CN217007147U - Online sampling device and online detection system - Google Patents
Online sampling device and online detection system Download PDFInfo
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- CN217007147U CN217007147U CN202220303339.9U CN202220303339U CN217007147U CN 217007147 U CN217007147 U CN 217007147U CN 202220303339 U CN202220303339 U CN 202220303339U CN 217007147 U CN217007147 U CN 217007147U
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Abstract
The utility model belongs to the field of biochemical analysis, and discloses an online sampling device and an online detection system. The online sampling device comprises a filtering module, a liquid storage module and a sample injection module which are sequentially connected in a conducting manner; the filtering module is used for filtering the online sampling sample; the liquid storage module is used for storing the filtrate filtered by the filtering module; the sample introduction module is used for quantitatively conveying the filtrate to the testing device; the filtration module is a disposable device. The online sampling device can monitor the synchronous analysis of the yield and the quality in real time, has basis for optimization, shortens the analysis period, meets the sample detection requirements of high throughput and low cost, completes the separation and the test of filtrate under the state of not stopping the operation, and has high efficiency and convenience in the whole process and convenient control.
Description
Technical Field
The utility model belongs to the technical field of biochemical analysis, and particularly relates to an online sampling device and an online detection system.
Background
Cell culture is not required in all links of research and development and production of biological medicines, and a cell culture process needs to be continuously optimized for improving the yield and quality of target proteins. In the prior art, the off-line operation mode is often adopted when the culture medium is sampled and analyzed. The off-line operation is to suspend the reaction process of the bioreactor, then to take out the culture solution, and then to detect the sample. The whole process can affect the reaction process, and the environment of the culture medium can be affected after a large amount of culture solution is extracted. In addition, there is a possibility that the sampling process may bring in foreign bacteria, resulting in contamination of the culture medium.
Therefore, it is very necessary to provide an online sampling device and an online detection system.
SUMMERY OF THE UTILITY MODEL
The utility model aims to provide an online sampling device and an online detection system, which solve the technical problems of reaction process stop and mixed bacteria influence during offline sampling of a culture medium.
The technical scheme provided by the utility model is as follows:
the utility model aims to provide the following steps: an online sampling device comprises a filtering module, a liquid storage module and a sample introduction module which are sequentially connected in a conducting manner;
the filtering module is used for filtering an online sampling sample;
the liquid storage module is used for storing the filtrate filtered by the filtering module;
the sample introduction module is used for quantitatively conveying the filtrate to the testing device;
the filtration module is a disposable device. The filtering module is a disposable device, namely, the filtering is replaced by a new one every time, the single sample measuring can be removed after the completion, and the next sample measuring can be replaced by a new one.
As a further preference, the filtration module comprises a first cassette, a second cassette and a filtration membrane; the first clamping shell and the second clamping shell are correspondingly arranged to form a closed cavity; the filter membrane is arranged in the closed chamber, and the closed chamber is divided into a first chamber and a second chamber which are separated by the filter membrane;
the first clamping shell is provided with a liquid inlet and a stock solution outlet; and a filtrate outlet is formed in the second clamping shell. Namely, a filter membrane, a first chamber and a second chamber which are separated by the filter membrane are arranged in the filter module; the filtering module is provided with a liquid inlet and a stock solution outlet which are communicated with the first chamber, and is also provided with a filtrate outlet which is communicated with the second chamber. The filtering module adopts transverse filtering, utilizes shearing force, reduces deposition and increases filtering effect.
As a further preferable mode, the liquid storage module comprises a liquid storage tank and a power pump; the power pump is used for driving the filtrate to flow directionally; the liquid storage tank is provided with a liquid storage inlet and two liquid storage outlets, and a filtrate accommodating cavity is formed in the liquid storage tank; the liquid storage inlet is communicated with the filtrate outlet; and one of the two liquid storage outlets is in conduction connection with the sample injection module, and the other one of the two liquid storage outlets is connected with the power pump.
Preferably, the power pump is a first peristaltic pump, and a first waste liquid bottle is arranged at an outlet at the tail end of the first peristaltic pump.
As a further preferred option, the sample injection module comprises an injection pump, a cleaning solution bottle, a dilution solution bottle and a second waste solution bottle; the injection pump comprises a six-way selector valve and an injector; the injector, the cleaning liquid bottle, the diluting liquid bottle and the second waste liquid bottle are respectively connected with corresponding valve ports of the six-way selector valve through pipelines.
