JPWO2007080935A1 - Inspection and judgment method of host environment for bio product manufacturing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means capable of examining a host environment in a growth process over time when an influenza virus strain or the like which is a raw material for a vaccine or the like is propagated to a high titer in a host such as a hatched chicken egg. A host capable of producing a bioproduct is irradiated with light having a wavelength in the range of 400 nm to 2500 nm or a partial range thereof, and its reflected light, transmitted light or transmitted / reflected light is detected, and the absorbance spectrum is detected. After obtaining the data, it was possible to predict the host environment by analyzing the absorbance of all the measured wavelengths or specific wavelengths using an analytical model prepared in advance.

Description

The present invention relates to a test / determination method of a host environment for production of a bio product. For example, when an influenza virus strain, which is a raw material for vaccines, is grown as a bioproduct to a high titer in a host such as a hatched chicken egg, the host environment is inspected and judged by near-infrared spectroscopy. The present invention relates to a product manufacturing method and an apparatus used for the method.

Currently, the production of bioproducts such as antiviral vaccines is propagated in hosts such as hatched chicken eggs. The titer for determining the effect of proliferation is measured using focus forming activity as an index. However, because a large amount of samples must be measured, the suitability of individual hosts and the growth status must be tested. Only the final bulk is tested. However, if the host is not compatible, the growth is poor, the growth is insufficient, or the host is killed, the goal cannot be confirmed without seeing the final result. This has caused inefficient production of bioproducts.
Therefore, an improved method for monitoring the development status of bio-product raw materials such as viruses in a preferred host for the production of bio-products such as vaccines is greatly beneficial for the development and manufacturing process of bio-products such as anti-viral vaccines. is there.

Recently, component analysis using near infrared rays has been performed in various fields. For example,
Various kinds of specific components are quantitatively analyzed by irradiating a host with visible light and / or near infrared rays and detecting a wavelength band absorbed by the specific components. For example, a sample is injected into a quartz cell, and a near-infrared spectrometer (for example, NIRSystem 6500, a near-infrared spectrometer manufactured by Nireco).
) Is used to irradiate visible light and / or near infrared rays in a wavelength range of 400 nm to 2500 nm, and analyze the transmitted light, reflected light, or transmitted / reflected light. In general, the near infrared
Chemical and physical information can be obtained without damaging the sample because the substance has a very low extinction coefficient and is not susceptible to scattering and is a low-energy electromagnetic wave. Therefore, by detecting the transmitted light from the sample, obtaining the absorbance data of the sample, and performing multivariate analysis of the obtained absorbance data, the sample information can be obtained immediately, for example, the structure of the biomolecule And the process of functional change can be captured directly and in real time. Examples of conventional techniques related to such near-infrared spectroscopy include those described in Patent Documents 1 and 2 below. Patent Document 1 discloses a method for obtaining information from a subject using visible-near infrared rays, specifically, a method for determining a group to which an unknown subject belongs, a method for identifying an unknown subject, and a subject. A method for monitoring the change with time in real time is disclosed. In Patent Document 2, by using a water molecule absorption band in the visible light and / or near-infrared region, the obtained absorbance data is subjected to multivariate analysis, so that milk or breast somatic cells are measured and cow's A method for diagnosing mastitis is disclosed.

Patent Document 3 discloses a method for diagnosing changes induced by transmissible spongiform encephalopathy (TSE) in animal and human tissues by measuring the infrared spectrum of the tissues. This method is also a post-mortem examination, in which a post-mortem histopathological tissue piece is examined.

JP 2002-5827 A International Publication No. WO01 / 75420 Special table 2003-500168 gazette

The object of the present invention is to provide a means by which a host environment in a growth process can be assayed over time when an influenza virus strain, which is a raw material for a bio product, in particular a vaccine, is grown to a high titer in a host such as a hatched chicken egg. There is to do.

The present invention comprises:
1. Absorbance spectrum data was obtained by irradiating a host capable of producing a bioproduct with light in the wavelength range of 400 nm to 2500 nm or a partial range thereof, and detecting the reflected light, transmitted light or transmitted reflected light. Thereafter, a method of obtaining at least one of information selected from the following about the host environment by analyzing the absorbance of all wavelengths or specific wavelengths measured therein using a previously created analysis model;
1) Determination of host decay 2) Determination of freshness of host 3) Determination of suitability of host bioproduct as production carrier 4) Determination of titer and / or normality in bioproduct production in host 2. The method according to item 1 above, wherein the host is an egg or a cultured cell.
3. 3. The method according to item 2 above, wherein the egg is a growing chicken egg.
4). 4. The method according to item 3 above, wherein the raw material of the vaccine is inoculated as a bioproduct into the allantoic cavity of the egg.
5. 5. The method according to any one of items 2 to 4 above, wherein the egg is measured in the longitudinal direction.
6). 6. The method according to item 5 above, wherein the air chamber side of the egg is the light output unit side.
7). The method according to item 6 above, which is transmitted light.
8). 8. A method for producing a bio product product bio-monitored by the method according to any one of 1 to 7 above.
9. A light projecting means for irradiating the host with light in the wavelength range of 400 nm to 2500 nm or a partial wavelength range thereof;
Spectroscopic means for performing spectroscopy before or after light projection, and detection means for detecting reflected light, transmitted light or transmitted reflected light of the light irradiated on the host;
A data analysis means for quantitatively or qualitatively analyzing the host environment by analyzing the absorbance at all wavelengths or specific wavelengths in the absorbance spectrum data obtained by detection using an analysis model prepared in advance. Inspection / diagnosis device characterized by having.
10. The apparatus according to item 9 used in the method according to any one of items 1 to 8.

