Articles and methods for analyte concentration measurement

Abstract

Apparatus and related methods are generally provided. Advantageously, some of the apparatus described herein are capable of, and / or may be configured to, detect the concentration of an analyte in a flowing fluid, in a fluid containing a high concentration of an analyte, and / or in a plurality of fluids and / or fluid samples, in quick succession. Some methods may include detecting the concentration of an analyte that is advantageous for one or more of the foregoing reasons.

Description

Technical Field

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 334,452, filed on April 25, 2022, entitled "Articles and Methods for Analyte Concentration Measurements", and U.S. Provisional Application No. 63 / 408,279, filed on September 20, 2022, entitled "Advanced Control Strategies for Continuous Capture of Monoclonal Antibodies Based on Biolayer Interferometry", each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Technical Field Apparatuses and related methods for assaying analyte concentration are generally described.

Background Art

[0003] Background Scientific apparatuses may be used to determine the concentration of analytes in various fluids. However, such apparatuses may have difficulty measuring analytes with high specificity at high concentrations with low latency or at high speed. As a result, new apparatuses and methods are needed.

Summary of the Invention

[0004] Summary The present disclosure generally describes apparatuses and methods. The subject matter described herein, in some cases, involves related products, alternative solutions to specific problems, and / or multiple different uses of one or more systems and / or articles. In some embodiments, a method is provided. The method includes contacting a probe with a fluid over a first period of time, detecting a variation in a signal over the first period of time, determining a first concentration based on the variation in the signal over the first period of time, and sending an instruction to a bioprocessing system based on the determination of the first concentration. The fluid flows over the probe. An analyte is present in the fluid at the first concentration. At least a portion of the analyte becomes immobilized on the probe.

[0005] In some embodiments, the method includes contacting a probe with a fluid over a first period of time. The fluid flows over the probe. An analyte is present in the fluid at the first concentration. At least a portion of the analyte becomes immobilized on the probe. The method further includes detecting a variation in an optical signal over the first period of time. The method further includes determining a first concentration based on the variation in the optical signal over the first period of time. The optical signal includes light reflected from an interface inside the probe and light reflected from an end of the probe.

[0006] In some embodiments, a system is provided. The system includes a first instrument including a probe and a detector configured to detect a variation in a signal over a first period of time, and a bioprocessing system. The system is configured to supply a fluid from the bioprocessing system to the first instrument. The first instrument is configured to determine a first concentration of an analyte in the fluid while the fluid contacts and flows over the probe based on the variation in the signal over the first period of time. The system is configured to send an instruction to the bioprocessing system based on the determination of the first concentration. In some embodiments, a first instrument is provided. The first instrument includes a probe and an optical detector configured to detect fluctuations in an optical signal over a first period. The first instrument is configured to determine a first concentration of an analyte in a fluid that contacts the probe and flows over the probe based on fluctuations in the optical signal over the first period. The optical signal includes light reflected from an interface inside the probe and light reflected from an end of the probe.

[0007] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the present invention when considered in conjunction with the accompanying figures. In cases where this specification and the incorporated references include conflicting and / or inconsistent disclosures, this specification shall prevail. In cases where two or more references incorporated by reference include conflicting and / or inconsistent disclosures, the reference having the later effective date shall prevail.

Brief Description of the Drawings

[0008] Brief Description of the Drawings Non-limiting embodiments of the present invention are described by way of example with reference to the accompanying figures. These are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component that is illustrated is typically represented by a single number. Not every component is labeled in every figure, nor is every component of each embodiment of the present invention shown, where illustration is not necessary to enable a person skilled in the art to understand the present invention and is for clarity purposes only. In the figures:

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DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description Apparatuses, systems, and related methods are generally provided. Advantageously, some of the apparatuses and systems described herein are capable of and / or may be configured to detect the concentration of an analyte in a flowing fluid, in a fluid containing a greater concentration of analyte than that which saturates the immobilization ability of a probe (e.g., the immobilization ability of a probe in an equilibrium state), and / or in a plurality of fluids and / or fluid samples in a rapid succession. Some methods may include detecting the concentration of an analyte that is advantageous for one or more of the foregoing reasons. Among the apparatuses, systems, and methods described herein are those that lack any of the foregoing advantages and / or are implemented under conditions other than those recited above (e.g., on a static fluid, on a fluid containing a low concentration of analyte, and / or on a single fluid).

[0016] Some methods relate to employing a probe to measure one or more properties of a fluid supplied by a bioprocessing system and then supplying an instruction to the bioprocessing system based on such measured property(ies). Some of the systems described herein are capable of and / or may be configured to perform such methods. Such methods may be performed with respect to an upstream bioprocess operation and / or a downstream bioprocess operation. Such instructions may include modifying one or more properties of a fluid within the bioprocessing system (e.g., returning them to a specific range), supplying the fluid to a different location (e.g., when the location where the fluid is supplied can no longer receive such fluid or it is no longer beneficial to supply such fluid), pausing (e.g., to allow an operator to make a repair, to stop the supply of liquid until an operator activates it), and / or not performing any operation (e.g., when the measurement indicates that the state within the bioprocessing system is acceptable and it is desirable to continue supplying the fluid to the same location).

[0017] Advantageously, methods related to measuring one or more characteristics of a fluid supplied by a bioprocessing system and then supplying instructions to the bioprocessing system based on such measured characteristic(s) may enable one or more characteristics of the bioprocessing system to be monitored during its operation and / or one or more characteristics of the bioprocessing system to be maintained within a particular range during operation of the bioprocessing system. These operations may be performed in an automated manner (e.g., without the need for operator attention and / or intervention). Enabling the characteristics of the bioprocess system to be monitored and / or controlled in an automated manner allows for repair, recording of the conditions present during any particular bioprocessing run, and / or quality control. It may also simplify laboratory operations by eliminating the need for an operator to perform functions that are automatically carried out.

[0018] Some methods relate to measuring the concentration of an analyte in multiple fluid samples using a single probe. Some of the instruments described herein are capable of performing and / or are configured to perform such measurements. The ability to perform such measurements beneficially enables multiple measurements to be made without the need to change probes, which may enhance the rate at which measurements are performed and / or reduce the cost of performing multiple measurements by reducing the number of probes required. Performing such measurements is particularly beneficial when many measurements need to be made relatively rapidly, such as when the change in concentration of an analyte in a fluid over time is determined and / or when the concentration of an analyte in fluids supplied from various instruments is determined.

[0019] In some embodiments, the method and / or apparatus may utilize components that facilitate rapid analysis of multiple fluid samples. As an example, in some embodiments, the apparatus includes one or more valves that can be reversibly switched between multiple positions. Each valve may be capable of, and / or configured to, reversibly place one or more fluid sources in fluid communication with a probe. Some apparatuses may include a valve that can reversibly switch between placing two or more fluid sources in fluid communication with a probe (e.g., at least two of the positions to which the valve can be reversibly switched are in fluid communication with different fluid sources). Some apparatuses may include one or more valves that supply fluid directly to a probe. The switchable valve, and the valve that supplies fluid directly to the probe, may increase the rate and / or concentration at which fluid can be supplied to the probe, as compared to other methods by which fluid can be supplied to the probe, which may increase the rate at which a signal will be obtained from the interaction between the fluid and the probe. As an example, such a valve may supply fluid directly to the probe without being further diluted by other fluid sources that supply different (e.g., diluting) fluids to the probe.

[0020] Some methods relate to employing multiple probes. Some of the apparatuses described herein are capable of and / or configured to employ multiple probes. Employing multiple probes advantageously enables measurements to be made on samples continuously obtained from a continuous source of fluid. As an example, measurements may be made on a continuous flow of fluid where the concentration of an analyte is expected to vary. The continuous fluid flow may be split into multiple samples and the samples may be supplied to the probes in such a way that measurements are made on each sample. The presence of multiple probes allows one (or more) probe(s) to be employed to contact the sample and immobilize the analyte present therein while one (or more) probe(s) may be subjected to a regeneration process so that it (or they) can be employed to contact a different sample and immobilize any analyte present therein. The regenerated one (or more) probe(s) may then be employed to contact the sample and immobilize the analyte present while the probe(s) employed previously to contact the sample(s) is (are) being regenerated. In some such embodiments, any sample generated may be supplied to the regenerated probe and any probe exposed to the sample may be subjected to a regeneration process while another probe is being exposed to a different sample, which may enable measurement of any sample generated at the time of generation.

[0021] Some methods relate to measuring the concentration of an analyte based on the rate at which the analyte is immobilized on a probe. Some of the apparatuses described herein are configured to perform such measurements and / or are capable of performing them. By measuring the rate at which the analyte becomes immobilized on the probe, it may be possible to advantageously measure relatively high analyte concentrations, such as the analyte concentration at which the ability of the probe to immobilize the analyte saturates, an analyte concentration close to such concentration, or an analyte concentration above such concentration, with relatively high accuracy. As a result, analytes at concentrations that result in the same or a similar amount of equilibrium immobilization on the probe may be immobilized on the probe at different rates and / or rates that are relatively easy to distinguish from one another. Consequently, measuring the rate at which the analyte becomes immobilized on the probe may present a way to measure the concentration of the analyte with an accuracy that may not be possible with other techniques.

[0022] In addition, measuring the rate at which the analyte becomes immobilized on the probe may be a relatively rapid way to measure the concentration of the analyte in a fluid and / or in a sample of the fluid. In some embodiments, the measurement of the rate at which the analyte becomes immobilized on the probe is performed based on data obtained before the analyte reaches a steady-state level of immobilization on the probe and / or before the analyte reaches a steady-state level of immobilization on the probe. In some embodiments, this period is relatively short, which may enable the concentration of the analyte in the fluid and / or in the sample of the fluid to be determined relatively quickly.

[0023] Some methods relate to performing measurements while a fluid and / or a sample of the fluid is flowing past a probe. Some of the apparatuses described herein are capable of and / or are configured to perform measurements on a fluid and / or a sample of the fluid flowing past the probe. Performing measurements on a fluid and / or a sample of the fluid flowing past the probe may be desirable for applications where measurements are made on a fluid and / or a sample of the fluid supplied from a source such as an apparatus and / or a component of an apparatus. The supplied fluid and / or sample of the fluid may flow past the probe and then to another or the same apparatus, another or the same component of an apparatus, or a waste container. Thereby, the probe may be incorporated into a flow-through system and / or a fluid flow loop in which measurements are made. In addition, the probe may be employed to perform multiple measurements and / or to undergo multiple processes (such as a measurement process, a regeneration process) without being moved and / or with the use of a relatively small number of moving parts.

[0024] It should also be understood that the methods described herein may also include performing measurements while the fluid and / or a sample of the fluid is stationary. As an example, the method may include flowing the fluid and / or a sample of the fluid to a location where it contacts the probe and then performing the measurement while the fluid and / or a sample of the fluid is stationary. Thereafter, the fluid and / or a sample of the fluid may be flowed such that it no longer contacts the probe. In one or more of the methods described herein, there may be no fluid flow. As described above, in some aspects, an apparatus is provided. In some aspects, a system including the apparatus is provided. An overview of some exemplary apparatuses is shown below.

[0025] FIG. 1 depicts an example of an instrument. In FIG. 1, instrument 100 includes a probe 102 and a detector 104, such as an optical detector. As described in more detail below, the probe may have one or more properties that enable and / or facilitate immobilizing one or more analytes thereon. The detector may be configured to detect a signal. If the detector is an optical detector, it may be configured to detect an optical signal, such as interference. In some embodiments, the instrument includes a detector coupled to the probe, such as an optical detector optically coupled to the probe. One such embodiment is shown in FIG. 2, which depicts an instrument 200 in which probe 202 is optically coupled to optical detector 204 by an optical cable 206. The optical cable may transmit light from the probe to the optical detector. In some embodiments, the instrument includes an optically transparent probe, and the optical signal to be detected passes through the probe and through an optical cable positioned on the side of the probe opposite the side on which the optical signal is generated, and is transmitted to the optical detector.

