Method for referencing analyte measurements performed in purification system

Abstract

In a method in which an analyte measurement performed in a purification system is referenced during a process step in a biopharmaceutical process, the following sub-steps are performed during the same process step: directing a medium through a purification unit (10) in which the analyte is removed from the medium or added to the medium; measuring, at a first measurement location upstream of the purification unit (10), at least one parameter related to the presence and / or amount of the analyte with a first measurement system (16); measuring the at least one parameter at a second measurement location downstream of the purification unit (10) with a second measurement system (18); and using the upstream measurement as a reference to the downstream measurement, or the downstream measurement as a reference to the upstream measurement.

Description

[0001] The present invention relates to a method for using as a reference an analyte measurement performed in a purification system during a process step in a biopharmaceutical process. The present invention also relates to a purification system comprising a measuring device configured to perform the method.

[0002] The concentration of analytes in the medium, such as the concentration of certain proteins and DNA, is an important control parameter in downstream processing. Standard equipment for detecting the presence of analytes and determining the amount of analytes includes ultraviolet visible (UV / Vis) spectrophotometers, which typically detect specific wavelengths, typically between 220 nm and 390 nm, with an accuracy of 1 nm to 3 nm.

[0003] Typical measurement setups record spectra in transmission mode. This means that the sample to be measured is located between the excitation light source and the detector. The light source and the detector can be directly connected via classical optics such as mirrors and lenses or via optical fibers.

[0004] The spectral data are recorded in absorbance units (AU, dimensionless quantity) and are defined as A = log10 (Φ 0 / Φ), where Φ 0 = radiant flux received by the material, Φ = radiant flux transmitted by the material, Φ 0 / Φ = T = transmittance of the material. For a typical chromatography unit (column or membrane) device, I 0 is the intensity of the incident light, and I is the intensity of the transmitted light.

[0005] In the following, a distinction will be made between absorbance measurements of process samples without analyte (reference matrix) and absorbance measurements of samples containing analyte. In particular, the absorbance of the matrix recorded by the spectrophotometer will be referred to as "I 0 ”, and the absorbance of the sample containing the analyte will be referred to as “I”.

[0006] The general procedure for UV-Vis measurements is as follows: (1) Record I 0 This is usually done with a sample similar to the process sample but without the analyte. Usually a pure buffer is used. (2) Record I, which is done with a process sample containing the analyte. Both measurements are standard measurements in a UV-Vis spectrophotometer. Then, reference I 0 It is used as a reference for I in a standard manner to allow the contribution of the matrix itself, the detector, etc. to be subtracted from the measurement of the sample containing the analyte in order to obtain the actual (corrected) absorption measurement result.

[0007] In theory, when I 0 When the isolated measurement is 0However, challenges arise when the measurement system is integrated into process equipment and used during the running process, especially during downstream processes.

[0008] A typical and exemplary integration of a transmission measuring UV-visible spectrophotometer in a downstream process is shown in FIG1 . The following description relates to a chromatography system, but is also applicable to other purification systems. Here, the process step N to be monitored is a capture step. In process step N-1, the cell culture fluid of the bioreactor is filtered and sent to a chromatography unit (column or membrane) with a protein A membrane or resin. The target protein binds to the membrane or resin, while the contaminants pass through the membrane or resin. The contaminants can be detected by a UV-visible spectrophotometer downstream of the chromatography unit (first detection situation).

[0009] The composition of the buffer flowing through the Protein A capture step is then changed to allow the protein to be washed or resolved (eluted) from the adsorbent. The target protein can now be detected using a UV-Vis spectrophotometer (second detection scenario).

[0010] Both tests are performed downstream of the chromatography unit, i.e. downstream of process step N. For both test cases (contaminant and target protein), the data used to record I 0 The reference matrix (medium without analyte) is different and must be acquired individually to achieve optimal absorbance measurements.

[0011] In the second detection case, the reference for the target protein measurement would be the elution buffer without the target protein. Unfortunately, the first thing the UV-Vis spectrophotometer sees is the buffer containing the washed-out target protein. It is not until the target protein is completely released from the chromatography unit that the UV-Vis spectrophotometer has a chance to perform proper I 0 Measurement.

