an immobilized enzyme reaction, and in a broader sense as an actor that utilizes a biochemical reaction including fermentation, culture, etc. Operational management of the reactor is extremely important when producing useful biological components using a bioreactor. Physical and chemical indicators such as pH, temperature, and dissolved oxygen have traditionally been used as indicators for operational management, but in recent years biosensors using immobilized enzymes have been developed, Detect generation etc. online,
Various control methods are being studied.

However, online monitoring and control systems using biosensors have the drawback that stable output cannot always be obtained for a long period of time due to deactivation of immobilized enzymes, adsorption of components in the reactor to electrode membranes, and the like. Also protein and carbohydrates.

At present, there are almost no biosensors that can be used for macromolecular compounds such as lipids and nucleic acid-related substances. Therefore, if you want to make the above compound into mercumalum, you generally manually collect samples from the reactor over time, perform pretreatments such as dilution, pH adjustment, centrifugation, and filter filtration, and then separate them using high-performance liquid chromatography. Currently, the results are analyzed and fed back to bioreactor operation.

High-performance liquid chromatography has rapidly advanced in recent years, and it has become possible to separate and analyze complex biological components in a short time and with good reproducibility. The suspended matter had to be removed manually, which was a bottleneck for online automated analysis.

[Problems to be Solved by the Invention] The purpose of the present invention is to solve the above-mentioned problems and provide a bioreactor online automatic analysis device using a high-performance liquid chromatograph that automates and continuousizes a series of pretreatment steps. It is in.

[Means for Solving the Problems] The present invention includes a means for collecting a sample of a product produced from a bioreactor, a means for diluting the collected sample and adjusting the pH, and a turntable incorporating the sample after pH adjustment. A bioreactor online automatic analyzer comprising means for filtering with a filter, means for obtaining a chromatogram using high-performance liquid chromatography for the sample after filtration, and means for washing the sample line, and the automatic analyzer further includes a specific image of the chromatogram. This is a bioreactor online automatic analyzer that includes a means for calculating the area value of the product and a means for converting the area value into functional properties of the product.

FIG. 1 shows an example of the apparatus of the present invention. The apparatus and operation of the present invention will now be described in detail with reference to FIG.

A sample of the product produced by the bioreactor 8 is sucked and held in the sample loop 7 via the low-pressure six-way switching valve 6 while the sample electromagnetic valve 5 is opened and the exhaust pump 22 is evacuated.

The dilution solution is a buffer solution of the desired PL, which is stored in a diluent tank 1, passes through a filter 2, is switched by a pump 3 using a solenoid valve 4, and a fixed amount is retained in a sample loop 7 via a low-pressure six-way switching valve 6. The sample is sent to the dilution tank 9 while being pushed out, and both are mixed uniformly by the magnetic stirrer 1O, and the sample is diluted and pH adjusted. In many cases, the sample taken from the outlet is immediately filtered, but as in the above procedure, it is necessary to adjust the PL (preparation method) for applying it to liquid chromatography, and filter it with the following filtration means, including the precipitate that is generated at that time. The preferred filtration means is a feature of the analyzer of the present invention, and is a turntable 1) incorporating a plurality of disposable filters 32. An example of this is shown in FIG. Figure 2 is a partial cross-sectional view for explaining the incorporation of the filter and the flow of the processing liquid.The turntable 1) has a plurality of small holes 31 passing through the turntable concentrically with respect to the rotating shaft 30. are provided, and a closed top-shaped filter 32 is attached to the top surface of the turntable in close contact with each other. Furthermore, for each filter, one turn is connected on another concentric circle. The turntable 1) is provided with a fixed platen 34 in contact with the turntable 1), and a plurality of pairs of small holes provided in the turntable are provided with a pair of small holes 36.3 that are perfectly aligned when the turntable is rotated.
7 are provided, and the small holes 36 and 37 are connected to the dilution tank 9 and column 18 shown in FIG. 1 through valves and the like, respectively. In order to ensure that the small holes of the turntable and fixed plate match and that there is no leakage during filtration, the contact surfaces should be rubbed together, for example, the bottom surface of the turntable and the exposed surface of the piping in small holes 36 and 37 should be rubbed together. It is desirable to perform the following processing. The filter 32 is preferably of a disposable type and can be easily attached and detached, and the filter used preferably has a pore diameter of 045 μm or less.

The liquid in the dilution tank to be filtered is once transferred to the sample loop 24 via the switching solenoid valve 23 by the pump 14, and then transferred to the small hole 36 by the pump 14 again via the switching solenoid valve 23.
33 via piping and pressure filtration with filter 32, the filtrate passes through small holes 31, 37 and is sent to sample loop 17 via high pressure sampling six-way valve 16, and then to column 18. The gradient solvent is stored in a storage tank 20 and sent to the column 18 by an HPLC pump 19. The above filtration may be suction filtration using the suction of a pump.