[0026] In some embodiments, the instrument includes a plurality of probes and / or detectors (e.g., a plurality of optical detectors) (not shown). In such embodiments, it is also possible for each detector (e.g., an optical detector) to be associated with (e.g., optically coupled to) a different probe, or for two or more probes to be coupled to (e.g., optically coupled to) a common detector (e.g., a common optical detector). It is also possible for the instrument to be configured such that one or more probes are reversibly associated with (e.g., optically coupled to) a detector (e.g., an optical detector) (also not shown). In such embodiments, a detector (e.g., a single optical detector) may be capable of, and / or may be so configured as to, be reversibly associated with (e.g., optically coupled to) two or more probes.

[0027] In some embodiments, the instrument includes one or more components in addition to and / or instead of the probe and the detector (e.g., an optical detector). As an example, in some embodiments, the instrument includes a light source (e.g., in addition to the probe, in addition to the probe and the optical detector). FIG. 3 shows an example of an instrument that includes a light source. In FIG. 3, instrument 300 includes light source 308. The light source may be capable of illuminating and / or configured to illuminate one or more portions of the instrument, such as the probe. In some embodiments, the light source is capable of supplying and / or is configured to supply light to the probe that is transmitted through the probe. The light source may supply light to the probe via an optical cable. As an example, in some embodiments, an optical cable couples the light source to the probe (e.g., may transmit light from the light source to the probe). FIG. 4 shows one non-limiting example of such an instrument. In FIG. 4, light source 408 is optically coupled to probe 402 by optical cable 410.

[0028] In some embodiments, the instrument includes a plurality of light sources (not shown). In such embodiments, it is possible for each light source to be associated with (e.g., optically coupled to) a different probe, or for two or more probes to be associated with (e.g., optically coupled to) a common light source. It is also possible for the instrument to be configured such that one or more probes are reversibly optically coupled to a light source (also not shown). In such embodiments, a single light source may be capable of and / or may be configured to be reversibly associated with two or more probes. This may be achieved, for example, by an optical switch configured to switch which probe the light source is associated with.

[0029] As another example, in some embodiments, the instrument includes a housing in which a probe and / or a detector (e.g., an optical detector) is positioned. FIG. 5 shows an example of an instrument 500 that includes a housing 512 in which the probe is positioned but the detector (e.g., an optical detector) is not positioned. The housing may and / or may be configured to contain one or more fluids and / or samples of fluids, such as one or more fluids contacted by the probe and / or a sample of one or more fluids contacted by the probe. In some embodiments, the probe is in contact with, capable of contacting, and / or configured to contact a fluid and / or a sample of a fluid positioned within the housing. The fluid may flow into the housing through an inlet and / or out of the housing through an outlet. FIG. 6 depicts an instrument 600 that includes an inlet 614 and an outlet 616. In some embodiments, the fluid flows into the inlet (and, in some embodiments, into the housing), past the probe, and out of the outlet (and, in some embodiments, out of the housing).

[0030] Some housings may also mechanically support and / or position the probe at a location where the probe will contact the fluid and / or sample of fluid. As an example, in some embodiments, the housing includes epoxy that provides strong support to the probe and / or protects the probe from damage. In some embodiments, the housing includes an opening into which the probe may be inserted. Additionally or alternatively, it is also possible for the probe to be mechanically coupled to the housing (e.g., by use of a clamp).

[0031] In some embodiments, the housing may form part of a microfluidic chip and / or part of a manifold. When the housing forms part of a manifold, the manifold may be configured to supply fluid from a fluid source to the probe, may be configured to supply fluid to the probe, and / or may be capable of supplying fluid to the probe. In some embodiments, the housing forms part of a manifold that includes one or more microfluidic channels. A probe positioned within such a housing may be in fluid communication with the microfluidic channel. The microfluidic channels present within the manifolds described herein may have various suitable shapes. In some embodiments, the manifold includes straight microfluidic channels. It is also possible for the manifold to include microfluidic channels that include steps and / or bends. One non-limiting example of such a microfluidic channel is shown in FIG. 7. FIG. 7 depicts an instrument 700 that includes a probe 702 and a microfluidic channel 718. The microfluidic channel shown in FIG. 7 includes a step 720. Some microfluidic channels may be perpendicular to the optical axis of the probe that is in fluid communication with it.

[0032] The manifold may further include one or more components that facilitate fluid communication between one or more instrument components and the manifold. By way of two examples, the manifold may further include openings and / or tube connectors (e.g., to facilitate fluid communication between a fluid source and the manifold, to facilitate fluid communication between a valve and the manifold). By way of one example, the manifold may include an O-ring and / or may be employed in conjunction with an O-ring (e.g., to seal the surface between the manifold and the probe). By way of a third example, the manifold may further include a luer connection (e.g., to facilitate a mechanical connection between the probe and the manifold).

[0033] The manifold may be formed from a variety of suitable materials such as plastics (e.g., molded plastics, machined plastics), and / or metals (e.g., steels such as stainless steel). In some embodiments, the manifold includes two plastic pieces attached to each other (e.g., via an adhesive, via double-sided tape). One or more microfluidic channels may be formed in the upper plastic piece. The bottom plastic piece may assist in insulating the microfluidic channels.

[0034] Another example of an additional component that may be included in the devices described herein is a fluid source. The fluid source may be associated with, capable of being associated with, and / or may be associated with the devices described herein. As an example, the fluid source may be configured to provide a fluid (and / or one or more samples thereof) to the housing and / or to the probe (e.g., they may be positioned upstream of the housing and / or the probe). As another example, the fluid source may be in fluid communication with, capable of being in fluid communication with, and / or may be configured to be in fluid communication with the interior of the probe and / or the housing. The association, provision, and / or fluid communication of the fluid may occur via an inlet. As an example, as shown in FIG. 8, the fluid source may be configured to be provided to the probe and / or to the interior of the housing in which the probe is positioned via an inlet. In FIG. 8, the fluid source 822 is in fluid communication with the inlet 814 via a conduit 824. A variety of suitable conduits may be employed, one non-limiting example of which is a tube. In some embodiments, the tube may be relatively short. This may facilitate rapid analysis of the fluid and / or fluid sample supplied to the probe and / or the housing, and / or may reduce the amount of fluid required to supply the probe.

[0035] In some embodiments, the instrument includes a plurality of fluid sources (e.g., two or more fluid sources). As an example, the instrument may include two sources of different fluids and / or two sources of the same fluid. In such embodiments, the instrument may include a plurality of inlets (e.g., two or more inlets). As an example, the instrument may include an inlet for each fluid source and / or for each type of fluid source. In some embodiments, the instrument includes exactly one inlet and / or more fluid sources than inlets. In such embodiments, the fluid source may be reversibly associated with the inlet and / or may be capable of being reversibly associated. Some inlets may be capable of being reversibly associated with a fluid source and / or with two or more fluid sources.

[0036] In some embodiments, the instrument includes a plurality of inlets and a plurality of probes, and each inlet is in fluid communication with a probe. Such inlets may also be in reversible fluid communication with one or more fluid sources. An instrument including such probes and inlets may be configured to bring the plurality of probes into contact with one or more fluids supplied through the inlet(s) and / or may be capable of bringing them into contact. In some embodiments, the instrument includes a plurality of probes and a plurality of inlets, and the instrument is configured to alternately bring each probe into contact with a different fluid, and each fluid is alternately supplied by an inlet that is in fluid communication with that probe. The instrument may also be configured to alternately bring each probe into contact with a different fluid, and each fluid is supplied by a different inlet that is in fluid communication with that probe.

[0037] Similarly, the instrument can include one outlet or can include two or more outlets. The outlet(s) can be associated with a location where the probe and / or housing is configured to discharge fluid and / or is capable of discharging fluid (e.g., a location downstream of the outlet). As an example, the outlet can place the interior of the probe and / or housing in fluid communication with such a location. As an example, in some embodiments, the outlet places the interior of the probe and / or housing in fluid communication with a waste container (e.g., a waste container downstream from the outlet). As another example, in some embodiments, the outlet places the interior of the probe and / or housing in fluid communication with a container in which fluid can be stored (e.g., as a final product, for further processing). As a third example, in some embodiments, the outlet places the interior of the probe and / or housing in fluid communication with a source of fluid. In such embodiments, the instrument can serve to recirculate fluid back to the original source after contact with the probe. As a fourth example, in some embodiments, the outlet places the interior of the probe and / or housing in fluid communication with an additional instrument (e.g., one that may further process and / or analyze the fluid). It is also possible for the outlet to be reversibly associated with and / or capable of being reversibly associated with one or more locations (e.g., one or more waste containers).

[0038] If the device includes one or more sources of fluid that may be reversibly associated with an inlet and / or two or more locations that may be reversibly associated with an outlet, the use of a valve may facilitate the reversible association. As an example, the device may include a valve configured to switch between a plurality (e.g., two or more, three or more, four or more, five or more, six or more) of positions. Each position (or some positions) may be associated with a source of fluid. As an example, each position may place a source of fluid in fluid communication with a probe with which the valve is in fluid communication. By switching the valve to a position associated with a source of fluid, that source of fluid is placed in fluid communication with the probe. By switching the valve from a position associated with a source of fluid, that fluid source may be removed from fluid communication with the probe. By switching the valve between two positions, one source of fluid may be removed from fluid communication with the probe and a different source of fluid may be placed in fluid communication with the probe.

[0039] The valve may be positioned downstream of the source(s) of fluid (e.g., the valve may be capable of placing the probe in fluid communication and / or the source(s) of fluid configured to place the probe in fluid communication may be positioned upstream of the valve), and thus may be positioned upstream of the inlet. In some embodiments, such a valve directly supplies fluid to the probe when in an appropriate position to do so. In other words, when the valve places a source of fluid in fluid communication with the probe, the fluid may flow directly to the probe. While flowing directly from the valve to the probe, the fluid may pass through minimal dilution (or no dilution) and / or mixing with other fluids.

[0040] Figure 9 shows one non-limiting example of an instrument that includes a valve 926 (labeled "selector valve" therein). Figure 9 depicts an instrument that includes three fluid sources (a sample source 932 labeled "sample intake" and "chromatography column", a neutralizing fluid source 928 of a buffer labeled "buffer", and a regeneration fluid source 930 labeled "regeneration", each of which will be described in more detail below), a probe 902 (labeled "BLI sensor"), an optical detector 908 (labeled "BLI optics"), and a waste container 934 (labeled "waste"). Figure 9 also depicts a pump 936 (labeled "peristaltic pump") that is employed to facilitate fluid flow from the housing to the waste container.

[0041] In some embodiments, the instrument includes two or more valves. As an example, in some embodiments, the instrument includes a plurality of valves. Each such valve is switchable between a plurality of positions (as described above by way of example), and each position (or some positions) may place the probe in fluid communication with a fluid source. It is also possible to make some or all of such valves switchable simply between an open position (i.e., a position that places the probe in fluid communication with a fluid source) and a closed position (i.e., a position that does not place the probe in fluid communication with a fluid source).

[0042] Figure 10 shows one non-limiting example of an instrument suitable for use with a plurality of such valves. In Figure 10, inlets 1038, 1040, and 1042 are each positioned upstream of probe 1002. Each such inlet may be in fluid communication with a valve that includes a position that places the probe in fluid communication with a different fluid source (e.g., inlet 1038 may be in fluid communication with a valve that includes a position that places probe 1002 in fluid communication with a sample source, inlet 1040 may be in fluid communication with a valve that includes a position that places probe 1002 in fluid communication with a regeneration fluid source, and / or inlet 1042 may be in fluid communication with a valve that includes a position that places probe 1002 in fluid communication with a neutralization fluid source). Figure 10 also depicts two outlets (outlets 1044 and 1046) that may also be in fluid communication with a valve that includes a position that places probe 802 in fluid communication with a waste container. The plurality of valves are shown in Figure 10 as valves 1048 - 1056.