According to the present invention, the environment of a host capable of producing a bio product can be easily and quickly examined and determined with high accuracy, and can be widely used for manufacturing various bio products or determining the suitability of a host. Can do. Since it is simple and rapid, it is useful when it is necessary to examine a large number of hosts all at once. Also, because the test can be performed non-invasively on the host,
For example, a sample can be selectively collected only from a host with an increased titer and used for production of a target bioproduct. Moreover, because of its excellent speediness and simplicity, it can be applied not only for the purpose of producing bioproducts, but also for testing the host eggs such as eggs themselves, or as a bioprocess monitor during mass culture in the production of various bioproducts. is there.

It is a figure which shows the structure of a chicken egg. It is a figure of the apparatus which uses a chicken egg as a host, irradiates from the air chamber side, receives light on the lower egg white side, and acquires absorbance spectrum data. The PLS model at the dilution stage is shown for 24 hours after H5 inoculation. It is a figure which shows the result of having performed Factor Select. It is a figure which shows the total partial regression coefficient of the multiple regression type created as a quantitative model. It is a figure which shows the prediction value with time in each dilution stage about a 24-hour hen egg after H5 inoculation. It is a figure which shows the prediction value with time in each dilution stage about a 24-hour hen egg after H5 inoculation. It is a figure which shows the PLS model in the dilution stage by performing regression analysis by PLS method and inoculating for 24 hours after PR8 inoculation. It is a figure which shows the result of having performed Factor Select. It is a figure which shows the total partial regression coefficient of the multiple regression type created as a quantitative model. It is a figure which shows the prediction value with time in each dilution stage about a chicken egg for 24 hours after PR8 inoculation. It is a figure which shows the prediction value with time in each dilution stage about a chicken egg for 24 hours after PR8 inoculation. It is a figure which shows the comparison of the regression coefficient of H5 and PR8.

One object of the present invention is to irradiate a host capable of producing a bio product with near-infrared light having a wavelength in the range of 400 nm to 2500 nm or a partial range thereof, and its reflected light and transmitted light. Alternatively, after obtaining the absorbance spectrum data by detecting the transmitted / reflected light, the information on the host environment is selected from the following by analyzing the absorbance of all wavelengths measured or the specific wavelength using a previously created analysis model. This is a method of obtaining at least 1.
1) Determination of degree of host decay 2) Determination of freshness of host 3) Determination of compatibility of host bioproduct as production carrier 4) Determination of titer and / or normality in bioproduct production in host

In the present invention, a bioproduct refers to a biological product that can be prepared using a host. A typical biological product is a vaccine, or various proteins in vivo. Specifically, influenza vaccine, HIV, hepatitis viruses such as hepatitis C virus, hepatitis B virus, BDV (Borna disease virus: Borna disease virus), SARS coronavirus, adult T cell leukemia virus, human parvovirus, enterovirus, Examples of various causative viruses of human or domestic viral diseases such as adenovirus, coxsackie group A / group B virus, echovirus, herpes simplex virus, influenza virus, norovirus, rotavirus, poliovirus, measles virus, rubella virus be able to. Bioproducts using organisms are produced not only in the agriculture, forestry and fisheries industry using animals and plants but also in engineering fields such as brewing, food, chemistry, and medical production. For example, bioactive organic compounds such as TPA and vitamins extracted from plants, insulin produced in large quantities in E. coli, growth hormones, bioactive proteins such as IFN and TGF, and penicillin recovered from bacterial culture supernatants such as fungi And bioactive proteins such as antibodies recovered from the culture supernatant of mammalian cells such as hybridomas.

In the present invention, the host means a host capable of producing a wide range of bioproducts such as chicken eggs (growing eggs) and cultured cells, and includes prokaryotes such as E. coli, eukaryotes such as yeast, multicellular organisms such as silkworm, bovine And mammals such as sheep. In addition, primary cultured cells derived from these organisms, established cells, eggs, sperm, ova, etc. also correspond to the host, and in the case of humans, individuals cannot be used as bioproduct production sites for ethical reasons. It is valid. In this way, pharmaceutical raw materials are mass-produced using cell engineering, genetic engineering, and developmental engineering techniques.

In the present invention, the degree of host rot is that the egg or tissue cell that is the host used for the production of the bio product has suffered damage that cannot be used as a bio product production host. It is a broad concept that includes the meaning of corruption in a meaningful way. Near-infrared irradiation is performed from a healthy or normal host to an unhealthy or abnormal host to obtain model absorbance spectrum data, and the host environment is confirmed in comparison with the model absorbance spectrum data. Needless to say, the present invention is not limited to the assay of the host for producing the bioproduct, but also includes a means for assaying the degree of perturbation and abnormality of general commercial chicken eggs, for example.

In the present invention, the freshness of the host means the degree of novelty of the egg or tissue cell, which is a host used for the production of the bio product, as the bio product production host. A model absorbance spectrum data is obtained by performing near-infrared irradiation on a host after a certain period of time from a host immediately after fractionation or immediately after production, and the environmental change of the host is confirmed as freshness in comparison with that. . Of course, the present invention is not limited to the assay of the host for production of the bio product, but also covers, for example, a means for assaying the freshness of general commercial chicken eggs.

In the present invention, the compatibility of a host bioproduct as a production carrier means compatibility of a chicken egg or tissue cell, which is a host used for production of a bioproduct, as a bioproduct production host. By performing near-infrared irradiation on a non-matching host from a host whose suitability has been determined in advance, model absorbance spectrum data is obtained, and the possibility of host matching is confirmed as suitability in comparison with the model absorbance spectrum data.