Preferably, the six-way selector valve is provided with a valve port for connecting the liquid storage module and the testing device respectively.
As a further preference, the online sampling device further comprises a control module; the first peristaltic pump, the injector and the six-way selector valve are respectively electrically connected with the control module.
The second object of the present invention is to provide: the detection system of the online sampling device also comprises a bioreactor and a testing device; the bioreactor is provided with an inlet and an outlet which are respectively communicated and connected with a liquid inlet and a stock solution outlet of the filtering module through pipelines, and a circulating passage is formed between the first chambers; the circulation passage is provided with a second peristaltic pump for pushing liquid to flow in series; and the valve port of the six-way selector valve is in conduction connection with the testing device through a pipeline.
Further preferably, the test device is a LC-MS, i.e. HPLC-MS or LC-MS, and the HPLC is combined with the MS or the LC is combined with the MS.
As a further preference, the second peristaltic pump is arranged between the stock solution outlet of the filtration module and the inlet of the bioreactor.
Has the beneficial effects that: in the detection method in the prior art, when a culture medium is sampled and analyzed, an off-line operation mode is usually adopted, the reaction process of a bioreactor is suspended, then culture solution is taken out, and then a sample is taken out for detection. The whole process can affect the reaction process, and the environment of the culture medium can be affected after a large amount of culture solution is extracted. The sampling process can bring in mixed bacteria, which causes the pollution of the culture medium. The whole process is not convenient, safe and efficient enough.
The online sampling device for the bioreactor can complete separation and test of filtrate without stopping operation, and the whole process is efficient and convenient and is convenient to control.
1) According to the online sampling device for the bioreactor, a circulation passage is formed between the bioreactor and the first chamber of the filtering module, filtered filtrate can be directly injected into the testing device through the six-way selector valve, and the test of relevant data is completed. The on-line sampling and analysis of the cell culture solution are realized in the true sense, the cell culture monitoring process is greatly simplified, the analysis and detection time is shortened, the monitoring cost is reduced, and the influence and pollution of the sampling on the sample and the culture process are avoided; the filtering module is a disposable device and is replaced after one-time cell culture sampling is finished, so that cross contamination among different batches of cell cultures is avoided;
2) the filtration module is replaceable, filtration pores can be selected by replacing the filtration modules with different specifications, only the components to be detected in the culture solution in the bioreactor are controlled to permeate the filtration membrane to enter the liquid storage device, and other culture solution components do not pass through but all return to the bioreactor along the circulation passage. Unnecessary components are not brought out in the process, the change of the culture volume and the culture environment is not caused, and the culture process is not influenced;
3) the transverse filtration adopts a tangential force filtration technology, and the setting direction of the filter membrane is parallel to the flowing direction of the liquid phase, but is different from the common vertical state; compared with the traditional longitudinal vertical filtration, the horizontal filtration speed is high, and the blockage of the membrane is not easy to cause; when the filtrate flows to generate shearing force, the filter cake is not easy to accumulate, and the membrane pollution rate is slowed down to a certain extent;
4) the components to be detected in the filtering module and the liquid storage tank all continuously flow and change in real time, so that real-time online sampling and detection are realized;
5) the culture medium environment in the bioreactor is an integral body, and real-time cell culture medium consumption and metabolic waste poison data can be obtained through online sampling and detection of the culture medium, so that culture optimization has a basis and a direction, and optimization efficiency is greatly improved;
6) by on-line sampling and detection of the protein, the synchronous analysis of the yield and the quality of the protein can be realized, the analysis period is greatly shortened, the cost is reduced, and the sample detection requirements of high throughput and low cost of a cell culture department are met.
Drawings
The above features, technical features, advantages and implementations of an online sampling device and an online detection system will be further described in the following detailed description of preferred embodiments in a clearly understandable manner with reference to the accompanying drawings.
FIG. 1 is a schematic view of the present invention with an on-line detection system.
Description of the reference numerals
1-a bioreactor;
2-an online sampling device; 21-a filtration module; 211-inlet of liquid; 212-stock solution outlet; 213-a filtrate outlet; 214-a filter membrane; 215-a second peristaltic pump;
221-a liquid storage tank; 222-a first peristaltic pump; 223-a first waste bottle; 231-six-way selector valve; 232-syringe; 233-second waste bottle;
3-testing the device.
