Automated system and method for analyzing samples from a bioreactor

Abstract

In one aspect, provided herein is a method for automated analysis of samples from a bioreactor, the method comprising withdrawing at least one sample from the bioreactor, applying pressure to the withdrawn at least one sample to form a sample stream, purifying at least one target protein in the sample stream using a first liquid chromatography device to produce a purified sample stream, separating the purified sample stream into a purified sample substream and an effluent stream, and analyzing the at least one target protein in the purified sample substream using a second liquid chromatography device. The subject invention is advantageous in that it provides a two-stage liquid chromatography process, using a first dimension liquid chromatography for purification and a second dimension liquid chromatography for analysis.

Description

Background Art

[0001] As a prior art, it is known to analyze a sample from a bioreactor by using a first-dimensional liquid chromatography for purification and a second-dimensional liquid chromatography for identification of a target protein. However, due to various constraints and sensitivities, the second-dimensional liquid chromatography can utilize only a small portion of the eluate of the first-dimensional chromatography in order to remove the lack of peak resolution related to the size of large protein peaks entering the second-dimensional liquid chromatography apparatus. As one approach, for use in the second-dimensional liquid chromatography, the purified sample is reduced to a small sample (typically 10%). Compared with the conventional approach, this innovative approach shortens the analysis time by a factor of 10. Based on the conventional approach, the purified sample is automatically divided into small portions in order to utilize high-resolution peak separation, each portion of the purified sample is collected in a sample loop, individually analyzed by the second-dimensional liquid chromatography, and the resulting data are combined to provide the final result. FIG. 2 shows a representative second-dimensional ion exchange chromatography of individual first-dimensional protein A peaks obtained by high-resolution peak separation. This approach works, but the analysis time is 10 times that of the innovative approach of the flow separation method.

Summary of the Invention

[0002] In one embodiment, an automated system for analyzing at least one sample from a bioreactor is provided. The system comprises a probe for drawing at least one sample from a bioreactor, a pump for pressurizing the drawn at least one sample to form a sample flow, a first conduit connected to the pump for carrying the sample flow, a first liquid chromatograph having a main inlet and a main outlet, wherein the main inlet is connected to the first conduit to receive the sample flow, and the first liquid chromatograph is configured to purify at least one target protein in the sample flow to produce a purified sample flow, the purified sample flow being discharged from the main outlet, a second conduit connected to the main outlet for carrying the purified sample flow, and a flow splitter having a splitter inlet, a branch outlet and a splitter outlet. The invention comprises a flow splitter, a flow restrictor associated with a branch outlet, the splitter inlet of which is connected to a second conduit to receive a stream of purified sample, the flow restrictor being able to discharge a portion of the stream of purified sample from the branch outlet as a portion of the stream of purified sample, and the stream of purified sample not discharged from the branch outlet being discharged from the splitter outlet as an outflow stream; a second liquid chromatography apparatus having a third conduit connected to the branch outlet to carry a portion of the stream of purified sample, and a second inlet connected to the third conduit to receive a portion of the stream of purified sample, wherein the second liquid chromatography apparatus is configured to analyze at least one target protein in the stream of a portion of the stream of purified sample. The invention of the subject is advantageous in that it provides a two-stage liquid chromatography process using first-dimensional liquid chromatography for purification and second-dimensional liquid chromatography for analysis.

[0003] In another embodiment, a method for automated analysis of at least one sample from a bioreactor is provided. The method includes withdrawing at least one sample from the bioreactor; pressurizing the withdrawn at least one sample to form a sample flow; purifying at least one target protein in the sample flow using a first liquid chromatography apparatus to generate a purified sample flow; separating the purified sample flow into a portion of the purified sample flow and an outflow flow; and analyzing at least one target protein in the portion of the purified sample flow using a second liquid chromatography apparatus.

[0004] These and other features of the subject invention can be better understood through the following detailed description and examination of the accompanying drawings. [Brief explanation of the drawing]

[0005] [Figure 1] This is a schematic diagram of the system formed according to the invention of the subject. [Figure 2] Representative second-dimensional ion-exchange chromatography images of individual first-dimensional protein A peaks obtained by high-resolution peak separation are shown. [Figure 3] This is a representative chromatogram of protein A, generated using size exclusion chromatography (SEC) by the system described in the subject invention. [Figure 4-5] This is a representative chromatogram of protein A, produced by the second liquid chromatography apparatus of the invention described in the subject, using cation exchange chromatography (CEX) along with weak and strong cation exchange columns, respectively. [Figure 6] This is a representative profile of the online bioreactor titration concentration (titer) from day 7 to day 15, measured by the first liquid chromatography apparatus of the system according to the subject invention. [Figure 7]This is a representative online amino acid analysis profile generated using the o-phthaldialdehyde derivatization method (OPA) with the first liquid chromatography apparatus of the system according to the subject invention. [Figure 8] The results of the addition and recovery test of amino acids added to cell culture media, derivatized using the o-phthalidaldehyde derivatization method (OPA) combined with the invention of the subject, compared to the carbamic acid 6-aminoquinolyl-N-hydroxysuccinimidi derivatization method (AQC or AQ) combined with offline processing, are shown. [Modes for carrying out the invention]