[0043] Fluid flow may also flow directly from the valves, across the probe, and then to one of two waste containers, through the instrument shown in Figure 10. Thus, it should be understood that fluid may flow in both directions through the instrument shown in Figure 10 (e.g., from valve 1040 or 1042 to waste container 1044, from valve 1038 to waste container 1046). Additionally, as can be understood from Figure 10, fluid may flow directly from the valves across the probe without undergoing dilution or mixing. In some embodiments, such flow may push a plug of another type of fluid (e.g., fluid supplied from different valves) downstream and / or to the waste container without substantial (or any) mixing between two types of fluids.

[0044] Figures 9 and 10 depict an instrument suitable for use with a valve positioned upstream of the inlet, although it should also be noted that the instrument can include a valve that is upstream of the probe but positioned downstream of the inlet. In other words, the instrument can include a housing that includes an inlet(s), and one or more valves may be positioned between the inlet(s) and the probe. In some such embodiments, fluid flows into the housing through the inlet, but contacts the probe only when the valve is in a position that places the inlet, through which the fluid enters the housing, in fluid communication with the probe.

[0045] In some embodiments, the instrument includes a probe, an inlet, and an outlet, which are arranged in a manner that aids in preventing the formation of air bubbles. It is also possible for an article to include an instrument that includes a probe, an inlet, and an outlet in this manner. One such design is shown in FIGS. 11 and 12. FIG. 11 depicts an article that includes a probe, an inlet, an outlet, and a housing. In FIG. 11, housing 1112 surrounds probe 1102. In addition, inlet 1114 is in fluid communication with probe 1102 and is configured to provide fluid to the probe. Similarly, outlet 1116 is in fluid communication with probe 1102 and is configured to remove fluid from the probe. As also shown in FIG. 11, probe 1102 includes an optical axis 1158, the inlet includes an inlet flow axis 1160, and the outlet includes an outlet flow axis 1162. The optical axis may be aligned along a path through which light is transmitted through the probe and / or along the longest major axis of the probe. The inlet flow axis may be aligned along the direction in which fluid flows through the inlet and / or along the longest major axis of the inlet. The outlet flow axis may be aligned along the direction in which fluid flows through the inlet and / or along the longest major axis of the outlet. In some embodiments, as in the embodiment shown in FIG. 11, the probe, the inlet, and the outlet may be arranged such that the optical axis, the inlet axis, and the outlet axis are positioned in a common plane. It is also possible to orient the optical axis of the probe substantially perpendicular.

[0046] The pump is a further example of additional components that can be included in the devices described herein. As described above, FIG. 9 depicts an example of a device that includes a pump. The pump may be employed at various suitable locations (by way of example, upstream of the probe, downstream of the probe, upstream of the inlet, downstream of the outlet) to facilitate the flow of fluid through the device. One non-limiting example of a suitable type of pump is a peristaltic pump. Some pumps may have a relatively small size (by way of example, they may be miniature pumps).

[0047] Another example of an additional component that can be included in the devices described herein is a temperature control system. The temperature control system may be associated with one or more parts (singular or plural) of the device, such as the probe, the manifold, the tubes fluidly connecting the valves to the inlet and / or the manifold, the tubes fluidly connecting the valves to the fluid source, and / or a detector (by way of example, an optical detector). It is also possible for the device to include two or more temperature control systems, each of which is associated with a different part (singular or plural) of the device. The temperature control system may be employed to heat and / or cool the associated part (singular or plural) of the device. Without wishing to be bound by any particular theory, it is thought that cooling a fluid (and / or a sample of the fluid) may advantageously increase the solubility of gases therein and reduce the formation of gas bubbles. Similarly, cooling a detector (by way of example, an optical detector) is thought to assist it in functioning correctly. Non-limiting examples of suitable temperature control systems include cooling fans, heat sinks, and insulation materials.

[0048] Further examples of additional components that may be included in the instrument described herein are filters such as purification filters and / or degassing filters. In some embodiments, one or more filters are positioned between the sample source and the probe (e.g., between the sample source and the inlet, between the sample source and the valve, between the inlet and the probe, between the valve and the probe). The filter may remove components of the fluid sample that would otherwise interact undesirably with the probe, such as impurities (e.g., in the case of a purification filter) and / or gases (e.g., in the case of a degassing filter). Non-limiting examples of impurities include particulates such as cell microparticles. An example of a purification filter is a filter suitable for crossflow filtration.

[0049] When a sample of pressurized fluid is supplied from an additional pressurized instrument to the instrument, gas may undesirably be present. A pressure drop as the fluid sample flows into the instrument may result in the formation of bubbles (e.g., microbubbles), which, if they contact the probe, may undesirably affect the generated signal (e.g., an optical signal). A degassing filter may employ atmospheric pressure and / or a pressure below atmospheric pressure to degas the fluid sample.

[0050] In some embodiments, the instrument includes a degassing device in addition to the degassing filter. As an example, in some embodiments, the instrument includes a vacuum degassing device, an ultrasonic degassing device, a heater configured to cause degassing, and / or a cooler configured to perform degassing. A vacuum degassing device may include a gas permeable membrane and a vacuum source positioned on the permeable membrane side opposite the fluid to be degassed. An ultrasonic degassing device includes a source of ultrasonic waves and a gas sink. A heater configured to cause degassing may include a heating element and a gas sink.

[0051] In some embodiments, the fluid (and / or a sample of the fluid) passes through a degassing device at a relatively high temperature (e.g., the temperature received from the source of the sample) and is then cooled prior to being contacted with the probe. This may degas at a temperature where the solubility of the gas is low and then advantageously enable contact between the fluid and the probe at a temperature where the solubility of the gas is high. The control device and the computer are further examples of additional components that may be included in the instruments described herein. In some embodiments, the control device may deliver power and / or instructions to one or more other instrument components (e.g., a light source, a valve, a pump, a detector, an optical detector). Such instructions may be provided periodically (e.g., according to a preselected schedule) and / or upon request (e.g., by an operator). In some embodiments, the control device delivers instructions regarding the flow of the fluid (e.g., start the flow of the fluid, stop the flow of the fluid, modify the rate at which the fluid flows, change where the fluid flows to, change where the fluid flows from, switch the position of a valve). In some embodiments, the control device delivers instructions regarding the detection of a signal (e.g., an optical signal) (e.g., turn on a detector and / or an optical detector, turn off a detector and / or an optical detector, adjust how a detector and / or an optical detector performs detection and / or optical detection). The computer may also deliver instructions to the control device and / or receive one or more signals from the control device. The control device and the computer may communicate with each other and / or with other instrument components in various ways, including via a USB cable and / or via Ethernet communication.

[0052] In some embodiments, the instrument is surrounded within a housing. As an example, in some embodiments, the instrument is surrounded within a housing that includes a base, a cover, and / or side walls. Some or all of these components may be formed of and / or include a light shield. In some embodiments, the housing includes a connector (e.g., an SMA connector such as an SMA905 connector) that enables an optical connection to be formed between one or more portions of the instrument and a device external to the instrument. Such portions of the instrument may include a light source, an optical detector, and / or a probe. FIG. 13 depicts one non-limiting example of an instrument surrounded by a housing.

[0053] In some embodiments, the instrument includes two or more parts surrounded by separate housings. An example of such an instrument is shown in FIG. 14. In FIG. 14, the instrument includes a first housing that surrounds a computer, a lamp, and a spectrometer. The first housing also includes a display and supports three fluid sources. The instrument shown in FIG. 14 further includes a second housing that supports a manifold and a probe. The first and second housings shown in FIG. 14 are in fluid communication, optical communication, and electrical communication, respectively, via tubes, one or more optical cables, and one or more electrical cables. The tubes and cables shown in FIG. 14 are positioned within a flexible sleeve. In some embodiments, the instrument includes first and second housings that each include a connector (e.g., an SMA connector such as an SMA905 connector) configured to form an optical connection. Such an instrument may further include an optical cable optically connected to both housings via these optical connectors. It is also possible for an instrument that includes two or more housings to include a first housing that differs in one or more ways from that shown in FIG. 14. As an example, in some embodiments, the instrument includes, in addition to or in place of, a first housing that surrounds electronics, one or more pumps, a power source, one or more light sources, and / or one or more detectors (e.g., one or more optical detectors). As another example, the instrument may include a first housing that includes a number of fluid sources other than three (e.g., exactly one fluid source, exactly two fluid sources, four or more fluid sources).

[0054] FIG. 14 also depicts any mechanical support for the second housing. In some embodiments, the instrument includes a second housing that surrounds a manifold, one or more probes, one or more valves, a temperature control system, one or more filters (e.g., a degassing filter, a purification filter), and / or one or more pumps. It is also possible for one or more (or all) of these components to be positioned outside of any housing present within the instrument.

[0055] Some of the devices described herein may be related to other devices. It is also possible for a device to be a source of a fluid supply and / or a source of one or more fluid samples. An example of a device is shown in FIG. 19, where the device is related to a chromatography column (labeled "chromatography column" and designated by reference numeral 932). In some embodiments, the devices described herein are in fluid communication with additional devices. Fluid communication may be accomplished in the manner described elsewhere in this specification for other fluid supply sources and / or fluid sample supply sources, such as tubes. Association may include on-line and / or at-line association.

[0056] In some embodiments, the devices described herein are capable of being related to, configured to be related to, and / or related to two or more additional devices. Similarly, such devices may be capable of being in fluid communication with, configured to be in fluid communication with, and / or in fluid communication with two or more additional devices. In some embodiments, a system includes both a device described herein and additional devices. The devices (by way of example, a device and additional devices) may be of the same type (by way of example, two or more bioreactors) or of different types (by way of example, a chromatography system and a bioreactor). The association may be reversible. In some such embodiments, a reversible association may be facilitated by the use of valves to switch between different positions related to different additional devices and / or the use of valves to select additional devices that are in fluid communication with and / or supply fluid to a device that includes a probe.

[0057] Some additional instruments may be capable of supplying fluid (and / or a sample of one or more fluids) to an instrument that includes a probe and / or may be configured to do so. Some methods may include supplying fluid (and / or a sample of one or more fluids) to an instrument that includes a probe. The fluid may be supplied to the instrument (and / or brought into contact with the probe) as an output from an additional instrument. As an example, the fluid may have the same composition as that within the additional instrument. It is also possible for the fluid as an output from the additional instrument to have a composition that is slightly different relative to that within the additional instrument (for example, the concentration of each component in the fluid supplied to the instrument and / or in contact with the probe may differ from the concentration in the fluid within the additional instrument by 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, or 0.1% or less; the concentration of each component in the fluid within the additional instrument may differ from the concentration in the fluid supplied to the instrument and / or in contact with the probe by 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, or 0.1% or less). In some embodiments, the fluid supplied to the instrument as an output from the additional instrument and / or brought into contact with the probe will not undergo any filtration step, any purification step, any centrifugation step, sterilization step, and / or any other step that would remove one or more components from the fluid and / or cause a chemical or biological reaction within the fluid after being removed from the additional instrument. In some embodiments, the fluid is supplied from the additional instrument to the instrument in an automated manner. As an example, the fluid may be supplied from the additional instrument to the instrument without any input by an operator and / or without requiring the operator to perform any steps.

[0058] In some embodiments, the fluid flowing out of the additional instrument is divided into a plurality of samples prior to flowing into an inlet. The additional instrument may be configured to divide the fluid flowing out therefrom into a plurality of samples, or the instrument including the probe may be configured to perform this division. As described above, some aspects relate to methods. Some methods may be implemented, in part and / or in whole, by one or more of the apparatuses described herein. Additionally, some apparatuses may be implemented in and / or configured to implement one or more of the methods described herein. An overview of some exemplary methods and steps is provided below.