Detailed Description
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description will be made with reference to the accompanying drawings. It is obvious that the drawings in the following description are only some examples of the utility model, and that for a person skilled in the art, without inventive effort, other drawings and embodiments can be derived from them.
Example 1
As shown in fig. 1, an online sampling device includes a filtering module 21, a liquid storage module and a sample injection module which are sequentially connected in a conducting manner;
the filtering module 21 is used for filtering the online sampling sample;
the liquid storage module is used for storing the filtrate filtered by the filtering module 21;
the sample introduction module is used for quantitatively conveying the filtrate to the testing device 3.
In practical use, the filtering module 21, the liquid storage module and the sample injection module are sequentially connected, and the reactor, the filtering module 21, the liquid storage module and the sample injection module are sequentially connected with the testing device 3. The filtering module 21 is used for completing a filtering task while sampling in the reactor, and then conveying the sample to the liquid storage module through a pipeline; the liquid storage module is used for transferring in a sample injection path, when the sample injection module is circulated to a sample injection detection stage according to the control of the control module, samples of the liquid storage module are conveyed to the sample injection module along with a pipeline, and then are conveyed to the testing device 3 through the sample injection module. The testing device 3 detects the sample and completes the measurement of the content or concentration of the sample.
In one embodiment, the filtration module 21 comprises a first cartridge, a second cartridge, and a filtration membrane 214; the first clamping shell and the second clamping shell are correspondingly arranged to form a closed cavity; the filter membrane 214 is arranged in the closed chamber, and the closed chamber is divided into a first chamber and a second chamber which are only separated by the filter membrane 214;
the first clamping shell is provided with a liquid inlet 211 and a stock solution outlet 212; the second cartridge is provided with a filtrate outlet 213. The filter module 21 adopts transverse filtration, and utilizes shearing force to reduce deposition and increase filtering effect.
In practical use, the online sampling device 2 can still stably take out a sample to be measured under the condition that the bioreactor 1 does not stop working. The filtering module 21 is composed of a first clamping shell, a second clamping shell and a filtering membrane 214, the first clamping shell and the second clamping shell are symmetrically arranged, and the first clamping shell and the second clamping shell are buckled to form a closed accommodating cavity for placing the filtering membrane 214. Separated into a first chamber and a second chamber by a filter 214. The filter membrane 214 is arranged in parallel with the flowing direction of the solution in the circulating passage, and the flowing of the filtrate generates shearing force, so that filter cakes are not easy to accumulate, and the membrane pollution rate is slowed down to a certain extent. A circulation path is formed between the solution flowing through the bioreactor 1 in parallel, and the circulation path can ensure that the solution passing through the filter membrane 214 has the same components as the solution in the bioreactor 1, thereby ensuring the accuracy and timeliness of detection. The on-line sampler comprises a filtration module 21 for providing filtration membranes 214 of different pore sizes, from coarse to fine being Microfiltration (MF), Ultrafiltration (UF), Nanofiltration (NF) and Reverse Osmosis (RO). When the target protein needs to be sampled, a PES microfiltration membrane with the pore size of 0.22 micron is used; however, ultrafiltration membrane 214 is used to sample the components of the medium.
The filter module 21 is composed of an upper and a lower clamping shells and a filter membrane 214 arranged between the two clamping shells. The upper and lower clamping shells are provided with corresponding positioning bulges and grooves which are correspondingly buckled to form a closed chamber, the filter membrane 214 is arranged between the upper and lower clamping shells and tightly buckled in front of the upper and lower clamping shells, and no gap is left at the joint. The closed chamber is divided into a first chamber and a second chamber by the filter membrane 214, and the first chamber forms a perfect closed loop with the bioreactor 1 through a pipeline. The second chamber is located on the other side of the filter membrane 214, and the liquid in the chamber is the liquid filtered by the filter membrane 214, and the liquid moves directionally under the action of the first peristaltic pump 222.
In one embodiment, the filtering module 21 is a disposable device, that is, each time the filtering is replaced with a new one, the single sample can be discarded after the single sample measurement is completed, and the next sample measurement can be replaced with a new one.