Before each sample is collected and analyzed, the diluent in the diluent tank 1 is used to pump the pump 3. +4. Switching solenoid valve 4.15
The first thing is that you can clean the flow path by operating the
It is clear from the figure. In the figure, 13 is a waste liquid solenoid valve;
Reference numeral 21 is a waste liquid bottle, and the piping between the switching solenoid valve 15 for cleaning the flow path of the high-pressure system and the diluent tank 1, as well as the piping to the drain tank (not shown), etc., are omitted and only shown by arrows. Ta.

When performing analysis and determination using the apparatus of the present invention, chromatography techniques used include ion exchange chromatography, molecular sieve chromatography, hydrophobic chromatography, affinity chromatography,
Any method used for high performance liquid chromatography such as reverse phase chromatography may be used.

As described above, the device of the present invention has the above-mentioned filter 3.
All steps except attachment and detachment in step 2 can be performed automatically and continuously. The filters need to be replaced once every 12 hours if 8 filters can be set on the turntable, although this depends on the properties of the analysis sample and the frequency of analysis, so it can be used as a bioreactor online automatic analyzer. It is.

Furthermore, if the area values of specific two fractions of the obtained chromatogram are highly correlated with the characteristic values of the product produced by the bioreactor, a method for calculating the area value of that fraction and a characteristic of the area value are provided. By adding a means to convert the value into a value and feeding it back to the operating program, automatic control of bioreactor operation becomes possible.

[Example] The product of a bioreactor in which wheat gluten is partially hydrolyzed using an immobilized enzyme was analyzed using the apparatus shown in FIG. The concentration of wheat gluten as a raw material was 5%. A sample of 0.5 mj2 was taken from a bioreactor operated at a reaction temperature of 40°C and a space velocity of 2 hr-' and diluted at a dilution rate of 30.
Adjust the pH to 4.2 and filter using a 0.45 μm hydrophilic membrane filter with an inner diameter of 4.6 mm and a length of 25 cm.
0DS-120T column (manufactured by Tosoh), 1 m
High-pressure liquid phase chromatography was performed using a gradient elution method at a flow rate of I2/min. The solvent system used was 0.
1% (volume %, same below) trifluoroacetic acid, 0.1% trifluoroacetic acid in B solution, 19.9% water, 60
A solution consisting of 120% Improper Tools in 120% Acetonitrile was used.

The time required from sample collection to completion of analysis was 20 minutes. A combination of conventional pretreatment and chromatography performed in parallel with this analysis required 90 minutes.

[Effects of the Invention] According to the present invention, by providing a filtration means built into a turntable, online automation of a bioreactor that is linked to chromatography is possible simply by automating pump operations, valve switching operations, etc. By introducing this, the dilution rate, sample injection amount, etc. can be set arbitrarily. Furthermore, columns used in liquid chromatography can be arbitrarily selected to be suitable for biological component analysis, and various biological component analyzes are possible.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing an example of the apparatus of the present invention, and FIG. 2 is an explanatory diagram of a filtering means. 1... Diluent tank 2... Filter 3... Pump 4... Switching solenoid valve 5... Sample solenoid valve 6... ...Low pressure six-way switching valve 7 ...Sample loop 8 ...Bioreactor 9 ...Dilution tank IO ...Magnetic stirrer 1) ...・・・
... Turntable 12 ... Motor 13 ... Waste liquid solenoid valve 14 ... Pump 15 ... Switching solenoid valve 16 ... High pressure sampling Six-way valve 17...
... Sample loop 18 ... Column 19 ... HPLC pump 20 ... Storage tank 21 ... Waste liquid bottle 22 ... Exhaust pump 23 ... ...Switching solenoid valve 24...Sample loop 30...Rotary shaft 31...Small hole 32...Filter 33... Small hole 34... Fixed plate 36, 37... Small hole

Claims (4)

[Claims] (1) A means for collecting a sample of the product produced by the bioreactor, a means for diluting the collected sample and adjusting the pH, a means for filtering the pH-adjusted sample using a filter built into a turntable, and a means for filtering the sample after filtration. A bioreactor online automatic analyzer comprising a means for obtaining a chromatogram of a sample by high-performance liquid chromatography and a means for cleaning a sample line. (2) A bioreactor online automatic analyzer, which further comprises means for calculating the area value of a specific fraction of a chromatogram and means for converting the area value into functional characteristics of a product, to the automatic analyzer according to claim 1. (3) The bioreactor online analysis device according to claim 1 or 2, wherein the product produced by the bioreactor is a protein. (4) The bioreactor online analysis device according to claim 1 or 2, wherein the product produced by the bioreactor is an enzymatic partial decomposition product of wheat gluten.