[0012] As already indicated, the typical integration of the measuring system is downstream of the chromatography unit in order to allow detection of the target protein in the eluate. Detection of DNA and HCP (host cell proteins) can also be performed during filling and column / membrane breakthrough of the chromatography unit (among other detection scenarios).

[0013] In another measuring arrangement as shown in FIG2 , another UV-visible spectrophotometer is inserted to achieve a transmission measurement upstream of the chromatography unit. The additional UV-visible spectrophotometer can monitor the filling of the chromatography unit by quantifying the protein concentration. In the depicted example, the filtered cell culture fluid passes through the UV-visible spectrophotometer before the target protein is adsorbed in the chromatography unit. The ideal reference would be a cell culture fluid without the target protein. However, this reference never passes through the measurement position upstream of the chromatography unit. Therefore, in this use case, there is also the problem of a compromised reference for the measurement.

[0014] To date, reference spectra (I 0 ) is simply considered suboptimal, or it may be obtained from old batches or laboratory experiments, but this practice is also suboptimal.

[0015] In some applications, precise reference to the right buffer is not mandatory, especially when only qualitative information is needed (e.g. absence or presence of a protein). In this case, common practice is to simply monitor the deviation from a certain reference time point (at which the reference was made). However, for quantitative predictions, this necessitates a more precise selection of the right sample for reference. This problem has not been solved to date.

[0016] WO 2010 / 151214 A1 describes a method for determining the binding capacity of a chromatography column. A feed signal representing the composition of the feed material supplied to the inlet of the column is detected by a first detector positioned somewhere along the feed line. An outflow signal representing the composition of the effluent from the column is detected by a second detector positioned somewhere along the outflow line. A determination unit analyzes the signals detected by the first detector and the second detector in order to determine the binding capacity of the chromatography column. In particular, a Δ (delta) signal is calculated, which is defined as the feed signal minus the outflow signal. The Δ signal is used to determine mutation points and saturation points, which in turn can be used to control the start and stop of certain chromatography process steps. However, WO 2010 / 151214 A1 does not solve the problem of precise reference. As explained above, in process applications, the spectrophotometer at the feed line cannot see the correct or ideal matrix to measure I 0 .

[0017] In conclusion, quantitative predictions require a correct reference, which cannot be given by a setup with a single absorbance measurement. Even in the case of two measurement systems installed upstream and downstream of the chromatography unit, an autonomous measurement system cannot acquire spectra based on an optimal reference.

[0018] It is an object of the present invention to provide an improved reference for analyte measurements in purification systems, especially in downstream applications.

[0019] The above problem is solved by a method according to claim 1. Advantageous and alternative embodiments of the invention are evident from the dependent claims.

[0020] The present invention provides a method for using as a reference an analyte measurement performed in a purification system during a process step in a biopharmaceutical process. According to the present invention, the following sub-steps are performed during the same process step: guiding a medium through a purification unit, in which an analyte is removed from the medium or added to the medium; measuring at least one parameter related to the presence and / or amount of the analyte with a first measurement system at a first measurement position upstream of the purification unit; measuring the at least one parameter with a second measurement system at a second measurement position downstream of the purification unit; and using the upstream measurement as a reference for the downstream measurement, or vice versa.

[0021] In the sense of the present invention, a "purification system" can be a chromatography system, or a filtration system, or a selective affinity purification system, or any other separation / retention system used in a process step, especially a downstream process step, of a biopharmaceutical process to retain the target product or contaminants. In the sense of the present invention, a "medium" is a fluid or a gas. Depending on the actual application, it can be a cell culture fluid, a solution, an eluent, etc.

[0022] The present invention is based on the following discovery: using a single measurement system or several autonomous measurement systems integrated in the downstream process, it is not possible to provide an ideal reference for the measurement. However, in purification systems, especially in chromatography systems, where the analyte is either removed from the medium during the filling of the chromatography unit, or the analyte is added to the medium during the elution of the chromatography unit, this problem can be solved by performing two measurements and using one measurement as a reference for the other measurement. In particular, a measurement is performed at a position where the analyte is not present in the medium, and the measurement is used as a reference for the actual measurement related to the analyte, which is performed at another position where the analyte is present in the medium. This means that the two measurements are not just two measurements of the same parameter performed at different positions. On the contrary, according to the present invention, the measurement of one measurement system is directly associated with the reference measurement of another measurement system.