In one embodiment, the liquid storage module comprises a liquid storage tank 221 and a power pump; the power pump is used for driving the filtrate to flow directionally; the liquid storage tank 221 is provided with a liquid storage inlet and two liquid storage outlets, and a filtrate accommodating cavity is arranged in the liquid storage tank; the liquid storage inlet is connected with the filtrate outlet 213 in a conduction way; and one of the two liquid storage outlets is in conduction connection with the sample injection module, and the other one of the two liquid storage outlets is connected with the power pump.
In practical use, a small amount of filtrate can be buffered by the arrangement of the liquid storage module, and the liquid in the pipeline slowly and quantitatively moves forwards under the action of the first peristaltic pump 222. Therefore, the liquid to be tested entering the testing device 3 through the six-way selector valve 231 can be stable, cannot be precipitated due to long-time waiting, cannot be out of operation for a long time, and still has the data obtained by testing as filtrate left in a pipeline due to the time lag of the previous test, so that the test data is influenced, and the test has hysteresis.
The liquid storage tank 221 is internally provided with an accommodating cavity and is also provided with a liquid storage inlet and two liquid storage outlets; the liquid storage inlet is connected with the filtrate outlet 213 through a pipeline; two branch circuits are branched from two liquid storage outlets, and one branch circuit is connected with a six-way selector valve 231 and used for supplying liquid to the testing device 3; the other branch is connected with a first peristaltic pump 222 for providing power. The two branches are connected in parallel, the pressure at the accommodating cavity is constant, and the first peristaltic pump 222 forms low pressure when working, so that the filtrate in the filtering module 21 is guided to flow along with the constant working frequency of the first peristaltic pump 222.
In one embodiment, the power pump is a first peristaltic pump 222, and a first waste liquid bottle 223 is disposed at an outlet of a terminal end of the first peristaltic pump 222.
In one embodiment, the sample injection module includes an injection pump, a cleaning solution bottle, a dilution solution bottle, and a second waste solution bottle 233; the injection pump comprises a six-way selector valve 231 and an injector 232; the syringe 232, the cleaning solution bottle, the diluting solution bottle and the second waste solution bottle 233 are respectively connected to corresponding valve ports of the six-way selector valve 231 through pipelines.
In one embodiment, the six-way selector valve 231 has ports for connecting the liquid storage module and the testing device 3, respectively.
In actual use, the sample injection module is used for quantitatively and stably injecting a sample to be tested into the testing device 3. The six-way selector valve 231 is used here, and can be controlled manually or by a control module according to actual needs, and various functions provided by connecting pipelines at different valve ports can be completed by switching the passages of the valve. The first position of the six-way selector valve 231 is connected with the filtrate pipeline, the second position is connected with the diluent bottle, the third position is connected with the cleaning liquid bottle, the fourth position is connected with the sample inlet of the testing device 3, the fifth position is connected with the second waste liquid bottle 233, and the middle position is connected with the injector 232. The syringe 232 is a main component for quantitatively injecting the filtrate to be measured, diluting and injecting the sample into the liquid inlet. The valve position can be changed according to different requirements, so that different functions can be realized. When the first position is communicated with the middle position, the filtrate is injected into the injector 232; when the second position is communicated with the middle position, the diluent is injected into the injector 232; and when the fourth position is communicated with the middle position, the sample loading is finished. When the third position is connected with the middle position, cleaning liquid is injected into the injector 232 to clean the injector 232; when the sixth position is connected to the middle position, the waste liquid generated during cleaning or sample injection can be discharged into the second waste liquid bottle 233. The entire procedure is around the syringe 232 in the middle position. The actual positions of the positions can be arranged according to actual needs and the operational smoothness.
In one embodiment, the online sampling device 2 further comprises a control module; the first peristaltic pump 222, the syringe 232 and the six-way selector valve 231 are electrically connected to the control module respectively.
In practical use, besides manual control, the work flow of the online sampling device 2 can be controlled by a control module according to practical requirements. The rotation power of the second peristaltic pump 215 can be adjusted to control the mixing degree with the solution in the bioreactor 1, for example, one minute before the test, the working frequency of the second peristaltic pump 215 is increased, and the solution in the circulating pipeline is changed to the solution in the bioreactor 1. The working frequency of the first peristaltic pump 222 is increased, the liquid in the liquid storage tank 221 can be emptied in time and replaced by fresh filtrate, and the timeliness of the test can be ensured.
The use of the six-way selector valve 231 can effectively complete the injection, dilution, sample injection and cleaning of the liquid of the syringe 232. The control module can improve the working efficiency and increase the accuracy and the periodicity of the test.