[0059] In some aspects, a method includes performing one or more steps to determine the concentration of an analyte in a fluid and / or a sample of the fluid. The fluid may be provided by a bioprocessing system. In some aspects, such a method further includes sending an instruction to the bioprocessing system based on this concentration. Some methods include performing one or more steps to determine the affinity of an analyte for a probe and / or a species immobilized on the probe. An example of such a step is contacting the probe with a fluid containing the analyte, according to. The analyte may be present in the fluid at a particular concentration (e.g., a concentration of 0 M, a concentration higher than 0 M). Contact between the fluid and / or sample of the fluid and the probe may cause some or all of the analyte present in the fluid and / or sample of the fluid to become immobilized on the probe. The amount of analyte immobilized on the probe may be affected by the amount of analyte present in the fluid and / or sample of the fluid contacting the probe, the amount of analyte already immobilized on the probe, and / or the length of time the fluid and / or sample of the fluid and the probe are in contact.

[0060] The probe may be contacted with the fluid and a sample of the fluid in various suitable ways. In some embodiments, the probe is contacted with the fluid (and / or a sample thereof) by being positioned within a housing into which the fluid (and / or a sample thereof) is introduced in an amount such that contact occurs between the fluid (and / or a sample thereof) and the probe. In some embodiments, the probe is contacted with the fluid (and / or a sample thereof) by being positioned where the fluid (and / or a sample thereof) flows. The contact may be made with the entire probe or with a portion and not with other portions. As an example, in some embodiments, a portion of the probe to which one or more reagents (e.g., one or more reagents to which an analyte may be immobilized) are immobilized is contacted with the fluid and / or a sample of the fluid. As another example, in some embodiments, a portion of the probe distal to the portion of the probe that is in contact with the optical cable is contacted with the fluid and / or a sample of the fluid. As a third example, in some embodiments, a surface of the probe that is perpendicular to the optical axis of the probe (e.g., the bottommost such surface that is perpendicular to the optical axis of the probe and opposite the surface that contacts the optical cable) is contacted with the fluid and / or a sample of the fluid.

[0061] Contact between the fluid (and / or a sample thereof) and the probe may occur over various suitable periods of time. In some embodiments, the time may be relatively short (e.g., from a few seconds to a few minutes). There is also a possibility that the time for which the probe is in contact with the fluid may be relatively long (e.g., up to 1 hour, several hours, or more). Further details regarding the range of times for which the probe will be in contact with the fluid are provided below. The concentration of an analyte in a fluid (and / or a sample thereof) may be relatively constant over the period of contact with the probe, or may vary over that period. As an example of the latter, in some embodiments, an analyte immobilized on the probe may be removed from the fluid (and / or a sample thereof) when immobilized on the probe. As another example of the latter, in some embodiments, the fluid (and / or a sample thereof) may flow over the probe for a period of time. Different portions of the fluid (and / or a sample thereof) may have different concentrations of the analyte therein, such that the concentration of the analyte in the fluid contacting the probe may change as different portions of the fluid (and / or a sample thereof) continuously flow over the probe. In some embodiments, a relatively small amount of the analyte is immobilized on the probe compared to the total amount of the analyte in the fluid (and / or a sample thereof) contacting the probe. In such embodiments, the concentration of the analyte in the fluid may be relatively constant over the period that the probe is in contact with the fluid. Similarly, the fluid (and / or a sample thereof) flowing over the probe may have a relatively uniform concentration of the analyte therein.

[0062] In some embodiments, the method includes contacting the probe with a plurality of fluids and / or samples of a plurality of fluids. The samples of the plurality of fluids may be supplied by a sample source. The probe may contact the fluids sequentially and / or in an alternating manner. As an example, in some embodiments, the probe is contacted with samples of a plurality of fluids (e.g., supplied by a sample source), and between samples of the plurality of fluids, the probe is contacted with one or more fluids that are not fluid samples. The fluids that are not fluid samples may assist in removing analyte (e.g., analyte derived from the sample) immobilized on the probe during exposure to different fluid samples.

[0063] In some embodiments, the fluid to which the probe is exposed may be controlled by the position of a valve with which the probe is in fluid communication and / or by opening the valve with which the probe is in fluid communication. As an example, in some embodiments, the valve may be switched between positions that place the probe in fluid communication with different fluid sources. By switching such a valve from one position to another, one fluid source may be removed from fluid communication with the probe and another fluid source may be placed in fluid communication with the probe. As an example, in some embodiments, the valve may be switched to remove a sample source from fluid communication with the probe and place a regeneration fluid source in fluid communication with the probe. As another example, in some embodiments, the valve may be switched to remove a regeneration fluid source from fluid communication with the probe and place a neutralization fluid source in fluid communication with the probe. As a third example, in some embodiments, the valve may be switched to remove a neutralization fluid source from fluid communication with the probe and place a sample source in fluid communication with the probe.

[0064] Some of the valves described herein may be switched between an open position and a closed position. By switching the valve from the closed position to the open position, a probe with which the valve is in fluid communication may be placed in fluid communication with a fluid source. By switching the valve from the open position to the closed position, a probe with which the valve is in fluid communication may be removed from fluid communication with a fluid source. Some methods may include placing a fluid source in fluid communication with a probe and removing it from fluid communication, respectively, by opening one or more valves and closing one or more valves.

[0065] Another example of a step that may be performed during the methods described herein is detecting a signal, such as an optical signal. The signal (e.g., an optical signal) may be associated with a fluid (and / or a sample of the fluid) that contacts the probe. As an example, the signal (e.g., an optical signal) may be detected when the fluid (and / or a sample of the fluid) is in contact with the probe. As another example, the signal may be an optical signal that includes interference between light reflected from two or more interfaces, such as an interface within the probe (e.g., between an inner portion of the probe and a coating disposed thereon), an interface between the probe and an analyte that was present in an initial fluid (and / or a sample of the fluid) immobilized on the probe, and / or an interface between the analyte and the external environment of the probe (e.g., a fluid, a sample of the fluid). In some embodiments, the signal (e.g., an optical signal) is affected by the amount of analyte immobilized on the probe. As a result, analysis of the signal (e.g., an optical signal) may be employed to determine the amount of analyte immobilized on the probe and / or the concentration of the analyte present in the fluid and / or a sample of the fluid that contacts the probe.

[0066] An optical signal may include light, and / or interference of light (e.g., interference between light transmitted through optical paths having different optical thicknesses but supplied by a common light source), or a state where such interference does not exist. As two examples, an optical signal may include interference between light reflected from two different interfaces (e.g., the interface between the inner part of the probe and a coating disposed on the inner part of the probe, the interface between the analyte and the probe, the interface between the analyte and the external environment of the probe, the interface at the end of the probe) related to the probe and / or the analyte immobilized on the probe, or a state where such interference does not exist. The light reflected from the interface may be supplied from the light source to the probe. As described above, the light source is optically coupled to the probe, whereby light is transmitted from the light source beyond the probe (e.g., parallel to the optical axis of the probe). When reaching the end of the probe, the light may be transmitted out of the probe and / or reflected from the interface between the probe and the external environment of the probe (and / or from the end of the probe). If there is any immobilized analyte on the probe, some light may be reflected from the interface between the probe and the analyte, and / or some light may be transmitted through the analyte. The analyte may change the effective refractive index of the tip and / or change the effective optical path length of the light transmitted through the probe. The light transmitted through the analyte encounters the environment with which the analyte is in contact (e.g., the fluid in contact with the probe, a sample of the fluid in contact with the probe). Some of the light encountering this environment may be transmitted into the environment with which the analyte is in contact (e.g., the environment external to the probe), and / or may be reflected from the interface between the environment and the analyte.

[0067] It is also possible that the probe described herein has one or more internal interfaces where reflection may occur. As an example, some probes may include one or more internal interfaces where reflection may occur, such as the interface between a coating and the interior of the probe on which the coating is disposed. Light reflected from one or more (and / or any additional locations) of the locations described above may also travel back through the probe. When light is reflected from multiple locations (e.g., at the interface between the probe and the analyte immobilized on the probe, at the interface between the analyte immobilized on the probe and the environment external to the probe, at the interface between a coating disposed within the probe and the analyte immobilized on the probe, at the interface between the interior of the probe and the coating disposed thereon, from the end of the probe), such light may interfere with each other. The interference of light may cause the intensity of the interfering light to increase or decrease depending on whether the interference is constructive or destructive, which may depend on the phase shift between the multiple light sources that interfere. The phase shift may depend on the difference in the path lengths that the light has traveled prior to interference, the refractive index(es) of the material(s) that the light has passed through prior to interference, and / or the wavelength of the light. As a result, by obtaining information about the intensity of the interfering light over various wavelengths, information about the thickness of the layer containing the analyte immobilized on the probe and / or the refractive index of such layer may be provided. This information may be employed to determine the amount of analyte immobilized on the probe.

[0068] FIG. 15 schematically depicts an example of a process for detecting the amount of analyte immobilized on a probe. As shown in FIG. 15, light traveling down the probe may be reflected from the interface between the coating disposed within the probe and from the interface between the analyte disposed on the probe and the external environment of the probe. The phase shift between these two sources of reflected light depends on the amount of analyte immobilized on the probe and the wavelength of the reflected light, which may affect the intensity of the reflected light that is measured. Thus, analysis of the intensity of the reflected light as a function of wavelength may be employed to determine the amount of analyte immobilized on the probe.

[0069] The optical signal can also include signals other than optical interference. As an example, in some embodiments, the optical signal includes fluorescence emitted from species immobilized on the probe and / or fluorescence generated from species immobilized on the probe (e.g., via reaction with species present in the fluid contacting the probe). As another example, in some embodiments, the optical signal includes reflected light. As an example, the amount of light reflected at different angles, different angular ranges, and / or a limited angular range may be detected. The intensity of such light at one or more specific angles, the intensity of such light over one or more different angular ranges, and / or the angle(s) at which the intensity of such light is lower may be employed to detect the angle at which surface plasmon resonance occurs. This angle may be affected by the immobilization of the analyte on the probe.

[0070] In some embodiments, a mechanical signal is detected. As one non-limiting example, in some embodiments, the variation in the resonance of a quartz resonator is the signal that is detected. The measurement of the amount of analyte immobilized on the probe may be performed with respect to a signal (e.g., an optical signal) detected over time. The period during which the signal (e.g., an optical signal) may be detected may be the same as the period during which the probe is contacted with the fluid (and / or its sample), or a different period (e.g., a subset of that period). The period may be the period during which the analyte is removed from the probe (e.g., during a regeneration step, during a neutralization step). Detecting a signal (e.g., an optical signal) over time may include detecting a single value of the signal (e.g., an optical signal) from a measurement performed over a period (e.g., detecting a value that is the average of the signal and / or optical signal over that period), and / or detecting multiple values of the signal and / or optical signal from different (overlapping or non-overlapping) measurements occurring over different periods.

[0071] In some embodiments, detecting a signal (e.g., an optical signal) over time includes detecting its variations over time. Variations include increases, decreases, or the absence of variations. In some embodiments, the variations include the first derivative of the signal (e.g., an optical signal). The variations of a signal (e.g., an optical signal) over a period may be determined from a plurality of measurements that result in a plurality of values of the signal (e.g., an optical signal) over that period, taken for a single signal (e.g., an optical signal) over that period. The period during which the signal (e.g., an optical signal) is measured may include various suitable points in time during immobilization of the analyte on the probe and / or removal of the analyte from the probe. By way of example, the variations may be measured when the amount of analyte immobilized on the probe is at one or more specific percentages of the amount of analyte immobilized on the probe in a steady state, when the amount of analyte immobilized on the probe is at a steady state value, or at any point in time therebetween, at the initial contact of the probe with the fluid containing the analyte and / or a sample of the fluid, and at the initial removal of the contact of the probe with the fluid containing the analyte and / or a sample of the fluid.