In the present invention, the titer and / or normality determination in the bioproduct production in the host is to cultivate or grow the egg or tissue cell which is the host used for the production of the bioproduct for the production of the bioproduct, In the process of culturing or growing, it means determining the titer (activity) of the bio product and / or the normality of the host itself. Immediately after the start of culture or growth, the host is irradiated with near-infrared light at regular intervals during the culture or growth period to obtain model absorbance spectrum data, and in comparison with this, the production status of bioproducts in the host is confirmed. It is. In addition, the model absorption spectrum is obtained by irradiating the host with near-infrared irradiation at regular intervals during the culture or growth period immediately after the start of the culture or growth, and comparison with the model confirms environmental changes such as normality of the host itself. Is.

One aspect of the present invention is application to bioproduct production using chicken eggs, particularly growing chicken eggs. Chicken eggs are stored at a constant temperature for several days after egg laying, for example, 4 to 10 days, preferably 6 to 9 days, 30 to 40 ° C., preferably 35 to 38 ° C., and inoculated with biomaterials as a host. The structure of the hen's egg is as shown in FIG. 1 consisting of the air chamber (3), egg white (1), amniotic cavity (2), allantoic cavity (4), and yolk (5). In the cavity (4). The inoculation amount is, for example, Influenza A virus A / PR / 834 (H1N1), Influenza A vir
Various absorbances of us A / crow / Kyoto / 53/2004 (H5N1), etc. are adjusted to create model absorbance spectrum data.
The host internal environment is estimated from the absorbance spectrum data by near-infrared irradiation by irradiating the host with light having a wavelength in the range of 400 nm to 2500 nm or a part of the wavelength before inoculation and at regular intervals after inoculation. Irradiation is performed in the vertical direction, for example, in the case of a chicken egg. Then, light irradiation is performed from the air chamber (4) side with the air chamber (3) in FIG. 1 facing up. For light irradiation, any of transmitted light, reflected light, and transmitted / reflected light can be used, but more generally transmitted light. FIG. 2 shows an apparatus that uses a chicken egg as a host to irradiate from the air chamber (4) side, receive light on the lower egg white (1) side, and acquire absorbance spectrum data. The near-infrared measurement conditions do not need to be exceptional, and general conditions as wide-ranging near-infrared measurements are applied as they are.

As described above, the absorbance spectrum data obtained by irradiation with near-infrared light can be assayed for changes in the host environment non-invasively by comparison with the standard absorbance spectrum data, and biomonitoring becomes possible. If this method is used, an efficient method for producing a bioproduct product is provided.

The apparatus used in the present invention includes a light projecting means for irradiating the host with light having a wavelength in the range of 400 nm to 2500 nm, or a part of the wavelength range, a spectroscopic means for performing spectroscopy before or after the light projection, and irradiating the host. The detection means for detecting the reflected light, transmitted light or transmitted / reflected light of the measured light, and the absorbance at all wavelengths or specific wavelengths in the absorbance spectrum data obtained by the detection are analyzed using an analysis model created in advance. And a data analysis means for analyzing the host environment quantitatively or qualitatively. The definition of each wording is as described above.

As an embodiment, an embodiment of the present invention in the production of vaccines such as influenza virus will be described.

Outline of spectrum measurement Inspection / diagnosis using this device is as follows: (a) The host is irradiated with light in the wavelength range of 400 nm to 2500 nm or a partial range thereof, and (b) the reflected light, transmitted light or transmitted reflected light is detected. (C) growth of the vaccine raw material such as influenza virus in the host by analyzing the absorbance at all wavelengths or the specific wavelength using the analytical model prepared in advance. Quantitatively or qualitatively examine and judge the situation or host environment.

The first feature of the present invention is that information in the host can be obtained simply and quickly with high accuracy, and can be used in vaccine production using chicken eggs, etc., or for characterization of chicken eggs themselves. The range of the wavelength irradiated to the host is in the range of 400 nm to 2500 nm or a part thereof (for example, 600 to 1000 nm). The wavelength range can be set as one or a plurality of wavelength ranges including wavelength light necessary for inspection / determination by the analysis model after the analysis model is created.

As the light source, a halogen lamp, LED, or the like can be used, but is not particularly limited. The light emitted from the light source is irradiated to the host directly or through light projecting means such as a fiber probe. A pre-spectral method of performing spectroscopy with a spectroscope before irradiating the host may be employed, or a post-spectral method of performing spectroscopy after irradiation may be employed. In the case of the pre-spectral method, there are a method in which the light from the light source is simultaneously dispersed by a prism and a method in which the wavelength is continuously changed by changing the slit interval of the diffraction grating. In the case of the latter method, the host is irradiated with continuous wavelength light whose wavelength is continuously changed by decomposing light from the light source with a predetermined wavelength width. For example, it is possible to decompose light having a wavelength in the range of 600 to 1000 nm with a wavelength resolution of 1 nm and to irradiate the host with light whose wavelength is continuously changed by 1 nm.

Reflected light, transmitted light or transmitted / reflected light of the light irradiated to the host is detected by the detector, and raw absorbance spectrum data is obtained. The raw absorbance spectrum data may be used as is for inspection / determination using an analysis model. However, data conversion such as decomposing peaks in the obtained spectrum into element peaks by spectroscopic or multivariate analysis techniques. Process
It is preferable to perform inspection / determination based on the analysis model using the absorbance spectrum data after conversion. Examples of spectroscopic techniques include second order differential processing and Fourier transform. Examples of multivariate analysis techniques include weblet transform and neural network methods, but are not particularly limited.

In the spectrum measurement by this apparatus, perturbation can be given to the host by adding a predetermined condition.