Example 2
As shown in fig. 1, a detection system of an online sampling device further comprises a bioreactor 1 and a testing device 3; the bioreactor 1 is provided with an inlet and an outlet which are respectively communicated and connected with the liquid inlet 211 and the stock solution outlet 212 of the filtering module 21 through pipelines, and a circulating passage is formed between the first chambers; the circulation passage is provided with a second peristaltic pump 215 for pushing the liquid to flow in series; the valve port of the six-way selector valve 231 is in conduction connection with the testing device 3 through a pipeline.
In one embodiment, the testing device 3 is a HPLC-MS or LC-MS, HPLC with mass spectrometry or LC with mass spectrometry.
In practice, the detection system comprises a bioreactor 1 and a LC-MS in addition to the on-line sampling device 2. The reaction state of the bioreactor 1 is observed from the reaction process, and the content of the substance to be detected and the molecule are determined by the LC-MS, so that the whole process is efficient and stable.
In one embodiment, a second peristaltic pump 215 is provided between the dope outlet 212 of the filtration module 21 and the inlet of the bioreactor 1.
The detection method of the detection system with the bioreactor online sampling device comprises the following steps:
s1, preparing a standard solution of a target substance of the sample to be tested according to the test requirement, and drawing a peak area-concentration standard curve;
s2, the control module controls the second peristaltic pump 215 to operate, and the culture solution in the bioreactor 1 is driven to flow along the circulation path; after being filtered by the filtering module 21, the filtrate to be detected is conveyed to the six-way selector valve 231;
s3, controlling and communicating the passages between the filtrate inlet and the injector 232 and between the diluent bottle and the injector 232 by the control module, and closing other passages; after the diluent and the filtrate to be tested are uniformly mixed in the injector 232, switching the six-way selector valve 231, closing the above channel, conducting the channel between the injector 232 and the testing device 3(HPLC-MS or LC-MS), and completing sample injection;
s4, the peak area of the target substance in the filtrate to be tested is measured by the testing device 3 and is substituted into the labeling curve drawn in the step S1 to calculate the concentration of the target substance;
s5, circularly measuring samples, and repeating the steps S2-S4; and repeating the steps S1-S4 when the object to be tested is replaced with a new object to be tested.
In practical use, the filter module 21 with a proper pore size needs to be replaced according to a project to be tested; the filter module 21 can be designed as a disposable device, with different pore sizes of the filter membrane 214. For sampling target protein, PES microfiltration membrane with 0.22 micron pore size is used; the medium was sampled for the components using an ultrafiltration membrane.
Because the testing device 3 is a liquid chromatograph-mass spectrometer, a standard curve needs to be prepared at present, and the content or concentration curve is tested according to actual needs.
The standard curve is prestored and stored after being manufactured, and the standard curve can be conveniently called out during later data calculation. The first peristaltic pump 222, the second peristaltic pump 215, and the syringe 232 are controlled by the control module at appropriate time periods as required by the test. The rotation power of the second peristaltic pump 215 can be adjusted to control the mixing degree with the solution in the bioreactor 1, for example, one minute before the test, the working frequency of the second peristaltic pump 215 is increased, and the solution in the circulation line is changed into the solution in the bioreactor 1. The working frequency of the first peristaltic pump 222 is increased, the liquid in the liquid storage tank 221 can be emptied in time, fresh filtrate is replaced, and the timeliness of the test can be guaranteed.
The use of the six-way selector valve 231 can effectively complete the injection, dilution, sample injection and cleaning of the liquid of the syringe 232. The control module can improve the working efficiency and increase the accuracy and the periodicity of the test.
In one embodiment, the control module controls the second peristaltic pump 215 to operate while controlling the first peristaltic pump 222 to operate; the first peristaltic pump 222 drives the directional flow of the filtrate, so that the filtrate flows into the first waste liquid bottle 223, and the other outlet pipeline connected in parallel flows to the six-way selector valve 231.
In one embodiment, after step S3, the control module controls to close the path between the syringe 232 and the testing device 3, and to open the path between the cleaning solution bottle and the syringe 232, thereby completing the cleaning of the syringe 232; after the above steps are completed, the above path is closed, and the path between the injector 232 and the second waste liquid bottle 233 is conducted, so that the discharge of the waste liquid is completed; after the above steps are completed, the six-way selector valve 231 is reset to the initial state.