[0023] The method according to the present invention is not limited to chromatography systems. On the contrary, it can be used in almost any purification system as defined above, wherein, contrary to absolute measurement, the analytical method and the matrix, i.e. medium, which does not have analyte (after removing analyte from the medium or before analyte is added to the medium) establish a relationship. The typical relative measurement using the fixed ground state (matrix) as a reference is absorbance measurement, but the method according to the present invention can also be used for other types of relative measurements. When the same position of analyte cannot be present in the medium to perform the measurement of the medium without analyte (matrix) (before removing analyte or after adding analyte), the method according to the present invention is particularly useful.

[0024] In an exemplary first use case, both measurements are performed during a filling step in which the analyte is adsorbed in a purification unit (e.g., a chromatography unit). Thus, the analyte is present in the medium before it reaches the purification unit, but is not present in the medium when the medium leaves the purification unit. In this case, when the measuring system is a UV-visible spectrophotometer, the upstream measurement comprises measuring the absorbance of the medium containing the analyte (before the medium reaches the purification unit), and the downstream measurement comprises measuring the absorbance of the medium without the analyte (after the medium passes through the purification unit).

[0025] In an exemplary second use case, both measurements are performed during an elution step in which the analyte is released from the adsorbent in the purification unit. Compared to the first use case, the presence of the analyte is reversed relative to the purification unit. Thus, when the measuring system is a UV-visible spectrophotometer, the measurement downstream of the purification unit here comprises measuring the absorbance of a medium containing the analyte, and the other measurement upstream of the purification unit comprises measuring the absorbance of a medium without the analyte.

[0026] It should be pointed out that, in the sense of the present invention, transmission measurement shall also include measurement in transflection mode, wherein both reflection and transmission are taken into account.

[0027] The invention also provides a purification system comprising a measuring device configured to perform the method according to the invention.

[0028] In particular, the purification system according to the present invention comprises at least one purification unit, a feed line leading to the inlet of the purification unit, and an outflow line leading from the outlet of the purification unit. The purification system also comprises a first measurement system arranged at the feed line for measuring at least one parameter of the medium related to the presence and / or amount of the analyte, and a second measurement system arranged at the outflow line for measuring the at least one parameter. The purification system also comprises a reference device, which is configured to use the measurement of the first measurement system as a reference for the measurement of the second measurement system or to use the measurement of the second measurement system as a reference for the measurement of the first measurement system.

[0029] Preferably, both the first and second measuring systems are UV-Vis spectrophotometers. However, other types of measuring systems capable of measuring at least one parameter of the medium related to the presence and / or amount of the analyte may also be used, such as for example NIR spectrophotometers.

[0030] To obtain an optimal measurement reference, the two UV / VIS spectrophotometers should be as identical as possible in terms of light flux and optical path. This can be ensured, for example, by aligning the spectral intensities of the two hardware systems during a factory acceptance test using the same or similar samples.

[0031] If a measuring system cannot use measurements from another measuring system as a reference, the reference means of the measuring arrangement can be part of a (separate) control unit.

[0032] Other features and advantages of the present invention will become apparent from the following description and from the accompanying drawings, in which:

[0033] - Figure 1 shows a series of downstream processing steps with an absorbance measurement with one insertion;

[0034] - Figure 2 shows the same sequence of downstream processing steps with two intervening absorbance measurements; and

[0035] - Figure 3 Relevant components of a purification device for carrying out the method of reference analyte measurement according to the invention are shown.

[0036] As already described above, FIG. 1 and FIG. 2 illustrate a typical sequence of sub-steps in a downstream process with one intervening absorbance measurement or two intervening absorbance measurements, respectively.

[0037] Figure 3Schematically illustrated is a purification system including a measuring device used in a downstream process step. The purification system can be a filtration system such as a membrane filtration system (e.g., cross-flow filtration), or a pre-coat filtration system, or a selective affinity filtration system, or a chromatography system for recovering biosynthetic products such as target protein from a cell culture fluid and purifying the product. Of course, the chromatography system is not limited to this application, and can also be used for virus-based therapy, plasmid DNA (pDNA), microbial advanced therapy, microcarrier process, cell-free gene therapy, or other applications. However, for simplicity, the following description relates to a purification system in the form of a chromatography system for recovering the target protein.