[0006] Referring to the figure, system 10 is provided for the automated analysis of one or more samples obtained from a bioreactor 12. The terms used herein are for the sole purpose of describing a particular embodiment and are not intended to limit the invention. As used herein, the term “sample” encompasses both singular and plural, without any particular limitation. The bioreactor 12 may be any standard bioreactor for growing a sample of biological proteins. A probe 14 may be provided in a position to draw a sample out of the bioreactor 12. A pump 16 may be provided to the probe 14 having an inlet 18 that leads, for example, through an extraction conduit 20. The pump 16 is configured to generate negative pressure to draw a sample out of the bioreactor 12 through the probe 14 and the extraction conduit 20.

[0007] Pump 16 may be any known design and may be configured as a manifold capable of connecting multiple inlets in parallel. Pump 16 may be a peristaltic pump configured to operate on the extraction conduit 20 or on a conduit leading to the extraction conduit 20 without contact with the sample. One or more intermediate vials 22 may be provided with pump 16 to collect the sample drawn from the bioreactor 12. The sample is drawn from the intermediate vials 22 and may be pressurized by the pump to provide a flow of sample out of pump 16. A first conduit 24 may be provided leading to the discharge port 26 of pump 16 to carry the generated flow of sample. In addition, a sample collection loop may be associated with pump 16, the extraction conduit 20 and / or the first conduit 24 to collect the sample in preparing the flow of sample.

[0008] A control system may be provided to control the pump 16. The control system may include a computer processing unit having non-temporary memory for storing instructions. The control system may be configured, for example, to start the pump 16 at predetermined intervals or other start times based on instructions stored in memory. In automatic operation, the pump 16 may behave as an automatic sampler.

[0009] A first liquid chromatography apparatus 28 is provided, which is preferably configured as a first-dimensional liquid chromatography apparatus configured to purify at least one target protein in a sample stream in order to generate a purified sample stream. A first conduit 24 carries the sample stream to a main inlet 30 of the first liquid chromatography apparatus 28. The flow rate of the sample stream through the first conduit 24 may be in the range of 0.5 to 5 mL / min. One or more main vials 29 may be used within the first liquid chromatography apparatus 28 to collect the sample stream for purification. Any known design of liquid chromatography apparatus may be appropriately used to purify the target protein in the sample stream.

[0010] The first liquid chromatography apparatus 28 includes a main outlet 32 ​​connected to a second conduit 34. The purified sample stream is discharged from the first liquid chromatography apparatus 28 through the main outlet 32 ​​into the second conduit 34. The purified sample stream may be discharged in amounts ranging from 1 to 100 μl, or from 1 to 80 μl, or from 1 to 60 μl, or from 1 to 40 μl, or from 1 to 20 μl, or from 1 to 10 μl. The first liquid chromatography apparatus 28 may include a pump to apply pressure to the discharged purified sample stream and, if used, to draw the sample from the main vial 29. The purified sample stream contains an increased proportion of at least one target protein purified by the first liquid chromatography apparatus 28.

[0011] A flow splitter 36 is provided to the system 10 having a splitter inlet 38, a branch outlet 40, and a splitter outlet 42. The splitter inlet 38 is connected to a second conduit 34 to receive a flow of purified sample. The flow restrictor 44 is associated with the branch outlet 40 so that only a portion of the flow of purified sample is discharged from the branch outlet 40 as a portion of the flow of purified sample. The portion of the flow of purified sample is any portion of the flow of purified sample and may include 50% or less of the flow of purified sample entering the splitter inlet 38, or 33.3% or less of the flow of purified sample entering the splitter inlet 38, or 10% or less of the flow of purified sample entering the splitter inlet 38. The portion of the flow of purified sample may represent an amount in the range of 40 μl or less. Any portion of the flow of purified sample that is not discharged from the branch outlet 40 is discharged from the splitter outlet 42 as an outflow that can be collected in a waste container 46. As those skilled in the art will understand, the flower restrictor 44 may be positioned within the splitter outlet 42, or multiple flower restrictors 44 may be used to be positioned within one or both of the branch outlet 40 and the splitter outlet 42.