In practical use, the first position of the six-way selector valve 231 is connected with the filtrate pipeline, the second position is connected with the diluent bottle, the third position is connected with the cleaning liquid bottle, the fourth position is connected with the sample inlet of the testing device 3, the fifth position is connected with the second waste liquid bottle 233, and the middle position is connected with the injector 232. The syringe 232 is a main component for quantitatively injecting the filtrate to be measured, diluting and injecting the sample into the liquid inlet. The valve position can be changed according to different requirements, so that different functions can be realized. When the first position is communicated with the middle position, the filtrate is injected into the injector 232; when the second position is communicated with the middle position, the diluent is injected into the injector 232; and when the fourth position is communicated with the middle position, the sample loading is finished. When the third position is connected with the middle position, the cleaning liquid is injected into the injector 232 to complete the cleaning of the injector 232; when the sixth position is connected to the middle position, the waste oil during cleaning or sample injection can be discharged into the second waste liquid bottle 233. The entire process is around the syringe 232 in the middle position. The actual positions of the positions can be arranged according to actual needs and the operational smoothness.
Content and differential analysis of target protein in culture solution of bioreactor 1:
the online sampling device 2 is adopted to sample the target protein, and the PES microfiltration membrane with the pore diameter of 0.22 micron is used as the filter membrane 214 in the filter module 21. During sampling, the target protein in the culture solution passes through the filtration membrane 214 of the filtration module 21, and other components such as cells cannot pass through the filtration membrane 214. The target protein solution passes through the liquid storage tank 221 and the reciprocating injection pump, and directly enters the HPLC-MS for detection without dilution. The yield (Titer) of the target protein is measured by HPLC-MS, the molecular weight and the glycoform of the protein are identified, the Titer of the target protein expressed in the culture solution is calculated by an external standard curve method by adopting a standard substance of the target protein (monoclonal antibody) with known concentration, and the Titer is measured to be 5.2 g/L. The theoretical molecular weight of each glycoform is calculated through the theoretical amino acid sequence of the target protein, the actual molecular weight measured by MS is compared with the theoretical molecular weight, so that the glycoforms of the protein in the culture solution are identified, the relative proportion of each glycoform is calculated through the peak area, and the identification and analysis of the target protein are completed, wherein the glycoforms, the theoretical molecular weight, the actual molecular weight and the relative content are shown in the following table 1.
TABLE 1
| Sugar type | Theoretical molecular weight Da | Actual molecular weight Da | Difference Da | Relative content% |
| G0F/G0F | 148059 | 148058 | 1 | 52 |
| G0F/G1F | 1480221 | 1480220 | 1 | 48 |
And (3) analyzing nutrient consumption of the culture medium in the culture solution:
the online sampling device 2 is adopted to sample the components of the culture medium, and the ultrafiltration membrane 214 in the filtration module 21 adopts an ultrafiltration membrane with the cut-off molecular weight of 10K. During sampling, the culture medium components in the culture solution pass through the ultrafiltration membrane in the filtration module 21, and other components such as cells and proteins cannot pass through the filtration membrane. The nutrient solution of the culture medium passes through a liquid storage tank 221 and a reciprocating injection pump, and enters HPLC-MS for detection after being diluted. The content of amino acids, vitamins and other nutrient components in the culture solution is measured by HPLC-MS, the amino acid test results and consumption trend of the cells on 0 th, 3 rd, 5 th, 7 th and 9 th days are shown in Table 2, and the vitamin test results and consumption trend are shown in Table 3.
TABLE 2
TABLE 3
Comparative experiment 1 (influence on the Environment inside the bioreactor 1)
On-line sampling and testing are carried out according to the steps described above, a bioreactor 1 of 1 liter is taken, the actual culture solution volume is about 600ml, 14 days of cell culture are carried out, and sampling is carried out twice a day to determine the nutrient consumption of the culture medium and protein Titer and quality analysis.