Data analysis method (creation of analysis model)
In the present invention, the apparatus analyzes the absorbance of a specific wavelength (or all measured wavelengths) in the obtained absorbance spectrum data using an analysis model, thereby performing a host status test and a bioproduct growth status test by the host. Do. In other words, in order to perform the final test, it is preferable that an analysis model is created in advance. Of course, this analysis model may be created at the time of spectrum measurement.

It is desirable to prepare the analysis model in advance before measurement, but the spectrum data acquired at the time of measurement is divided into two for analysis model creation and verification, and the analysis model obtained based on the data for creation of the analysis model You may test using. For example, when a large number of hosts are tested simultaneously, a part of the host is used for creating an analysis model. In this case, an analysis model is created at the time of measurement. With this method, an analysis model can be created without teacher data. It can handle both quantitative and qualitative models.

The analysis model can be created by multivariate analysis. For example, when predicting the raw material titer (concentration) in the host for the growth of influenza virus vaccine raw material, the data matrix storing the absorption spectra of all wavelengths obtained by spectral measurement is decomposed into scores and loadings by singular value decomposition. Then, a main component that summarizes the variation in the amount of vaccine raw material in the host is extracted (principal component analysis). As a result, independent components with less collinearity (= high correlation between explanatory variables) can be used for multiple regression analysis. And score or load explanatory variables,
A multiple regression analysis with the target variable as the amount of vaccine material is applied. Thereby, the analysis model which estimates the amount of vaccine raw materials from the absorption spectrum of all the measurement wavelengths or a specific wavelength can be created. These series of operations (multivariate analysis) are performed by Principal Component Regressi (PCR).
on) or PLS (Partial Least Squares) regression method (references:
Yukihiro Ozaki, Akifumi Uda, Toshio Akai "Multivariate Analysis for Chemists-Introduction to Chemometrics",
Kodansha, 2002). Other regression analysis methods include CLS (Classical Least Squares) method and cross-validation method.

An analysis model using multivariate analysis can be created using self-made software or commercially available multivariate analysis software. In addition, quick analysis is possible by creating software specialized for the purpose of use.

An analysis model assembled using such multivariate analysis software is saved as a file, and this file is called when testing a host using a different bioproduct raw material. Perform qualitative or qualitative tests. This makes it possible to test the host environment for the production of simple and rapid bioproducts. Note that the analysis model stores multiple analysis models such as quantitative models and qualitative models as files,
Each model is preferably updated as appropriate.

When the analysis model is created, the wavelength light necessary for the verification by the analysis model is determined.
This apparatus can further simplify the apparatus configuration by irradiating the host with one or more wavelength ranges determined in this way.

Preferred host measurement method and data analysis method according to the present invention In the spectrum measurement according to the present invention, perturbation can be given to a host by adding a predetermined condition. In data analysis using this device,
Data analysis that draws out the effect of this perturbation is preferably exemplified.

Perturbation
“Perturbation” refers to obtaining a plurality of different spectral data by causing a change in absorbance of a sample by setting and measuring a plurality of types and conditions for a certain condition. Conditions include physical changes by changing the concentration (including concentration dilution), repeated light irradiation, extending the irradiation time, applying electromagnetic force, changing the optical path length, temperature, pH, pressure, mechanical vibration, and other conditions. Any of those that bring about chemical changes, or a combination thereof, can be mentioned, and is broadly divided into (1) the method of light irradiation and (2) the method of preparation and preparation of the host. . As for (1), repeated irradiation of light and (2) as an example of concentration dilution will be described below.

The repeated irradiation of light is a method for performing spectrum measurement of a host specimen by giving a perturbation of a plurality of measurements by irradiating light continuously or at regular time intervals. For example, by continuously irradiating light three times, the absorbance of the host slightly changes (fluctuates), and a plurality of different spectral data can be obtained. By using these spectral data for multivariate analysis such as SIMCA method and PLS method, analysis accuracy can be improved, and highly accurate inspection / diagnosis becomes possible. In addition, when measuring a normal spectrum, it is measured by irradiating light a plurality of times. This is intended to obtain an average value, and is different from “perturbation” here.

The change in the absorbance of the host due to the perturbation is considered to be caused by a change (fluctuation) in the absorption of water molecules in the host. That is, by repeating light irradiation three times as perturbation, the first time, 2
It is considered that slightly different changes occur in the response and absorption of water in the third and third times, resulting in fluctuations in the spectrum.

By carrying out regression analysis by the PLS method using the absorbance spectrum data obtained by each of these three repeated irradiations, the amount of the influenza virus vaccine material in each host was successfully quantified.

In addition, when the light is repeatedly irradiated three times in this way, each host is made good by performing class discrimination by the SIMCA method using at least two absorbance spectrum data among the obtained three absorbance spectrum data. Therefore, it is possible to perform high-precision inspection / diagnosis. The number of times of light irradiation is not particularly limited to 3 times, but about 3 times is preferable in consideration of the complexity of data analysis.

On the other hand, for perturbation by concentration dilution, a bioproduct raw material to be administered to a host is prepared by diluting it in several stages, and the spectrum of each host is measured. Thereby, a plurality of spectrum data is obtained for one specimen host, and by using these spectrum data for multivariate analysis, highly accurate examination / diagnosis becomes possible. As an example of multivariate analysis in this case, first, PLS regression analysis with the dilution degree as the target variable is performed for each specimen host, and then the obtained regression vectors are classified using pattern recognition such as SIMCA method. Examination / diagnosis is possible by discriminating and classifying which class of regression vector (pattern) is close to the class using the class discrimination model thus created.

The number of dilutions and the degree of dilution are not particularly limited. Since fluctuations need only occur in the spectrum acquired by perturbation due to concentration dilution, these numerical values can be arbitrarily set.