[0012] The third conduit 48 is connected to the branch outlet 40 to carry a portion of the purified sample flow.

[0013] The second liquid chromatography apparatus 50 is provided having a secondary inlet 52 connected to a third conduit 48 to receive a portion of the purified sample flow. The second liquid chromatography apparatus 50 is configured to analyze one or more target proteins in the portion of the purified sample flow. Any known design of liquid chromatography apparatus can be appropriately used for the analysis of target proteins, and this may include a second-dimensional liquid chromatography apparatus. Due to sensitivity and other constraints, the size of the sample for analysis by the second liquid chromatography apparatus 50 is limited. The flow splitter 36 allows for automatic diversion of a portion of the purified sample flow to the second liquid chromatography apparatus 50 for analysis by the second liquid chromatography apparatus 50.

[0014] The third conduit 48 and / or the second liquid chromatography apparatus 50 may be provided with at least one sample collection loop 56 for collecting a portion of the purified sample flow to accumulate a specific amount for injection into the second liquid chromatography apparatus 50. If multiple sample collection loops 56 are used, they may be arranged in parallel to continuously collect portions of the purified sample flow. Multiple sample collection loops 56 reduce the amount of flow discharged from the branch outlet. For example, a flow splitter 36 may be configured to discharge 10% of the purified sample flow entering the splitter inlet 38 as a portion of the purified sample flow. A predetermined amount, e.g., 40 μl, of the portion of the purified sample flow may be collected in a single sample collection loop 56 in preparation for injection. If multiple sample collection loops 56 are used, they may collectively collect a predetermined amount of the portion of the purified sample flow. A portion of the purified sample flow can be drawn into the second liquid chromatograph 50 and / or the sample collection loop 56 by a pump located in the second liquid chromatograph 50 and / or along the third conduit 48. The pump can be automated to activate when a predetermined amount is detected in the sample collection loop 56.

[0015] Optionally, a filter 54 may be introduced into the probe 14 and / or extraction conduit 20 to filter the sample drawn from the bioreactor 12.

[0016] Standard cleaning techniques may be used between analyses, which include operating the system 10 with a cleaning solution throughout one or more operating cycles.

[0017] For example, certain components of the system 10, such as probe 14, one or more conduits, and / or parts of the flow splitter 36, may need to be replaced after a certain number of cycles.

[0018] As those skilled in the art will understand, system 10 can be used, for example, for the purification and analysis of monoclonal antibodies (mAbs) and Fc fusion proteins.

[0019] As a non-limiting example, System 10 includes: The first liquid chromatography apparatus 28 may be any commercially available 1D-LC liquid chromatography apparatus capable of purifying a target sample, for example, a liquid chromatography apparatus sold by Agilent Technologies, Inc. in Santa Clara, California, under the names "1260 Infinity" or "1290 Infinity". The second liquid chromatography apparatus 50 may be any commercially available 2D-LC liquid chromatography apparatus capable of analyzing the purified sample, for example, a liquid chromatography apparatus sold by Agilent Technologies, Inc. in Santa Clara, California, under the name "1290 Infinity". Pump 16 may be any commercially available automated online sampling system, for example, one sold by Flownamics Analytical Instruments in Madison, Wisconsin, under the name "Seg-Flow 4800 (registered trademark)". Flow splitter 36 may be any commercially available flow splitter, for example, one sold by Mott Corporation in Farmington, Connecticut, under the name "PerfectPeak (registered trademark)".

[0020] The invention of the subject enables a continuous bioprocess and enables near real-time monitoring of titration concentration levels, important quality attributes (CQAs) of the processed sample, and amino acid levels within the bioreactor 12. For example, the first liquid chromatography device 28 can be configured to analyze the amino acid levels in the sample stream carried by the first conduit 24. In this manner, feedback control can be established to add depleted amino acids back to the bioreactor 12.

[0021] In addition, the first liquid chromatography device 28 and / or the second liquid chromatography device 50 can be configured to analyze the processed sample and the titration concentration levels within the CQAs.

[0022] example Using the system 10, it was found that the system 10 operates well and the results are comparable to those generated using the results of offline tests, from the results of online size and charge variant analysis generated using protein A chromatography to perform a 1:10 flow separation after the first dimension and before second dimension size exclusion chromatography (SEC) and cation exchange chromatography (CEX) analysis. Tables 1 and 2 show comparative data for SEC and CEX chromatography using system 10 (online) and offline (manual) processing. Samples 1-4 are monoclonal antibody (mAb) molecules obtained from the bioreactor and analyzed on different days.