Only the components to be detected in the culture solution in the bioreactor 1 penetrate through the filtering module 21 and enter the liquid storage tank 221, and the cells and other culture solution components cannot pass through the filtering membrane 214, so that the cells and other culture solution components are separated and then all return to the bioreactor 1 along the circulation path. The online sampling volume is about 0.1ml per time, and after 14 days of culture and sampling, the total sampling volume is about 2.8ml (the amount of the part which is driven by the first peristaltic pump 222 and flows to the first waste liquid bottle 223 and is discharged is very small and can be ignored), which accounts for 0.5% of the initial culture volume 600ml, and the influence is very small; and the sampling does not cause the reduction of the cell number, does not influence the cell density and the absolute yield of single-batch protein, and does not influence the process conditions such as the supplement amount, the dissolved oxygen, the pH and the like.
Comparative example was the use of off-line sampling, 14 days cell culture, twice daily sampling, 5ml each for media consumption and protein Titer and mass analysis. After 14 days of culture and sampling, a total of 140 ml of sample was taken, which accounted for 23% of the initial culture volume of 600ml, and there were only 460 ml of culture solution at the end of the culture. On the one hand, the cells are also lost with each sampling, and the absolute yield of the final protein is also reduced by about 25%. On the other hand, the culture volume is reduced, and culture process parameters such as feeding amount, dissolved oxygen, pH and the like are also influenced, so that corresponding adjustment is needed to influence the confirmation of process conditions.
Comparative experiment 2 (influence on protein content and quality)
Protein analysis experimental conditions:
1) content Titer experimental conditions:
offline sample processing
200. mu.l of the sample to be tested was transferred into a 1.5ml centrifuge tube, centrifuged at 13000rpm for 10 minutes, and 100. mu.l of the supernatant was transferred into a sample bottle.
Sample analysis
A chromatographic column: POROS A20 Column, 2.1X 30mm or equivalent
A mobile phase A: 20mM phosphate buffer
Mobile phase B: 100mM glycine solution, pH 3.0
Flow rate of mobile phase: 1ml/min
Temperature of the column: 25 deg.C
Detection wavelength: 280nm
Sample injection amount: 30 μ l
The mobile phase gradient settings are shown in table 4 below:
TABLE 4
| Time (min) | Flow rate (ml/min) | Mobile phase a ratio (%) | Mobile phaseB ratio (%) |
| 0.00 | 1 | 100 | 0 |
| 0.50 | 1 | 100 | 0 |
| 0.51 | 1 | 0 | 100 |
| 1.50 | 1 | 0 | 100 |
| 1.51 | 1 | 100 | 0 |
| 4.00 | 1 | 100 | 0 |
2) And SEC experimental conditions:
offline sample processing
The sample to be tested is centrifuged at 13000rpm for 10 minutes, and the supernatant is removed for sample injection and analysis. The sample injection volume (μ l) was 200(μ g)/concentration of sample to be measured (μ g/μ l).
Sample analysis
A chromatographic column: tosoh G3000SWXL, 7.8X 300mm or equivalent
Mobile phase: 200mM KH2PO4，250mM KCl，pH 6.2
Flow rate of mobile phase: 0.5mL/min
Temperature of the column: 25 deg.C
Detection wavelength: 280nm
3) And EX experimental conditions:
offline sample processing
The sample to be tested is centrifuged at 13000rpm for 10 minutes, and the supernatant is removed for sample injection analysis. The sample injection volume (μ l) was 200(μ g)/concentration of sample to be measured (μ g/μ l).
Sample analysis
A chromatographic column: MABPac SCX-10 or equivalent
Mobile phase A: 125mM NaH2PO4Solutions of
Mobile phase B: 125mM Na2HPO4Solution(s)
Mobile phase C: 1M NaCl solution
A mobile phase D: deionized water
Temperature of the column: 30 deg.C
Detection wavelength: 280nm
The mobile phase gradients are shown in table 5 below:
TABLE 5
| Time (min) | Flow rate (ml/min) | pH | NaCl(mM) |
| 0.00 | 0.85 | 5.6 | 0 |
| 3.00 | 0.85 | 5.6 | 0 |
| 20.00 | 0.85 | 7.2 | 30 |
| 25.00 | 0.85 | 7.2 | 100 |
| 25.10 | 0.85 | 5.6 | 0 |
| 35.00 | 0.85 | 5.6 | 0 |
The sample was taken on-line and tested as described in example 3, and the Titer content and quality (SEC purity and CEX charge isomer purity) of the protein of interest (monoclonal antibody used in this example) was analyzed on-line in real time according to the above protein analysis experimental conditions.