Similarly, for the perturbation conditions other than concentration dilution and repeated light irradiation, a plurality of types and conditions may be set for each condition so as to cause fluctuations in the acquired spectrum (Japanese Patent Application 2003-2003). 379517).

Data analysis method to extract perturbation effect “Data analysis to extract perturbation effect” is to create an analysis model using multiple spectral data obtained by perturbation for one host, and to use the analysis model. The following three methods can be given as specific examples of the data analysis method.

(A) Quantitative analysis: A method for quantifying a target substance in a host such as an influenza vaccine raw material amount using a quantitative model created by a regression analysis such as a PLS method. A plurality of quantitative models are obtained by perturbation per host. Created using spectral data. By quantifying the bioproduct raw material in the host, it is possible to predict the transition of bioproduct production, bioproduct production capacity, and the like.

(B) Qualitative analysis 1: Method for testing a host using a qualitative model created by class discrimination analysis such as SIMCA method The qualitative model uses a plurality of spectral data obtained by perturbation per host. create. By creating a class discrimination model of three or more classes, it is possible to predict host environment, host growth status, host normality, growth degree, freshness, host suitability, and the like.

(C) Qualitative analysis 2: (1) Perform regression analysis (PLS method etc.) with each value of perturbation such as concentration dilution value (dilution degree) (each value to which perturbation is given) as a target variable. (2) A method for testing a host using a qualitative model created by performing class discrimination analysis such as SIMCA method on the regression vector obtained by the same analysis. This is performed using a plurality of spectral data obtained by perturbation per host. By creating class discrimination models of three or more classes and classifying them by pattern recognition, a host test similar to the above can be performed.

Specific Configuration of Measuring Device in the Present Invention The configuration of the inspection / diagnosis system for an apparatus according to the present invention includes a probe (light projecting unit), a spectroscopic /
It can be configured with four elements, a detection unit, a data analysis unit, and a result display unit.

Probe (light projection means)
The probe is light from a light source such as a halogen lamp or LED (wavelength 400 nm to 2500 n).
m has a function of leading the entire range of m or a partial range thereof to the host to be measured. For example, a configuration in which a fiber probe is used and light is projected to a measurement target (host) through a flexible optical fiber can be used. In general, a probe of a near-infrared spectrometer can be manufactured at low cost and is low in cost.

In addition, although it is good also as a structure which projects the light emitted from the light source directly to the host which is a measuring object, a probe is unnecessary in that case, and a light source functions as a light projection means.

As described above, when an analysis model is created, wavelength light necessary for inspection / diagnosis using the analysis model is determined. This apparatus can further simplify the apparatus configuration by irradiating the host with one or more wavelength ranges determined in this way.

In addition, the present apparatus is preferably configured to perform spectrum measurement while providing perturbation, and preferably has a configuration necessary for perturbation.

Spectrometer / detector (spectrometer and detector)
This apparatus has a configuration of a near-infrared spectrometer as a measurement system. In general, a near-infrared spectrometer irradiates a host, which is a measurement object, with light, and a detection unit detects reflected light, transmitted light, or transmitted / reflected light from the object. Further, the absorbance of the detected light with respect to incident light is measured for each wavelength.

There are two types of spectroscopic methods: pre-spectroscopy and post-spectroscopy. Pre-spectrometry is performed before projecting on a measurement object. Post-spectroscopy detects and separates light from the measurement object. The spectroscopic / detection section of this device
Any spectroscopic method may be employed.

There are three types of detection methods: reflected light detection, transmitted light detection, and transmitted reflected light detection. In the reflected light detection and the transmitted light detection, the reflected light and the transmitted light from the measurement object are detected by the detector, respectively. In transmitted / reflected light detection, refracted light incident on the object to be measured is reflected in the object, and light emitted outside the object again detects light that interferes with the reflected light. The spectroscopic / detection unit of the present apparatus may employ any detection method of reflected light detection, transmitted light detection, and transmitted reflected light detection.

The detector in the spectroscopic / detection unit is, for example, a CCD (Charge Coupled Device) which is a semiconductor element.
However, the present invention is not limited to this, and other light receiving elements may be used. The spectroscope can also be configured by known means.

Data analysis unit (data analysis means)
Absorbance by wavelength, that is, absorbance spectrum data is obtained from the spectroscopic / detection unit. The data analysis unit tests for changes in the host environment using an analysis model created in advance based on the absorbance spectrum data.

As the analysis model, a plurality of analysis models such as a quantitative model and a qualitative model may be prepared, and different models may be used depending on whether quantitative evaluation is performed or qualitative evaluation is performed. In addition, the analysis model may be configured such that a model is created for each bioproduct raw material and / or for each host, and any inspection can be performed with one apparatus.

The data analysis unit can be configured by a storage unit that stores various data such as spectrum data, a program for multivariate analysis, an analysis model, and an arithmetic processing unit that performs arithmetic processing based on these data and programs. It can be realized by an IC chip or the like. Therefore, it is easy to reduce the size of the apparatus in order to make it portable. The above analysis model is also written in a storage unit such as an IC chip.

Result display section (display means)
The result display unit displays the analysis result in the data analysis unit. Specifically, the bioproduct concentration value such as the amount of the influenza vaccine raw material in the host obtained as a result of the analysis by the analysis model is displayed. Or, in the case of a qualitative model, “host normal”, “high possibility of host abnormality”, “host abnormality”, “raw material growth normal”, “raw material growth abnormality”, “high possibility of raw material growth abnormality”, etc. Is displayed. If the device is portable,
The result display unit is preferably a flat display such as a liquid crystal.