[0038] The chromatography system comprises a purification unit, here a chromatography unit 10 (column or membrane). A feed line 12 leads to the inlet of the chromatography unit 10 and an outflow line 14 leads from the outlet of the chromatography unit 10 .

[0039] A first measuring system 16 for measuring at least one parameter of the medium related to the presence and / or amount of the analyte is arranged at the feed line 12. A second measuring system 18 for measuring the same parameter is arranged at the outflow line 14. In the example described further below, the measuring systems 16, 18 are UV-visible spectrophotometers which are identical in terms of light throughput and optical paths.

[0040] Both measurement systems 16, 18 are connected to a control unit 20, which is configured to reference the measurements of the first measurement system 16 to the measurements of the second measurement system 18, or to reference the measurements of the second measurement system 18 to the measurements of the first measurement system 16. The control unit 20 is not necessarily a separate component. In fact, the control unit can be part of a superior control system, or it can be part of one of the measurement systems 16, 18.

[0041] The working principle of the measurement system including two measurement systems 16 and 18 will be explained below in conjunction with two typical use cases in the downstream process. For simplicity, it is assumed that the first measurement system 16 and the second measurement system 18 only perform one measurement during the corresponding process step. Of course, one or both of the measurement systems 16 and 18 can also perform repeated measurements.

[0042] For example, the downstream process includes a capture step in which insoluble matter is removed from the cell culture fluid (medium) by a filtration unit 22 or similar device. The filtered cell culture fluid is then supplied to the chromatography unit 10 via a feed line 12.

[0043] In the chromatography unit 10 , the target protein binds to a resin or membrane (adsorbent), such as a Protein A membrane. The target protein-depleted medium flows through the outlet of the chromatography unit 10 into an outflow line 14 .

[0044] This filling step is monitored by a first measurement performed at the feed line 14 with the first measuring system 16. This means that the first measurement is performed upstream of the chromatography unit 10, wherein the target protein is still present in the medium.

[0045] During the filling step, the second measuring system 18 performs a second measurement at the outflow line 16 downstream of the chromatography unit 10 , ie after the target protein has been removed.

[0046] The second measurement on the medium without the target protein is used as a reference for the first measurement.The difference in the signal (recorded spectrum) corresponds to the presence or amount of the target protein (analyte).

[0047] In particular, considering that both measuring units 16, 18 are measuring devices that are UV-visible spectrophotometers, the absorbance of the target protein, i.e., "I", is measured using the first measuring system 16 upstream of the chromatography unit 10. As a reference for this measurement, the absorbance of the medium (reference matrix) without the target protein, i.e., "I 0 A separate measurement is performed during the filling step with a second measuring system 18 downstream of the chromatography unit 10. The actual (corrected) absorbance of the medium with the target protein can then be obtained based on the two independent measurements.

[0048] After sending the first and second measurement values ​​to the control unit 20, a referencing procedure may be performed in the control unit 20. According to an alternative, the second measurement value is sent to the first measurement unit 16 which is configured to use the second measurement value as a reference for the first measurement value.

[0049] In the subsequent elution step of the downstream process, a different medium (buffer) is sent through the chromatography unit 10. The composition of the buffer is selected to allow the release of the target protein from the adsorbent. The buffer containing the target protein flows through the outlet of the chromatography unit 10 into the outflow line 14.

[0050] The first measurement and the second measurement are respectively carried out with a first measuring system 16 upstream of the chromatography unit 10 and a second measuring system 18 downstream of the chromatography unit 10. In this use case, the buffer in the feed line 12, in which the first measuring system 16 is arranged, does not contain the target protein, while the buffer in the outflow line 14, in which the second measuring system 18 is arranged, is enriched with the target protein.

[0051] The second measurement performed using the second measurement system 18 is used to monitor the elution step. For the reverse case, the second measurement uses the first measurement performed using the first measurement system 16 as a reference in a similar manner as described above. The deviation between the first measurement (reference spectrum) and the second measurement (actual spectrum) corresponds to the presence or amount of the target protein.