[0072] The point in time at which the variations of the signal (e.g., an optical signal) are measured may be selected as needed. In some embodiments, the variations of the signal (e.g., an optical signal) are measured when the amount of analyte immobilized on the probe is 0% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more of the amount of analyte immobilized on the probe in a steady state. In some embodiments, the variations of the signal (e.g., an optical signal) are measured when the amount of analyte immobilized on the probe is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amount of analyte immobilized on the probe in a steady state. Combinations of the ranges described above are also possible (e.g., 0% or more and 50% or less, 0% or more and 10% or less, or 0% or more and 5% or less). Other ranges are also possible.

[0073] As described above, the value of a signal (e.g., an optical signal) may indicate the amount and / or type of analyte immobilized on the probe. At equilibrium, the amount of analyte immobilized on the probe may indicate its affinity for the probe and / or species immobilized on the probe. Additionally, the variation of a signal (e.g., an optical signal) over time may indicate the rate at which the analyte becomes immobilized on the probe. The rate at which the analyte becomes immobilized on the probe may depend on the amount of analyte in the fluid to which the probe is exposed, and / or the interaction between the analyte and the probe. As an example of the latter, the rate at which the analyte becomes immobilized on the probe may depend on the affinity of the analyte for the probe (and / or its interfacial chemistry, and / or the reagent immobilized on the probe), and / or the rate at which the analyte binds to the probe (and / or its interfacial chemistry, and / or the reagent immobilized on the probe). As another example of the latter, the rate at which the analyte is removed from the probe when contacted with a fluid other than the fluid containing the analyte (e.g., a sample of a different fluid, a regeneration fluid, a neutralization fluid) may also indicate the affinity of the analyte for the probe (and / or its interfacial chemistry, and / or the reagent immobilized on the probe), and / or the rate at which the analyte binds to the probe (and / or its interfacial chemistry, and / or the reagent immobilized on the probe). As a result, the methods described herein may be suitable for determining the affinity of an analyte for a probe and / or species immobilized on the probe. The affinity of an analyte for a probe may be parameterized by its binding constant and / or its dissociation constant, as described in more detail below. While not wishing to be bound by any particular theory, it is believed that any given analyte may bind most rapidly to the probe upon initial contact of the fluid (and / or its sample) containing the analyte with the probe. As a result, it is also believed that measuring the variation of a signal (e.g., an optical signal) may provide more accurate information, and / or may provide information more rapidly, if the period during which the variation is measured includes the initial contact of the fluid (and / or its sample) with the probe.

[0074] Fluctuations in a signal (e.g., an optical signal) may indicate a change in the amount of analyte immobilized on the probe, which itself may be a measurement taken before the amount of analyte immobilized on the probe reaches a steady-state value, and / or a changing concentration of analyte in the fluid contacting the probe and / or in a sample of the fluid contacting the probe. In the former scenario, the rate at which the analyte immobilized on the probe approaches its steady-state value may vary with the concentration of analyte in the fluid (and / or sample of fluid) with which the probe is contacted, and thus may be employed to assess the concentration of analyte in that fluid and / or sample of fluid. In some embodiments, the variation in the rate at which the analyte immobilized on the probe approaches its steady-state value varies more than the amount of analyte immobilized on the probe at steady state. As a result, in some embodiments, measuring the rate at which the analyte becomes immobilized on the probe provides a more sensitive and / or more rapid way of determining the concentration of analyte than measuring the amount of analyte immobilized on the probe at the final steady-state value. As described above, it is possible for the fluid and / or sample of fluid contacting the probe to have a concentration in the analyte that changes over time. In such embodiments, a particular determination of the analyte concentration may also be made by determining the average amount of analyte in the fluid contacting the probe during the period in which the determination is made. Multiple determinations of the analyte concentration made on such fluid and / or sample of fluid may result in different analyte concentrations indicative of different analyte concentrations in the fluid and / or sample of fluid over time.

[0075] In some embodiments, signals and / or fluctuations in the signals are measured continuously. This is accomplished by continuously contacting the fluid supplied to the instrument with the probe therein and measuring the signal during the measurement of the continuous signal. In some embodiments, as described in more detail elsewhere herein, multiple probes may be employed to implement such a method. As an example, a first probe is employed to measure a first signal and / or a first fluctuation in the signal, while a second probe is provided for doing so. During the measurement of the first signal and / or the first signal fluctuation, the fluid may be contacted with the second probe and a second signal and / or a second signal fluctuation may be measured. Such a process may be employed using multiple probes such that the fluid supplied by the instrument (e.g., a plurality of its samples) is continuously contacted with the probes and its signal and / or fluctuations are continuously measured. In such embodiments, the fluid may be supplied in an on-line and / or at-line manner.

[0076] In some embodiments, a detected signal (e.g., an optical signal) is compared to a model signal profile. The model signal profile may be related to a desired and / or expected outcome (e.g., an expected signal profile related to the presence of an analyte in a fluid in contact with a probe; an expected signal profile at an expected time such as at the breakthrough of a column and / or chromatography medium with which the probe is in fluid communication to which the analyte is related), or an undesired and / or unexpected outcome (e.g., the presence of air bubbles in a fluid in contact with a probe, malfunction of an instrument). Comparing the signal (e.g., an optical signal) to the model signal profile is employed to assess whether the instrument and / or probe is functioning in a normal manner. As an example, if the signal (e.g., an optical signal) does not match the model signal profile related to the expected outcome, it may indicate the presence of air bubbles in the fluid contacted by the probe and / or malfunction of the instrument. On the other hand, if the signal (e.g., an optical signal) matches such a model signal profile, it may indicate that the instrument is functioning properly. As another example, if the signal (e.g., an optical signal) matches the model signal profile related to the presence of air bubbles in the fluid contacted by the probe and / or malfunction of the instrument, it may indicate the presence of such air bubbles and / or malfunction.

[0077] In some embodiments, two or more signals (and / or their variation over time) are detected. The different signals may be related to different samples of a common fluid and / or different fluids. The former may be useful for monitoring changes in one or more properties of the fluid present within an additional instrument to which the fluid is supplied. The latter may be useful for monitoring the properties of fluids present within different additional instruments. The signals may be of the same type or different types, and may be measured by the same probe or different probes. Similarly, the signals may be measured over different periods or the same period. In some embodiments, a first signal is employed to determine a first concentration of a first analyte in a first fluid, and a second signal is employed to determine a second concentration of a second analyte in a second fluid.

[0078] Some of the methods described herein relate to techniques that may be employed to determine the concentration of an analyte in a fluid flowing over a probe and / or in a sample of the fluid. As described elsewhere herein, some instruments include a fluid source, an inlet, an outlet, a pump, and other components that facilitate the flow of fluid (and / or a sample thereof) over the probe. Some of the methods described herein relate to techniques that may be employed to make two or more measurements using a single probe. In some embodiments, an analyte may be immobilized on the probe while the probe is being employed to perform a first measurement. While the analyte remains immobilized on the probe, it may be difficult or impossible to perform another measurement using that probe. Consequently, a method for removing the analyte from the probe may facilitate the use of the probe for multiple measurements.

[0079] In some embodiments, a regeneration fluid may be employed to remove an analyte from a probe. As an example, in some embodiments, the probe may be contacted with a regeneration fluid after (and optionally after a signal associated with the fluid and / or an optical signal has been detected) being contacted with a fluid (and / or a sample thereof) containing the analyte. The regeneration fluid may be configured to cause some or all (e.g., at least a portion) of any analyte immobilized on the probe, such as an analyte immobilized on the probe during contact with the fluid (and / or a sample thereof) containing the analyte and / or during signal (e.g., optical signal) detection, to be detached. The regeneration fluid may cause some or all (e.g., at least a portion) of any analyte immobilized on the probe to be detached in various suitable ways. As an example, in some embodiments, the regeneration fluid is capable of dissolving some or all of the analyte immobilized on the probe and / or is configured to do so. As another example, in some embodiments, the regeneration fluid is capable of causing a chemical reaction that cleaves the bond and / or binding interaction between the analyte and the probe and / or is configured to do so (e.g., reducing the affinity of the analyte for the probe, the interfacial chemistry of the probe, and / or a reagent immobilized on the probe). As a third example, in some embodiments, the regeneration fluid is capable of decomposing the analyte and / or is configured to do so in such a way that the decomposition products dissolve in the regeneration fluid. As a fourth example, in some embodiments, the regeneration fluid includes species that have a higher affinity for the probe (and / or its interfacial chemistry and / or a reagent immobilized thereon) than the analyte and / or are configured to move the analyte away from the probe.

[0080] The regeneration fluid may also perform one or more of the processes described above by causing a conformational change in the analyte and / or a reagent immobilized on a probe to which the analyte binds. The regeneration fluid may cause this change by changing the chemical and / or physical parameters of the fluid to which the probe is exposed, such as its pH, conductivity, and / or temperature. After contact with the regeneration fluid, sufficient amounts of the analyte may be detached from the probe so that the probe can be employed for another measurement. As an example, in some embodiments, the probe is contacted with a first fluid (or a sample of the first fluid) containing the analyte, then with the regeneration fluid, and then with a second fluid (or a second sample of the first fluid, or a sample of the second fluid). A signal (e.g., an optical signal) may be detected during and / or after the probe is contacted with the first and / or second fluid (and / or samples of the first and second fluid).

[0081] It should be understood that it is possible to make measurements on the first and second fluids, but referring to the "first" fluid does not necessarily imply the presence of the "second" fluid or any other fluid. Similarly, referring to the "first" fluid does not exclude the presence of the "second" fluid, the "third" fluid, or additional fluids not explicitly mentioned. It should also be understood that referring to other "first things" (e.g., a sample of the first fluid, a first signal, a first optical signal, a first concentration, a first analyte, etc.) does not imply the presence of a "second" item of the relevant kind, nor does it exclude the presence of a "second" item or additional such items.

[0082] In some embodiments, the probe is contacted alternately with a fresh sample and a regeneration fluid in a plurality of fluid samples. In other words, the process described above may be repeated for a plurality of fluid samples. In some embodiments, the probe is contacted with a plurality of fluids (e.g., a fresh sample and a regeneration fluid, two of the aforementioned fluids and a neutralization fluid) in a repeating cycle. By repeating the process described above, the concentration of an analyte in the fluid can be monitored over time. As an example, a case where signals (e.g., optical signals) associated with some or all of a plurality of samples are detected and the concentration of the analyte in the sample associated with each signal (e.g., optical signal) is determined. This may be desirable when it is expected that the concentration of the analyte in the fluid may change over time and / or may change when the fluid is sampled. In some embodiments, the process described above is performed in such a way that the concentration of the analyte in the fluid is repeatedly determined over a predetermined period, or until the concentration exceeds a predetermined limit (e.g., a predetermined minimum concentration, a predetermined maximum concentration, a predetermined concentration range), and / or until the variation of a signal (e.g., an optical signal) exceeds a predetermined limit (e.g., a predetermined minimum amount, a predetermined maximum amount, a predetermined range).

[0083] Some of the regeneration fluids are suitable for removing the analyte from the probe, but may leave the probe and / or the instrument on which the probe is positioned in a state that is disadvantageous for further measurements. As an example, in some embodiments, the regeneration fluid may have a pH that has an undesirable effect on the analyte and / or one or more species present in the fluid (and / or its sample) in which the analyte is positioned. As another example, in some embodiments, the regeneration fluid may contain species that have an undesirable effect on the analyte and / or one or more species present in the fluid (and / or its sample) in which the analyte is positioned. Such species may be deposited on the probe surface simultaneously with and / or after the analyte is removed from the probe surface. Contacting the subsequent fluid (or a sample of the subsequent fluid) with the probe may expose the fluid (and / or its sample) to such undesirable species. Similarly, contacting the fluid and / or a sample of the fluid with the probe may include flowing the fluid (and / or its sample) over the probe immediately after the regeneration fluid and / or flowing the regeneration fluid away from the probe in a manner such that the fluid (and / or its sample) contacts the regeneration fluid and is exposed to such undesirable species.