Examples of the present invention will be described below, but the present invention is not limited to the following examples.
Example 1: Examination by near-infrared spectroscopy Measurement of absorption spectrum In this example, the absorption spectrum of each host was measured by the following measurement method.

Obtained immediately after egg laying of healthy eggs, and kept at 37 ° C. for 7 days, and then stored at 37 ° C. for 1 day in a test laboratory. Bio-product ingredients were grown using eggs. In addition, embryo at the time of inoculation
Was the size of the tip of the thumb. Influenza Avirus A / PR / 8/34 (H1N
The stock solution 10 ⁸ FFU / ml of 1) is appropriately diluted with a PBS buffer solution, for example, 100 μl is inoculated into one host, and finally the final inoculation amount (10 ⁴ , 10 ^{3) is} increased 10 times step by step. , 10 ² ,
(10 ¹ , 10 ⁰ FFU) were performed in 3 cases each. As another flu, Influenza
A stock solution of A virus A / crow / Kyoto / 53/2004 (H5N1) 10 ⁷ FFU / ml is diluted with PBS buffer as appropriate. For example, 100 μl is inoculated into one host, and finally 10 times step by step. The final inoculation amount (10 ³ , 10 ² , 10 ¹ , 10 ⁰ , 10 ⁻¹ FFU) up to 3 times each was performed. For FFU, MDCK cells were used for measurement in the Focus Forming Unit.

After inoculation, chicken eggs as a host were stored at 37 ° C. at a constant temperature, and observed with an oocyte tester. 48 hours after inoculation in the H3N1 10 ³ , 10 ² , 10 ¹ PFU inoculation group and PR / 8 10 ⁴ PFU inoculation group
The embryo died. The chorioallantoic fluid was collected 72 hours after inoculation for each group, and the infectious titer was measured.

The infectivity titer (HA titer) was measured by the following method.
1. Homogenize chicken erythrocytes stored in Alzeba solution, take 2-3 ml, and add 5 times PBS (-)
Suspend in
2. Centrifuge the suspension of erythrocytes at 2k rpm for 5 minutes, discard the supernatant, and suspend with 5 volumes of PBS (-). Repeat this two more times.
3. Discard the supernatant and suspend 2-3 drops in 2 ml of PBS (-) with P1000 Pipetteman.
4. Dilute this solution 10 times and use it as a 0.5% chicken erythrocyte suspension.
5. Prepare 50 ml of 2-fold serial dilution of virus solution in a U-shaped or V-shaped 96-well plate.
6. Add 50 ml of 0.5% chicken erythrocyte suspension to each well and mix gently.
7. Check for erythrocyte aggregation after about 1 hour.
Determine the HA titer by observing red blood cell aggregation up to the power dilution of 8.2.

On the other hand, about each group, it measured in the vertical direction about the egg which is a host using near infrared rays. The air chamber side was the light output part side.
As shown in Fig. 2, the host is placed and a near infrared spectrometer (product name "Fruit-Tester-20 (FT-20) (J
apan Fantec Research Institute, Shizuoka, Japan) ”), and the measurement was performed with perturbation of repeated light irradiation. Specifically, the wavelength light of 600 to 1100 nm is continuously 3
The absorption spectrum was measured by detecting each transmitted light by irradiating the host twice.
The wavelength resolution is 2 nm. By sandwiching the egg between the light output part and the light detection part, the optical path length passing through the host was set to the size of the egg. The integration time is 8000 msec for eggs and 20 msec for chorioallantoic fluid.

Analysis of Absorption Spectrum In this example, as analysis 1, PLS (partia
l Create a least squares regression analysis) model, high FFU sample egg (PR8 10 ⁴ F
FU, H5 10 ³ FFU), over time to predict the low FFU sample eggs ^{(PR8 10 0 FFU, H5 10} -1 FFU), were studied.
As analysis 2, PLS models were prepared at each dilution stage for chicken eggs 24 hours after inoculation.
The ^{10 4 FFU~10 0 FFU, H5 10} 3 FFU~10 -1 FFU over time predicted, was investigated concentration dependence.
PLS analysis conditions are H5, cross validation, smoothing (17), Factor7, SECV = 0.8
2611, RVAL = 0.81604, PR8, crossvalidation, smoothing (17), Factor9, SE
CV = 0.6089, RVAL = 0.9049. Validation is a method of removing the sample and verifying the validity of the model. There are mainly step validation and cross validation. Step validati
“on” excludes a set of consecutive sample orders, “cross” excludes them in order, and verifies whether the excluded samples are correctly determined. Smoothing (17) indicates that “Smooth” is smoothed. The smoothing conversion is based on the principle of the polynomial filter of Savitzky-Golay, and it rotates to the explanatory variable in the window including the point of the central data and n points on one side, and that n = 17 is selected. Show. The Factor number indicates the number of main components used. SECV indicates the standard deviation of cross validation. RVAL represents a predictive correlation coefficient when cross-validation is performed.

Analysis method: PLS model was prepared at each dilution stage for chicken eggs 24 hours after inoculation. The obtained absorption spectrum was analyzed by PLS method for each dilution.

In order to create an analysis model, the amount of H5 antigen in each host (10-fold dilution) was measured by ELISA.
Regression analysis was performed by the PLS method, and a PLS model was prepared at the dilution stage with eggs for 24 hours after H5 inoculation (RVAL = 0.81). Fig. 3 shows the model diagram, and the horizontal axis shows the infectious titer FFU (Log10) using the existing measurement method.
The infectivity titer FFU (log10) predicted by near infrared spectroscopy was displayed on the vertical axis, and a model was created. H5 5-3 24h-2 is 100ul of H5N1 stock solution (10 ⁷ FFU / ml) diluted to the fifth power of 10
The third of three eggs inoculated (ie, 10 FFU inoculated) was measured after 24 hours of culture, indicating the second measurement of three consecutive measurements.