[0023]

Table 1

[0024]

Table 2

[0025] A representative chromatogram of protein A / SEC generated by system 10 is shown in Figure 3. Representative chromatograms of protein A / CEX generated by the second liquid chromatography apparatus 50 using weak and strong cation exchange columns are shown in Figures 4 and 5, respectively.

[0026] In addition to product quality results, the titration concentration results generated simultaneously from first-dimensional protein A chromatography are a further advantage. Representative profiles of online bioreactor titration concentrations from day 7 to day 15, measured by the first-dimensional liquid chromatography apparatus 28, are illustrated in Figure 6. Other possible chromatography techniques, such as RP-HPLC, HIC, HILIC, affinity chromatography, and denaturation SEC (reduced and unreduced), can be performed on bioreactor samples using system 10.

[0027] Amino acid analysis (AAA) System 10 can be used in conjunction with in-column o-phthaldialdehyde derivatization (OPA) and in-line sampling, thereby enabling System 10 to be fully automated to perform online amino acid analysis (AAA). A representative online amino acid analysis profile generated using OPA with a first liquid chromatograph 28 interacting with a pump 16 acting as an autosampler is shown in Figure 7.

[0028] A comparison of OPA using the subject invention with the carbamic acid 6-aminoquinolyl-N-hydroxysuccinimidi derivatization method (AQC or AQ) using a combination of offline (manual) first- and second-dimensional chromatography (e.g., AccQ-Tag, sold by Waters Corporation in Milford, Massachusetts, USA) demonstrates that any significant differences in the results between the two methods fall within the inherent variability of AAA. For example, Tables 3 and 4 show the results of a splice recovery test performed by adding amino acids to NaOH, demonstrating that sufficient recovery is achieved with both methods (OPA / subject invention vs. AQC / offline combination).

[0029] [Table 3]

[0030] [Table 4]

[0031] Figure 8 shows the results of an amino acid addition and recovery test in cell culture medium, demonstrating good and relatively sufficient recovery when comparing AQC / offline and OPA / the invention. Table 5 shows representative online AAA profiles of each amino acid during bioreactor operation from day 7 to day 16 using OPA / the invention.

[0032] [Table 5]

Claims

1. An automated system for analyzing at least one sample from a bioreactor, A probe for extracting at least one sample from the bioreactor, A pump that applies pressure to the at least one sample drawn out so that it becomes a sample flow, A first conduit connected to the pump to carry the flow of the aforementioned sample, A first liquid chromatography apparatus having a main inlet and a main outlet, wherein the main inlet is connected to a first conduit to receive the sample flow, the first liquid chromatography apparatus is configured to purify at least one target protein in the sample flow to generate a purified sample flow, and the purified sample flow is discharged from the main outlet, A second conduit connected to the main discharge port for carrying the flow of the purified sample, A flow splitter having a splitter inlet, a branch outlet, and a splitter outlet, wherein the splitter inlet is connected to a second conduit to receive the flow of the purified sample, a flow restrictor is associated with the branch outlet so as to be able to discharge a portion of the flow of the purified sample from the branch outlet as a portion of the flow of the purified sample, and the flow of the purified sample that is not discharged from the branch outlet is discharged from the splitter outlet as an outflow, and the second conduit is continuous between the main outlet and the splitter outlet, A third conduit connected to the branch outlet to carry a portion of the flow of the purified sample, A second liquid chromatography apparatus having a second inlet connected to a third conduit to receive a portion of the purified sample flow, wherein the second liquid chromatography apparatus is configured to analyze the at least one target protein in the portion of the purified sample flow, and the third conduit is continuous between the branched discharge port and the second inlet, Equipped with, system.

2. The at least one sample drawn is collected from the bioreactor into one or more intermediate vials. The system according to claim 1.

3. The pump extracts at least one of the extracted samples from the one or more intermediate vials, and then applies pressure to the at least one of the extracted samples so that it becomes a sample flow. The system according to claim 2.

4. The first liquid chromatography apparatus includes one or more main vials for collecting the sample flow. The system according to claim 3.

5. The first liquid chromatography apparatus is a first-dimensional liquid chromatography apparatus. The system according to claim 1.

6. The pump further comprises a filter for filtering the at least one sample before applying pressure. The system according to claim 1.

7. The flow of a portion of the purified sample is less than 50% of the flow of the purified sample. The system according to claim 1.

8. The system further comprises at least one sample collection loop for collecting a portion of the flow of the purified sample to accumulate a predetermined amount for analysis by the second liquid chromatography apparatus. The system according to claim 1.

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