[0084] To address the concerns described above, a neutralizing fluid may be employed. In some embodiments, the probe is contacted with the neutralizing fluid after being contacted with the regeneration fluid and / or before being contacted with the second fluid and / or a sample of the second fluid. The neutralizing fluid may be configured to remove any undesirable species deposited on the probe (and / or the housing on which the probe is positioned) from the regeneration fluid (and / or the second fluid and / or a sample of the second fluid) and / or to remove some or all of the regeneration fluid (and / or the second fluid and / or a sample of the second fluid) from the housing on which the probe is positioned. It is also possible for the regeneration fluid to adjust one or more physical and / or chemical properties of the probe (such as pH, conductivity, and / or temperature).

[0085] As described above, FIG. 9 shows one non-limiting example of an embodiment where the instrument includes both a source of neutralizing fluid and a source of regeneration fluid. Also, as shown therein, a valve may be employed upstream of the inlet to a housing containing a probe configured to switch between the source of regeneration fluid and the source of neutralizing fluid (e.g., switch between bringing the source of regeneration fluid into fluid communication with the probe and bringing the source of neutralizing fluid into fluid communication with the probe). The same valve may also be employed to switch between the positions associated with those fluids and one or more positions (singular or plural) associated with one or more sources of additional fluids (e.g., fluids that may include an analyte, fluids to be analyzed within the instrument).

[0086] Some of the methods described herein relate to techniques that may be employed for using multiple probes (e.g., a plurality of probes) together. The probes among the plurality of probes may be arranged in series and / or in parallel. As described above, in some embodiments, it may be desirable for one or more probes to be contacted with a series of fluids (e.g., the fluid to be analyzed and the regeneration fluid, the two aforementioned fluids and the neutralizing fluid, samples of a plurality of fluids to be analyzed, and one or both of the regeneration fluid and the neutralizing fluid). Even if any particular probe is in contact with a fluid that is not the fluid to be analyzed, it may be desirable to analyze the fluid and / or a sample from the fluid. Such measurements may be facilitated by using a plurality of probes.

[0087] In some embodiments, the method includes contacting a first probe with a first series of fluids, at least one of the first series of fluids including a fluid that is not the fluid to be analyzed. While the first probe is being contacted with a fluid that is not the fluid to be analyzed (e.g., a regeneration fluid, a neutralization fluid), the second probe may be contacted with the fluid to be analyzed and / or a sample of the fluid. Similarly, while the second fluid is being contacted with the fluid to be analyzed and / or a sample of the fluid to be analyzed, the second probe may be contacted with a fluid that is not the fluid to be analyzed (e.g., a regeneration fluid, a neutralization fluid). In some embodiments, the fluid to be analyzed and / or a sample of the fluid is continuously supplied to a plurality of probes. The probe to which any particular fluid or sample of fluid is provided may be one that has been contacted with a regeneration fluid, one that has been contacted with a neutralization fluid, and / or one on which a relatively small amount of analyte has been immobilized, and / or one having zero analyte. After any particular probe has been contacted with the fluid to be analyzed and / or a sample of the fluid to be analyzed, it may subsequently be contacted with a regeneration fluid and / or a neutralization fluid and then again contacted with the fluid to be analyzed and / or a sample of the fluid to be analyzed.

[0088] In some embodiments, samples of each fluid are contacted with a probe and a plurality of samples of fluids are supplied to a plurality of probes in such a way that each probe is continuously contacted with a sample of the fluid. As an example, in some embodiments, the following process may be continuously performed; first, a first probe is contacted with a first sample of fluid; then, while the first probe is being contacted with a regeneration fluid and / or a neutralization fluid, a second probe is contacted with a second sample of fluid; then, while the first probe is being contacted with a third sample of fluid, the second fluid is contacted with a regeneration fluid and / or a neutralization fluid; then, optionally, while the first probe is being contacted with a regeneration fluid and / or a neutralization fluid, the second probe is contacted with a fourth sample of fluid. This process may be repeated and / or may be performed continuously (e.g., while continuously increasing the sample of fluid).

[0089] It is also possible for two or more probes to be contacted with a sample of a common fluid simultaneously and / or for two or more probes to be contacted with samples of different fluids simultaneously. In some embodiments, while two or more probes are contacted with a common sample and / or different samples, one or more additional probes are not contacted with a sample of the fluid. Such probes may be contacted with a fluid other than the sample (for example, a regeneration fluid, a neutralization fluid). In some embodiments, while two or more probes are contacted with a common sample and / or different samples, one or more probes are regenerated. In some embodiments, the method includes a period in which the number of probes among a plurality of probes contacted with a common sample and / or different samples is greater than the number of probes among a plurality of probes not contacted with any sample. In some embodiments, contacting two or more probes with a common fluid (and / or a sample of a common fluid) may assist in detecting any abnormality regarding one or more probes. As an example, two or more probes may be contacted with a common fluid to generate two or more signals (for example, two or more optical signals), each generated from a different probe and related to the common fluid. Signals (for example, optical signals) generated from different probes may be compared with each other. If they are the same as each other or within a reasonable error range of each other, this may indicate that there is no abnormality related to any of the probes. On the other hand, if two signals (for example, two optical signals) are different from each other by an amount outside the normal error range, it may indicate that there is an abnormality related to one of the probes and / or a portion of the common fluid (and / or its sample) contacted by one of the probes. Such abnormalities may include, among others, gas bubbles.

[0090] As described above, the plurality of probes present within the instrument may only include probes that are different from each other, and / or may be capable of detecting a common analyte, and / or may only include probes configured to detect. It is also possible for the plurality of probes to include two or more probes that are different from each other at one or more points. As an example, the plurality of probes may be capable of detecting different analytes and / or may include two or more probes configured to detect. Such probes may be useful when it is desired to analyze the concentration of one or more analytes in a fluid (and / or a sample of the fluid), and / or when it is desired to analyze the concentration of different analytes in different fluids (and / or samples of the fluid) supplied to the probes among the plurality of probes. In some embodiments, the plurality of probes include both two probes that are not different from each other (e.g., two or more probes) and two probes that are different from each other (e.g., two or more probes).

[0091] Some of the instruments described herein are capable of outputting one or more signals and / or are configured to do so. Similarly, some of the methods described herein include outputting one or more signals. Advantageously, such signals may provide information regarding one or more characteristics of the fluid and / or sample of the fluid with which the probe is in contact. Various signals may be output from the instruments described herein. In some embodiments, the instrument is capable of outputting and / or is configured to output an electrical signal. Similarly, some methods may include outputting an electrical signal. The electrical signal may indicate the amount of analyte immobilized on the probe and / or the concentration of analyte in and / or in a sample of the fluid in contact with the probe. As another example, the electrical signal may indicate that the analyte immobilized on the probe and / or present in the fluid (and / or a sample thereof) exceeds a predetermined amount. The electrical signal may also indicate that the analyte immobilized on the probe and / or present in the fluid (and / or a sample thereof) exceeds the limit at which the instrument can accurately determine the amount of analyte in the fluid (and / or a sample thereof). In some embodiments, the electrical signal indicates that the variation in a signal related to the immobilization of the analyte on the probe (e.g., an optical signal, a signal related to the binding of the analyte to the probe) exceeds a predetermined amount.

[0092] As described elsewhere herein, some of the instruments described herein are capable of, are configured to, and / or are associated with and / or in fluid communication with an additional instrument. Some methods may include determining the concentration of an analyte in a fluid supplied by and / or received from an additional instrument and / or in one or more samples of a fluid supplied by and / or received from an additional instrument. Some systems may include an additional instrument.

[0093] Also, as described elsewhere in this specification, the devices described herein are capable of outputting one or more signals and / or can be configured to do so. In some embodiments, such signals are transmitted to additional devices. As an example, the signal may be transmitted to a computer in electrical communication with the devices described herein (e.g., via a standard identified in Open Platform Communications). The computer then displays information to an operator and / or records it to a file. As another example, the signal may be output to additional devices (e.g., additional devices with which the device is in fluid communication, additional devices with which the device is not in fluid communication). Such signals may include instructions for performing an action and / or may instruct an additional device to perform an action. In some embodiments, the devices described herein send instructions and / or the method includes sending instructions based on determination of the concentration of an analyte in a fluid contacting the probe.

[0094] Non-limiting examples of commands may include commands to do nothing, pause, stop temporarily, modify one or more properties of a fluid within an additional instrument, change the flow of fluid out of and / or within an additional instrument, supply fluid to another location, and / or provide fluid flowing out of an additional instrument to a different container (e.g., different probes, different inlets, waste containers, containers that temporarily contain fluid, different columns present within an instrument, and / or different housings that contain chromatographic media). It should be noted that when the fluid is present within the additional instrument, it may undergo one or more processes that modify one or more of its properties (e.g., the concentration of one or more species therein). Such processes may occur upstream of the probe but downstream of the location of the fluid that is the subject of the command sent to the additional instrument. Thus, commands sent to an additional instrument based on the concentration of an analyte in a fluid supplied by and / or received by the additional instrument may include modifying one or more properties of a fluid that is present within the additional instrument and that differs from the fluid whose concentration was measured in one or more respects, changing the flow of a fluid that flows out of and / or through the additional instrument and that differs from the fluid whose concentration was measured in one or more respects, supplying a fluid that differs from the fluid whose concentration was measured in one or more respects to another location, and / or providing a fluid that flows out of the additional instrument and that differs from the fluid whose concentration was measured in one or more respects to a different container. As an example, measurement of the concentration of an analyte in a fluid supplied by a column and / or chromatographic media within a chromatography system may form the basis for commands sent regarding whether the fluid upstream of the column and / or chromatographic media should continue to be supplied to the column and / or chromatographic media and / or whether it should be supplied to a different column and / or chromatographic media. The fluid upstream of the column and / or chromatographic media may differ from the fluid whose concentration was measured in one or more respects, such as in the concentration of an analyte therein (e.g., the concentration of the analyte may be much lower after passing through the column and / or chromatographic media than upstream of the column and / or chromatographic media).

[0095] Doing nothing may also include continuing to operate the additional instrument in the same way as it was operating prior to sending the command (e.g., supplying flow from the additional instrument at the same rate and to the same location, maintaining the same or different fluid properties as the fluid contacting the probe within the additional instrument).

[0096] Modifying one or more properties of the fluid within the additional instrument may include modifying the temperature, pressure, flow rate, pH, conductivity, dissolved oxygen content, and / or the concentration of one or more species (e.g., nutrients including sugars such as glucose, amino acids, and / or fatty acids; and / or metabolites such as lactate) of the fluid. As described above, this fluid may be the same as the fluid contacting the probe or may differ in one or more respects. In some embodiments, modifying one or more properties of the fluid within the additional instrument is performed as part of a process aimed at achieving a desired critical quality attribute by providing appropriate critical process parameters. By way of example, modifying one or more properties of the fluid within the additional instrument may include achieving and / or maintaining desired culture conditions, enhancing one or more characteristics (e.g., product yield, productivity, concentration) of a product produced by a bioprocess occurring within the additional instrument, and / or reducing impurities related to the product and / or process.

[0097] Supplying fluid to different locations can involve supplying it to different probes, to different locations instead of the probe (e.g., to a column or the medium of a column and / or chromatograph within another chromatography system, to a waste container, to a valve, to an outlet, to an analysis system), to a different location downstream of the same probe (e.g., to different columns and / or the chromatographic medium within a column, or to another chromatography system, to a column and / or the chromatographic medium within a column or to another chromatography system or a bioprocessing system instead of returning to a waste container, to a column and / or the chromatographic medium within a column or to another chromatography system instead of a waste container, returning to a bioprocessing system instead of a waste container and then to a valve, to an outlet, to an analysis system), or after passing through a different location upstream of the same probe (e.g., different columns, chromatographic media, combinations of columns, and / or combinations of chromatographic media present within a chromatography system). The various locations to which the fluid may be supplied may be in different bioprocessing systems or in different locations within the same bioprocessing system. As described above, this fluid may be the same as the fluid that contacts the probe or may differ in one or more respects.