Figure 4 shows the results of Factor Select. The horizontal axis represents the number of Factors and the vertical axis represents the error (SECV: Sta
ndard Error of cross-Validation: standard deviation of cross-validation). Since the PLS model with the smallest SECV is the best, the factor select will minimize the SECV.
r number 7 (correlation coefficient r is 0.82611 at this time) was selected, and analysis was performed using the PLS model at this time.

Figure 5 shows the partial regression coefficient (regression vector (Regression
Vector)). The horizontal axis indicates the wavelength (nm), and the vertical axis indicates the regression coefficient value. The wavelength used is 600 nm to 1100 nm, and the wavelength resolution is 2 nm. The regression coefficient is
Show which wavelengths are the most important and which are almost useless. Wavelengths with high regression coefficients are
The higher the infectivity value, the higher the absorbance at that wavelength. A negative regression coefficient indicates that the absorbance at that wavelength is higher as the infectivity value is lower. A regression coefficient of 0 indicates that the absorbance at that wavelength is not related to the level of infectivity.

Figures 6 and 7 show the change in the predicted value [NIRS method: FFU (log10)] calculated from the near-infrared spectrum obtained over time using the PLS model created with chicken eggs 24 hours after H5 inoculation. .
Figure 6 is the result of FFU10 ³ and FFU10 ^-1, in FIG H5 3-1 H5 3-2 H5 3-3 and
H5 7-1 H5 7-2 H5 7-3 means that three absorbance data respectively obtained by three consecutive irradiations were used for H5. Using the analytical model obtained by PLS analysis, the H5 titer of each host could be predicted with high accuracy. The analysis model assembled in this way is saved as a file, and this file is called when testing and diagnosing unknown samples, and the H5 titer of the target host is predicted using the analysis model. Thereby, a simple and quick inspection of the host environment becomes possible. “Log10” indicates that each explanatory variable is converted in common logarithm. In this analysis, to define the objective variable (Dependent variable) 10 ^1-fold dilution log10 (10 ¹⁾ = 1 and, Similarly, 10 ^two-fold dilution log10 (10 ²⁾ = 2,10 ³ fold dilutions are log10 (10 ³ ) = 3. For sample names, for example, H5 3-2 is the second of three eggs inoculated with H5N1 10 ³ FFU. H5 7-1 is the ^{first of} three eggs inoculated with H5N1 10-1 FFU.

^{7, FFU10 3, FFU10 2, FFU10} 1, and FFU10 the result of ^0, figure H5 3
-1 H5 3-2 H5 3-3, H5 4-1 H5 4-2 H5 4-3, H5 5-1 H5 5-2 H5 5-3, H5 6-1 H5 6
-2 H5 6-3 means that three absorbance data respectively obtained by three consecutive irradiations were used for H5. Using the analytical model obtained by PLS analysis, the H5 titer of each host could be predicted with high accuracy. The analysis model assembled in this way is saved as a file, and this file is called when testing and diagnosing unknown samples, and the H5 titer of the target host is predicted using the analysis model. Thereby, a simple and quick inspection of the host environment becomes possible. "Log10
"Indicates that each explanatory variable has been converted to a common logarithm. In this analysis, the objective variable (
The Dependent variable) 10 ¹ dilution is defined as log10 (10 ¹⁾ = 1, Similarly, 10 ^two-fold dilution log10 (10 ²⁾ = 2,10 ³ fold dilutions and log10 (10 ³⁾ = ³ Defined. For sample names:
For example, H5 4-2 is the ^{second of} three eggs inoculated with H5N1 10 ² FFU. H5 6-1 will show that this is the first of the three eggs inoculated with H5N1 10 ⁰ FFU.

In order to create an analysis model, the amount of PR8 antigen in each host (10-fold dilution) was measured by ELISA.
Regression analysis by the PLS method was performed, and a PLS model at the dilution stage was created with eggs 24 hours after PR8 inoculation (RVAL = 0.905). Fig. 8 shows the model diagram, and the horizontal axis shows the infectious tit FFU (Log10) using the existing measurement method.
The infectivity titer FFU (log10) predicted by near infrared spectroscopy was displayed on the vertical axis, and a model was created. PR8 5-3 24h-2 is 100ul of PR8 undiluted chorioallantoic fluid (10 ⁸ FFU / ml) diluted to the fifth power of 10
A third of three eggs inoculated (ie, inoculated with 10 ² FFU) was measured after 24 hours of culture, indicating the second measurement of three consecutive measurements.

Fig. 9 shows the results of Factor Select. The horizontal axis represents the number of Factors and the vertical axis represents the error (SECV: Sta
ndard Error of cross-Validation: standard deviation of cross-validation). Since the PLS model with the smallest SECV is the best, this Factor Select is the factor that minimizes the SEV
Formula 9 (correlation coefficient r is 0.6089 at this time) was selected, and analysis was performed using the PLS model at this time.

Fig. 10 shows the partial regression coefficient (regression vector (Regressi
on Vector)). The horizontal axis indicates the wavelength (nm), and the vertical axis indicates the regression coefficient value. The wavelength used is 600 nm to 1100 nm, and the wavelength resolution is 2 nm. The regression coefficient is
Show which wavelengths are the most important and which are almost useless. Wavelengths with high regression coefficients are
The higher the infectivity value, the higher the absorbance at that wavelength. A negative regression coefficient indicates that the absorbance at that wavelength is higher as the infectivity value is lower. A regression coefficient of 0 indicates that the absorbance at that wavelength is not related to the level of infectivity.