Comparative example 2 is an off-line sampling method, and after the off-line sampling, the protein is easy to adsorb and degrade, and the change of the protein content and the quality is analyzed according to the protein analysis experimental conditions.
The ratio of protein content and quality of the online and sampled two parallel batch cell culture processes online and offline is shown in table 6 below.
TABLE 6
| Item | Monitoring qualification standard | On-line | Off-line |
| Content Titer | Not less than 4g/l | 4.8g/l | 3.7g/l |
| SEC purity | Not less than 95 percent | 95％ | 90％ |
| CEX Charge isomers | The main peak is not less than 60 percent | Main peak 70% | Main peak 55% |
The data in table 6 show that, for the same sample, with the same analytical instrument and test method (protein analysis experimental conditions), the protein is adsorbed or degraded due to off-line sampling, the content is reduced by 23%, the SEC purity is reduced by 5%, the CEX main peak is reduced by 15%, the content and quality are significantly reduced, the Titer content and quality of the protein cannot be truly reflected, the accuracy of quality analysis and monitoring is affected, and the monitored protein content and quality are both changed from qualified to unqualified due to sampling problems. Online monitoring may well address this issue.
In conclusion, the online sampling device 2 of the bioreactor 1 can obtain real-time cell culture medium consumption and metabolic waste poison data, so that culture optimization has basis and direction, and optimization efficiency is greatly improved; through the on-line sampling and detection of the protein, the synchronous analysis of the yield and the quality of the protein can be realized, the analysis period is greatly shortened, the cost is reduced, and the sample detection requirements of high throughput and low cost of a cell culture department are met.
It should be noted that the above embodiments can be freely combined as necessary. The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An online sampling device, its characterized in that: comprises a filtering module, a liquid storage module and a sample injection module which are sequentially connected in a conducting manner;
the filtering module is used for filtering an online sampling sample;
the liquid storage module is used for storing the filtrate filtered by the filtering module;
the sample introduction module is used for quantitatively conveying the filtrate to the testing device;
the filtration module is a disposable device.
2. The online sampling device of claim 1, wherein: a filter membrane, a first chamber and a second chamber which are separated by the filter membrane are arranged in the filter module; the filtering module is provided with a liquid inlet and a stock solution outlet which are communicated with the first cavity, and is also provided with a filtrate outlet which is communicated with the second cavity.
3. The online sampling device of claim 2, wherein: the liquid storage module comprises a liquid storage tank and a power pump; the power pump is used for driving the filtrate to flow directionally; the liquid storage tank is provided with a liquid storage inlet and two liquid storage outlets; the liquid storage inlet is communicated with the filtrate outlet; and one of the two liquid storage outlets is in conduction connection with the sample injection module, and the other liquid storage outlet is connected with the power pump.
4. The on-line sampling device of claim 3, wherein: the power pump is a first peristaltic pump, and a first waste liquid bottle for collecting waste liquid is arranged at an outlet at the tail end of the first peristaltic pump.
5. The on-line sampling device of claim 1, wherein: the sample injection module comprises an injection pump, a cleaning liquid bottle, a dilution liquid bottle and a second waste liquid bottle; the injection pump comprises a six-way selector valve and an injector; the injector, the cleaning liquid bottle, the diluting liquid bottle and the second waste liquid bottle are respectively connected with corresponding valve ports of the six-way selector valve through pipelines.
6. The on-line sampling device of claim 5, wherein: the six-way selector valve is provided with valve ports which are respectively used for connecting the liquid storage module and the testing device.
7. The online sampling device of claim 4 or 5, wherein: the device also comprises a control module; the first peristaltic pump, the injector and the six-way selector valve are respectively electrically connected with the control module.
8. A test system with an on-line sampling device according to any of claims 1 to 7, wherein: the device also comprises a bioreactor and a testing device; the bioreactor is provided with an inlet and an outlet which are respectively communicated and connected with a liquid inlet and a stock solution outlet of the filtering module through pipelines, and a circulating passage is formed between the first chambers; the circulation passage is provided with a second peristaltic pump for pushing liquid to flow in series; the valve port of the six-way selector valve is connected with the testing device in a conduction mode through a pipeline.
9. The on-line sampling device of claim 7, wherein: the testing device is a liquid chromatograph-mass spectrometer.
10. The on-line sampling device of claim 7, wherein: the second peristaltic pump is arranged between the stock solution outlet of the filtering module and the inlet of the bioreactor.
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