[0098] Halting may also include halting one or more operations of a bioprocessing system and / or halting the supply of fluid to a probe. Halting may occur for a defined period or for an indefinite period (e.g., until an operator induces a restart, shutdown, change in one or more operations, change in one or more properties of the fluid within the bioprocessing system, and / or change in the location where the fluid is supplied). In some embodiments, halting enables one or more internal adjustments to be made to a bioprocessing system that supplies fluid to a probe, the bioprocessing system to stop supplying fluid to the probe, a new bioprocessing system to cause fluid to be supplied to the probe, and / or the fluid supplied to the probe to be switched from being supplied by one bioprocessing system to being supplied by another bioprocessing system.

[0099] In some embodiments, the devices described herein are capable of interfacing with additional devices and / or are configured to do so in a manner that does not adversely affect (or, in some embodiments, does not affect at all) the functionality of the additional devices. As an example, in some embodiments, the device is configured to receive and / or be capable of receiving fluid and / or a sample of fluid from an additional device in a manner that maintains the sterility of the fluid remaining within the additional device.

[0100] In some embodiments, the devices described herein are capable of performing a bioprocess and / or are so configured and / or are capable of interfacing with and / or are so configured to interface with additional devices in which a bioprocess, such as a bioprocessing system, is performed. In such embodiments, a first device (e.g., the device described above) can monitor a bioprocess and / or report the results of measurements performed on a fluid undergoing the bioprocess and / or obtained from a bioprocessing system, but which is in one or more respects different from a fluid present in one or more parts of the bioprocessing system, and / or can be so configured. The measurements can be performed on a sample of the fluid undergoing the bioprocess and / or on a sample obtained from a fluid present within the bioprocessing system but which is in one or more respects different from a fluid present in one or more parts of the bioprocessing system. In some embodiments, the reporting of the measurement results can have a relatively low time lag (e.g., low enough to enable bioprocess control).

[0101] A bioprocessing system can be a system in which a bioprocess is being performed, is capable of having a bioprocess performed therein, and / or is configured to perform a bioprocess. Non-limiting examples of bioprocesses include biotechnology processes and biopharmaceutical processes such as those involved in the production of a desired therapeutic biologic. Non-limiting examples of suitable therapeutic biologics include biologics, vaccines, components for cell or gene therapy, and non-therapeutic biologics such as dyes, biofuels, and / or dietary supplements. A bioprocess can involve the expression of a therapeutic biologic by a microorganism or mammalian cell and / or the cell itself can be the desired therapeutic biologic. In some embodiments, the desired therapeutic biologic can be the result of a cell-free production process based on one or more components of a natural or non-natural obtained cell.

[0102] In some embodiments, the bioprocess(es) implemented in a bioprocessing system (and / or a bioprocess that a bioprocessing system is capable of implementing and / or is configured to implement) is an upstream process. The upstream process may include operations and / or workflows implemented during the development, optimization, screening, and / or selection of stocks and / or cell lines, cell culture, the production of a desired product using cells and / or cell components, and / or processes implemented between such operations. The culture can be carried out at various scales (e.g., μL to thousands of L) using different reactor settings and geometries (e.g., rocking motion, stirred tank, bubble column, fixed bed) by applying different operating modes (e.g., batch, fed-batch, perfusion, continuous, and / or combinations thereof). The culture process may also be monitored, analyzed, and / or controlled, typically based on different sensor technologies (e.g., soft, electrochemical, biochemical, optical; offline, online, in-line, at-line). In some upstream processes, after culturing a biological agent, it is purified (e.g., via a downstream process). During purification, the culture broth may be separated from the desired product, which can be cells and / or other expression components (e.g., monoclonal antibodies, polyketides, enzymes, vaccines). One non-limiting example of a bioprocessing system that may be employed in an upstream process is a bioreactor. Non-limiting examples of suitable bioreactors include batch-fed bioreactors, fed-batch bioreactors, and perfusion bioreactors.

[0103] In some embodiments, the bioprocess(es) implemented in a bioprocessing system (and / or a bioprocess that a bioprocessing system is capable of implementing and / or is configured to implement) is a downstream process. The downstream process may include various techniques and methods for the recovery, purification, analysis, and / or characterization of a desired product. The downstream process may involve cell disruption, sedimentation, centrifugation, precipitation, crystallization, extraction, filtration, adjustment of the pH and conductivity of a liquid, enzymatic or chemical modification, dilution, buffer exchange, evaporation, adsorption, and / or chromatography. In some embodiments, the downstream process includes steps of analysis and / or characterization. Such steps assist in the recovery of a purified product that conforms to one or more quality characteristics (e.g., the glycosylation pattern of an antibody, the concentration of endotoxin). Before filling and packaging the purified product, formulation steps involving buffer exchange, drying, lyophilization, or crystallization may be performed to bring it to a state suitable for storage and distribution. Non-limiting examples of bioprocessing systems that may be employed in the downstream process include chromatography systems (e.g., batch chromatography systems, continuous chromatography systems), filtration systems (e.g., tangential flow filtration systems, ultrafiltration / diafiltration systems), centrifugation systems, and centrifuges.

[0104] As described elsewhere herein, in some embodiments, the devices described herein include a chromatography system and / or are related to additional devices that are chromatography systems. The chromatography system may include a column and / or a chromatographic medium (e.g., a chromatographic medium contained within a column). Non-limiting examples of suitable chromatographic media include resins, membrane adsorbents, and monoliths.

[0105] In embodiments involving a chromatography system, the chromatography system and / or one or more of its components (by way of example, the column therein, the chromatographic medium therein) may supply fluid to a probe. In some embodiments, the method includes performing one or more operations on the chromatography system and / or a part thereof. As an example, in some embodiments, the method includes loading a column and / or a chromatographic medium in a chromatography system. As another example in some embodiments, the method includes washing a column and / or a chromatographic medium (by way of example, after it has supplied fluid to a probe, to prepare it for elution, to prepare it for regeneration). As another example, in some embodiments, the method includes eluting a column and / or a chromatographic medium (such as after it has been supplied with a fluid containing an analyte, after it has supplied fluid to a probe, after an analyte has been loaded). As a third example, in some embodiments, the method includes regenerating a column and / or a chromatographic medium (such as to prepare it to load an analyte from a fluid after elution, after washing).

[0106] In some embodiments, the method includes detecting when an analyte first begins to elute / pass through from a chromatography system, a column within the chromatography system, and / or a chromatographic medium present within the chromatography system (e.g., a chromatographic medium present within a column). The analyte may be a species that it is desirable to recover from the chromatography system or an impurity that it is desirable to exclude from a sample of the fluid flowing through the chromatography system. The method may also include splitting the fluid flowing from the chromatography system (e.g., from a first column therein, from a first chromatographic medium therein) into a plurality of samples and then contacting each sample with a regenerated probe. Before any analyte is detected within a sample of the fluid, the sample of the fluid may be flowed to a first location (e.g., a container in which it may be stored, a waste container) after contacting the probe. When the analyte is first detected, the chromatography system may adjust the flow such that the fluid being supplied thereto and / or the fluid upstream of the first column and / or the chromatographic medium (and, according to circumstances, the fluid downstream of the first column, the fluid downstream of the first chromatographic medium, and / or the fluid being contacted with the probe, which may differ from the latter at one or more points) is supplied to a second column and / or a second chromatographic medium instead of the first column and / or the first chromatographic medium. At this point, the instrument may continue to be employed to determine the amount of analyte in a sample of the fluid eluting from the chromatography system as a whole (e.g., a sample of the fluid that has flowed through the second column and / or the second chromatographic medium).

[0107] In some embodiments, the processes described above are repeated multiple times. As an example, in some embodiments, a chromatography system includes three or more columns and / or chromatography media. After a breakthrough of the second column and / or chromatography media is detected in a sample of fluid received from the second column and / or chromatography media, the chromatography system may adjust the flow such that the fluid being supplied thereto and / or the fluid upstream of the second column and / or second chromatography media is supplied to a third column and / or third chromatography media instead of the second column and / or second chromatography media. At this point, the instrument may continue to be employed to determine the amount of analyte in a sample of fluid eluting from the chromatography system as a whole (e.g., a sample of fluid that has flowed through the third column and / or third chromatography media). In some embodiments, while the fluid is not being supplied to the first column and / or first chromatography media (e.g., when instead being supplied to the second and / or third column and / or chromatography media), the first column and / or first chromatography media may be washed, eluted, and / or regenerated. The processes described above may be repeated using two or more columns and / or chromatography media (e.g., four or more columns and / or chromatography media), and / or may be implemented such that samples of fluid exiting the columns are provided to three or more columns and / or chromatography media in a repeating sequence. The processes described above may be accomplished using countercurrent chromatography and / or simulated moving bed chromatography. This enables the implementation of continuous chromatography processes, which may in turn enable the implementation of integrated continuous bioprocesses (ICBs). For example, it may enable connecting a continuous USP to a continuous DSP as described herein.

[0108] Advantageously, the above-described process may also be capable of correcting for variations in the amount of analyte supplied to the chromatography system (e.g., variations in the analyte produced therein when the fluid is supplied by another bioprocessing system such as a bioreactor), and / or for variations in the volume of the column and / or chromatographic media. Both of these features may also be suitable for reducing losses of analyte during purification of the analyte, and / or for enhancing the use of resin present in the column and / or chromatographic media (e.g., for enhancing dynamic binding capacity).

[0109] In some embodiments, a sample of fluid contacted with a probe may be recycled by a chromatography system. Such a sample may be a sample containing an analyte and / or a sample not containing an analyte. The apparatus described herein may be employed in various suitable chromatography systems and at various suitable locations in a chromatography process. As an example, in some embodiments, the apparatus described herein may be employed to detect a breakthrough of a species (i.e., transport from a column and / or chromatographic medium loaded with the species) bound to a column and / or chromatographic medium. In such embodiments, the concentration of an analyte in a sample of fluid flowing through the column and / or chromatographic medium may be determined to assess when and / or whether any breakthrough occurs. As another example, in some embodiments, the apparatus described herein may be employed to detect the elution of an analyte from a column and / or chromatographic medium. In such embodiments, the concentration of an analyte in a sample of fluid flowing through a purification column and / or chromatographic medium may be determined to assess when elution begins and ends. As a further example, the apparatus described herein may be employed to determine the binding strength of a column and / or chromatographic medium to an analyte and / or to generate a breakthrough curve of the analyte.

[0110] One non-limiting example of a suitable chromatography system is a continuous simulated moving bed chromatography system. Various suitable columns may be employed, such as capture columns, purification columns, polishing columns, cation exchange columns, anion exchange columns, affinity columns, hydrophobic interaction columns, and / or mixed mode columns. The column may include a chromatographic medium such as a resin, a membrane absorber, and / or a monolith.

[0111] As also described elsewhere in this specification, in some embodiments, the apparatus described herein includes a bioreactor and / or is related to additional apparatus that is a bioreactor. In some embodiments, the method includes detecting the concentration of an analyte in a bioreactor over time. Advantageously, such method may be performed without an additional cell removal step prior to detecting the concentration of the analyte. A sample of the fluid present in the bioreactor may be obtained therefrom and then contacted with a probe. After contacting the probe, the sample of the fluid may be returned to the bioreactor or provided to a waste container. The former scenario allows for repeated sampling of a small-scale (e.g., mL-scale) bioreactor without consuming the full working volume of the bioreactor. In some embodiments, the sample of the fluid is received from the bioreactor in a manner that maintains the sterility of the fluid remaining in the bioreactor. It is also possible for the sample of the fluid to pass through an apparatus including a probe in a manner that receives the sample from the bioreactor and maintains its sterility (e.g., such that it can be returned to the bioreactor while still being sterile). It is also possible for the additional apparatus to include a container (e.g., a surge container) that receives fluid from one or more of the apparatuses described above.