Figures 11 and 12 show the estimated value [NIRS over time at each dilution stage for chicken eggs 24 hours after PR8 inoculation.
Method: FFU (log 10)].
Figure 11 is the result of FFU10 ⁴ and FFU10 ^0, drawing PR8 3-1 PR8 3-2 PR8 3-3
And PR8 7-1 PR8 7-2 PR8 7-3 were obtained for PR8 by three consecutive irradiations 3
Means that two absorbance data were used. Using the analytical model obtained by PLS analysis, the PR8 titer of each host could be predicted with high accuracy. The analytical model assembled in this way is saved as a file, and this file is called when testing and diagnosing unknown samples, and the PR8 titer of the target host is predicted by the analytical model. Thereby, a simple and quick inspection of the host environment becomes possible. “Log10” indicates that each explanatory variable is converted in common logarithm. In this analysis, to define the objective variable (Dependent variable) 10 ^1-fold dilution log10 (10 ¹⁾ = 1 and, Similarly, 10 ^two-fold dilution log10 (10 ²⁾ = 2,10 ³ fold dilutions are log10 (10 ³ ) = 3. For sample names, for example, PR8 3-2 is the second of three eggs inoculated with PR8 10 ⁴ FFU. PR8 7-1 will show that this is the first of the three eggs inoculated with PR8 10 ⁰ FFU.
FIG. 12 shows the results of FFU10 ⁴ , FFU10 ³ , FFU10 ² , and FFU10 ¹ , and PR in the figure
8 3-1 PR8 3-2 PR8 3-3, PR8 4-1 PR8 4-2 PR8 4-3, PR8 5-1 PR8 5-2 PR8
5-3, PR8 6-1 PR8 6-2 PR8 6-3 means that for PR8, three absorbance data respectively obtained by three consecutive irradiations were used. Using the analytical model obtained by PLS analysis, the PR8 titer of each host could be predicted with high accuracy. The analytical model assembled in this way is saved as a file, and this file is called when testing and diagnosing unknown samples, and the PR8 titer of the target host is predicted by the analytical model. Thereby, a simple and quick inspection of the host environment becomes possible. “Log10” indicates that each explanatory variable is converted in common logarithm.
In this analysis, to define the objective variable (Dependent variable) 10 ^1-fold dilution log10 (10 ¹⁾ = 1 and, Similarly, 10 ^two-fold dilution log10 (10 ²⁾ = 2,10 ³ fold dilutions are log10 (10 ³ ) = 3. For the sample name, for example, PR8 4-2 is the second of three eggs inoculated with PR8 10 ³ FFU. PR8 6-1 indicates the ^{first of} three eggs inoculated with PR8 10 ¹ FFU.

FIG. 13 compares the regression coefficients of H5 and PR8.
Wavelengths corresponding to peaks and valleys of the regression coefficient common to H5 and PR8 are wavelengths that change in common by inoculation of influenza virus eggs.
Wavelengths corresponding to peaks and valleys with different regression coefficients in H5 and PR8 are wavelengths that change specifically by inoculating eggs of the respective influenza virus strains.

(Example 2)
The model of the present invention is a PLS model created based on spectral data at 24 hours,
If the predicted value is calculated by including spectral data from 72 hours to 72 hours, it is possible to predict whether an influenza virus infection has occurred. Therefore, the spectral data not included in the model can be well predicted and can be applied to unknown samples.

As described above, the present invention irradiates a host capable of producing a bio product with light having a wavelength in the range of 400 nm to 2500 nm or a partial range thereof, and reflects the reflected light, transmitted light, or transmitted reflected light. After detecting and obtaining absorbance spectrum data, the host environment can be inspected and determined easily and quickly with high accuracy by analyzing the absorbance of all wavelengths measured or specific wavelengths using an analysis model created in advance. It can be widely used for monitoring bioproduct production hosts.

Claims (10)

Absorbance spectrum data was obtained by irradiating a host capable of producing bioproducts with light having a wavelength in the range of 400 nm to 2500 nm or a partial range thereof, and detecting the reflected light, transmitted light or transmitted reflected light. After that, the absorbance of all wavelengths or specific wavelengths measured in it,
A method of obtaining at least one of information selected from the following about the host environment by analyzing using an analysis model prepared in advance;
1) Determination of degree of host decay 2) Determination of freshness of host 3) Determination of suitability of host as bioproduct manufacturing host 4) Determination of titer and / or normality in bioproduct manufacturing process in host   The method of claim 1, wherein the host is an egg or a cultured cell.   The method of claim 2, wherein the egg is a growing chicken egg.   The method according to claim 3, wherein the raw material of the vaccine is inoculated as a bioproduct into the allantoic cavity of the egg.   The method according to claim 2, wherein the egg is measured in the longitudinal direction.   6. The method according to claim 5, wherein the air chamber side of the egg is the light output part side.   7. The method of claim 6, wherein the method is transmitted light. The manufacturing method of the bio product biomonitored by the method as described in any one of Claims 1-7. A light projecting means for irradiating a host for bioproduct production with light having a wavelength in the range of 400 nm to 2500 nm or a partial range thereof;
A spectroscopic means for performing spectroscopy before or after the light projection, and a detection means for detecting reflected light, transmitted light or transmitted / reflected light of the light irradiated on the sample;
A data analysis means for quantitatively or qualitatively analyzing the host environment by analyzing the absorbance at all wavelengths or specific wavelengths in the absorbance spectrum data obtained by detection using an analysis model prepared in advance. Inspection / diagnosis device characterized by having. Apparatus according to claim 9 used in the method according to any one of claims 1-8.