[0052] In this case of use, the absorbance of the medium containing the target protein, i.e., "I", is measured using a second measuring system 18 downstream of the chromatography unit 10. As a reference for this measurement, the absorbance of the medium without the target protein, i.e., "I 0 A separate measurement is performed during the elution step using the first measuring system 16 upstream of the chromatography unit 10. The actual (corrected) absorbance of the medium with the target protein can then be obtained based on the two independent measurements.

[0053] Downstream processing may include further process steps such as purification and product polishing, which are not relevant here for the analyte measurement.

[0054] The main field of use of the described reference procedure is UV-visible spectroscopy (absorbance measurements) in downstream applications. In principle, however, the method can be used for any production step in which a single analyte or even two or more analytes are enriched or depleted in a controlled manner.

[0055] For example, the reference method can also be used with fluorescence measurements (instead of absorbance measurements). In this case, the fluorescence spectrum (or even a fluorescence signal without spectral resolution) will not have the fluorescence spectrum (signal) of the analyte as a reference. The reference method is also suitable for Raman spectroscopy and other light scattering techniques, such as for particle detection when only particles of certain sizes are filtered and the distribution of the particle size of interest changes in the purification system. The method can also be applied in the case of non-optical measurements, such as in the case of dielectric spectroscopy for, for example, cell counting or vitality measurements. Measurements before the cell retention filter can be used as a reference for measurements in the cell-free medium after the retention filter, so that corrections can be made for changes in the medium over time. Another type of non-optical measurement is (multi-frequency) ultrasonic detection for particle detection.

[0056] Depending on the reference method before and after purification, the first measurement system can be operated in multiplex mode, or the first measurement system can be two of the I 0 A dual channel system in which the channels are connected to probes located on both sides of the purification system.

[0057] Reference numerals list

[0058] 10 chromatography units

[0059] 12 Feeding pipeline

[0060] 14 Outflow pipeline

[0061] 16First measurement system

[0062] 18 Second measurement system

[0063] 20Control Unit

[0064] 22 Filter units

Claims

1. A method for using as a reference an analyte measurement performed in a purification system during a process step in a biopharmaceutical process, wherein: The following substeps are performed during the same process step: - guiding the medium through a purification unit (10) in which the analyte is removed from the medium or added to the medium; - measuring at least one parameter related to the presence and / or amount of the analyte using a first measurement system (16) at a first measurement location upstream of the purification unit (10); - measuring the at least one parameter using a second measurement system (18) at a second measurement location downstream of the purification unit (10); as well as - Use upstream measurements as a reference for downstream measurements, or use downstream measurements as a reference for upstream measurements.

2. The method according to claim 1, characterized in that Both measurements are performed during a filling step in which the analyte is adsorbed in the purification unit (10).

3. The method according to claim 2, characterized in that The upstream measurement comprises measuring the absorbance of a medium containing the analyte, and the downstream measurement comprises measuring the absorbance of a medium without the analyte.

4. The method according to claim 1, characterized in that: Both measurements are performed during the elution step, in which the analyte is released from the adsorbent in the purification unit (10).

5. The method according to claim 4, characterized in that The downstream measurement comprises measuring the absorbance of a medium containing the analyte, and the upstream measurement comprises measuring the absorbance of a medium without the analyte.

6. A purification system comprising a measuring device configured to perform the method according to any of the preceding claims.

7. The purification system according to claim 6, comprising: at least one purification unit (10), a feed line (12) which leads to the inlet of the purification unit (10), an outflow line (14), the outflow line (14) leading from the outlet of the purification unit (10), a first measuring system (16) arranged at the feed line (12) for measuring at least one parameter of the medium related to the presence and / or amount of the analyte, a second measuring system (18) arranged at the outflow line (14) for measuring the at least one parameter, and A reference device is provided, wherein the reference device is configured to use the measurement of the first measurement system (16) as a reference for the measurement of the second measurement system (18) or to use the measurement of the second measurement system (18) as a reference for the measurement of the first measurement system (16).

8. The purification system according to claim 7, characterized in that: The first measuring system (16) and the second measuring system (18) are both ultraviolet-visible spectrophotometers.

9. The purification system according to claim 8, characterized in that Both UV-Vis spectrophotometers are identical in terms of light throughput and optical path.

10. The purification system according to any one of claims 7 to 9, characterized in that The reference device is part of the control unit (20).

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