[0112] To determine the production rate of an analyte, a fluid sample may be contacted with a probe as frequently as desired (for example, every few hours). This process can be employed to determine the point in time when the concentration of the analyte rises above a predetermined value and / or when the variation in a signal (for example, an optical signal) associated with the immobilization of the analyte on the probe exceeds a predetermined amount. As an example, the method may include determining the point in time when the concentration of the analyte reaches a level indicating that growth within a bioreactor can be terminated and from which a desired product (for example, the analyte) can be obtained. As another example, the method may include determining the point in time when the concentration of the analyte reaches a level that is undesirable for cell growth. In such cases, the method may include outputting a signal indicating this fact, thereby automatically inducing an adjustment of the growth conditions and / or warning an operator to act.

[0113] In some embodiments, the apparatus described herein is configured to be reversibly associated with, capable of being reversibly associated with, and / or is reversibly associated with two or more additional apparatuses such as two or more bioprocessing systems and / or two or more bioreactors. Such an apparatus is configured to be in fluid communication with, capable of being in fluid communication with, and / or is in fluid communication with two or more additional apparatuses, bioprocessing systems, and / or bioreactors. In some embodiments, the association and / or fluid communication is reversible. As an example, the association and / or fluid communication may occur through a single inlet. In such embodiments, a valve may be employed to switch the position of the probe between positions in fluid communication with two or more bioreactors. Supplying a single inlet using two or more bioreactors may be advantageous when it is desirable to determine the concentration of an analyte in the additional apparatuses, bioprocessing systems, and / or bioreactors at relatively infrequent intervals compared to the time required to determine the concentration of the analyte and / or regenerate the probe. In such embodiments, a single apparatus can be advantageously used to monitor over time the concentration of an analyte in two or more additional apparatuses, bioprocessing systems, and / or bioreactors, and fewer apparatuses may be required to perform these measurements than if each bioreactor were associated with a different apparatus.

[0114] Similarly, some methods include contacting the probe with fluid supplied from two or more additional apparatuses, and some systems may include two or more additional apparatuses each configured to supply and / or capable of supplying fluid (e.g., a first fluid output from a first additional apparatus and a second fluid output from a second additional apparatus) to the apparatus.

[0115] In some embodiments, the two or more additional instruments are two or more bioprocessing systems. As an example, in some embodiments, the method includes contacting a probe with fluid supplied from two bioprocessing systems, the system includes two bioprocessing systems, and / or the instrument is configured to be reversibly associated with, capable of being reversibly associated with, and / or reversibly associated with two or more bioprocessing systems. In some embodiments, one of the bioprocessing systems is an upstream bioprocessing system and one of the bioprocessing systems is a downstream bioprocessing system. As an example, in some embodiments, one of the bioprocessing systems includes a bioreactor and one of the bioprocessing systems includes a chromatography system. In such embodiments, the method can include performing a bioprocess that employs a bioreactor to produce an analyte as a result, and also includes capturing the analyte by employing a chromatography system. This can be performed in an automated manner and / or without operator input. As an example, based on a measurement of the concentration of an analyte in a fluid supplied by a bioreactor, a command to continue performing the bioprocess and / or supply the fluid to a chromatography system may be sent to the bioreactor. As another example, based on a measurement of the concentration of an analyte in a fluid supplied by a chromatography system, a command is sent to the chromatography system to continue loading a column and / or chromatographic medium therein, eluting the column and / or chromatographic medium, and / or directing the fluid (e.g., supplied by a bioreactor, by a column and / or chromatographic medium in a chromatography system) to a particular column and / or chromatographic medium. In some embodiments, a common probe may be employed to perform all such concentration measurements without the need for threshold adjustment according to.

[0116] It is also possible that two or more bioprocessing systems are upstream bioprocessing systems and / or that two or more bioprocessing systems are downstream bioprocessing systems.

[0117] In some embodiments where there are two or more additional instruments that supply fluid, are capable of supplying fluid, and / or are configured to supply fluid, a detector present within the instrument may be capable of detecting a signal and / or may be configured to detect a signal when the probe is contacted with the fluid supplied by each additional instrument. By way of example, the detector may be configured to detect and / or be capable of detecting a variation in a first signal when the probe is contacted with the fluid supplied by a first additional instrument, and may be configured to detect and / or be capable of detecting a variation in a second signal when the probe is contacted with the fluid supplied by a second additional instrument.

[0118] In some embodiments where there are two or more additional instruments that are capable of supplying fluid, supplying fluid, and / or are configured to supply fluid, the instrument and / or system may be capable of sending instructions to one of the additional instruments and / or be configured to send instructions based on the concentration of an analyte in a fluid supplied by another additional instrument. Similarly, some methods include determining the concentration of an analyte in a fluid supplied by a first additional instrument and sending instructions to a second additional instrument based on this determination. Such instructions may include the instructions described above (e.g., modifying one or more properties of a second fluid within a bioprocessing system, continuing to supply the second fluid to a probe, supplying the second fluid to a different location, and / or doing nothing). By way of non-limiting example, in some embodiments, instructions may be sent to a chromatography system based on the concentration of an analyte in a fluid supplied by a bioreactor (e.g., for supplying the fluid to different locations such as different columns and / or chromatographic media within a chromatography system, recycling the fluid, supplying the fluid to a waste container), instructions may be sent to a bioreactor based on the concentration of an analyte in a fluid supplied by a chromatography system, and / or instructions may be sent to one chromatography system based on the concentration of an analyte in a fluid supplied by a first chromatography system.

[0119] Non-limiting further examples of additional instruments that may be in fluid communication with the instruments described herein include filtration devices, centrifuges, pumps, and valves.

[0120] As described above, in some embodiments, the instrument includes a probe. The probe may assist in detecting the concentration of an analyte in a fluid and / or a sample of the fluid that contacts the probe. The probes described in this specification may have various suitable designs. In some embodiments, the probe is an optical probe. As an example, the probe may be an optical fiber probe and / or may include an optical fiber. FIG. 16 shows a non-limiting embodiment of a cross-section of a probe including an optical fiber. In some embodiments, the probe includes one or more components that enable optical coupling to an optical cable. As an example, in some embodiments, the probe includes components such as an SMA connector (e.g., an SMA905 connector), a BNC connector, a push, lock, and / or twist function, and / or a plastic hub compatible with a connector having a compression spring. FIGS. 17-18 show an example of such a component in combination with an optical cable. In some embodiments, the probe is coupled to the optical cable via a ferrule. The ferrule may include an optical fiber with a polished tip, which may facilitate optical communication with the probe. The optical cable may also include one or more components that assist in strain relief at the coupling location.

[0121] In some embodiments, the probe may transmit and / or convey light of multiple wavelengths (e.g., visible wavelengths, near-infrared wavelengths). This may be facilitated, in some embodiments, by the presence of one or more polished ends (e.g., a polished end perpendicular to the optical axis of the probe).

[0122] In some embodiments, the probe includes a surface having a functionalized and / or surface chemistry that aids in the immobilization of the analyte. As an example, the surface functionalization and / or chemistry may also facilitate the binding of one or more analytes thereto. In some embodiments, the probe includes a surface to which one or more reagents (e.g., one or more reagents suitable for immobilizing an analyte) are immobilized. The reagent(s) may be immobilized on the probe in various suitable ways. As an example, the reagent(s) may be bound to the probe. The binding may include covalent, ionic, polar, van der Waals, hydrophobic, and / or hydrogen bonds.

[0123] In some embodiments, the one or more reagent(s) are immobilized on the probe in such a way that no significant (and / or any) detachment from the probe occurs during contact with the fluid (and / or sample thereof) to be analyzed, contact with a regeneration fluid, contact with a neutralization fluid, and / or any other process typically carried out in one or more of the methods described above. As an example, the reagent(s) may be immobilized on the probe in a manner that is stable to water, aqueous solutions, buffers, acids, bases, and / or body fluids.

[0124] Additionally or alternatively, one or more reagent(s) can be immobilized on the probe in such a way that the probe can be regenerated. The method can also include re-functionalizing the probe. Re-functionalizing can sometimes include removing one or more reagent(s) from the probe. As an example, in some embodiments, re-functionalizing includes exposing the probe, on which one or more reagent(s) are immobilized, to a fluid (e.g., a buffer such as an acidic buffer) that peels off one or more of those reagent(s) from the probe. Subsequently, the probe may be exposed to a fluid containing one or more new reagent(s) to be immobilized on the probe. By re-functionalizing the probe, advantageously, the probe can be employed in multiple ways and / or immobilize two or more analytes. As an example, prior to determining the concentration of a first analyte in a fluid and / or a sample of the fluid, a first reagent and / or a set of reagents may be immobilized on the probe. In some situations, it may be desirable to reuse the probe to determine the concentration of a second analyte in a fluid and / or a sample of the fluid (e.g., the same fluid as before, a different fluid, the same sample as before, a different sample). Thus, after detecting the concentration of the first analyte, the probe is re-functionalized to yield a probe on which a new reagent and / or a set of reagents can be immobilized, such that the probe can then be employed to detect the concentration of the second analyte.

[0125] Some probes may not be re-functionalized and / or may not be capable of being re-functionalized.

[0126] Various suitable reagents may be immobilized on the surface of the probes described herein. Some reagents may be of a type that can perform one or more chemical reactions, such as one or more chemical reactions that result in immobilization of an analyte on the probe. By way of example, a probe may include a reagent that can bind (e.g., covalently, ionically, by polar interactions, by van der Waals interactions, hydrophobically, by hydrogen bonding, by complexation) to an analyte, absorb the analyte, and / or adsorb the analyte. In some embodiments, one or more of the aforementioned chemical reactions may also immobilize on the probe the analyte with which the reagent reacts. Selected non-limiting examples of suitable reagents include biomolecules (e.g., proteins, glycoproteins, peptides, nucleic acids (e.g., DNA, RNA, mRNA), antibodies (e.g., antibodies against exosomes such as anti-CD63 and / or anti-CD9, antibodies against proteins, antibodies against viruses, antibodies against virus-like particles), antibody fragments, antigens, polysaccharides, carbohydrates, hormones, streptavidin, glutathione), ligands (e.g., ligands for proteins such as protein A), small molecules, viruses, cells, inorganic compounds (e.g., aminopropylsilane), sequestering compounds, capsids, bacteria, resins (e.g., Ni-NTA), plasmids, nutrients, metabolites, metabolic by-products, and combinations thereof. Non-limiting examples of proteins include protein A, protein G, protein L, and lectins. One non-limiting example of a combination of two or more of the aforementioned reagent types is a reagent that includes protein A and an antibody against exosomes and / or viruses. The antibody may be immobilized on protein A immobilized on the probe surface and may be capable of immobilizing exosomes and / or viruses. In such embodiments, as well as in other embodiments, two or more reagents are immobilized on the probe (and, in some embodiments, one or more such reagents may be a combination of two or more reagents).

[0127] In some embodiments, the reagent immobilized on the surface of the probe is suitable for performing chemical and / or biological reactions including binding. It is also possible for the probe to be suitable for performing chemical and / or biological reactions that do not include binding. When present, binding may include a reaction between a target and a binding partner that specifically binds to the target (e.g., an agent or molecule that specifically binds to the target). Binding may also include immobilizing a target (e.g., an analyte) on the binding partner. In some embodiments, the binding partner may specifically bind to an epitope on a target molecule (e.g., an analyte). Non-limiting examples of specific pairs of binding partners and targets include antibodies and antigens, antibody fragments and antigens, antibodies and haptens, antibodies and peptides, antibodies and small molecules, antigens and fusion proteins, antibody fragments and haptens, enzymes and enzyme substrates, enzymes and inhibitors, enzymes and cofactors, binding proteins and substrates, carrier proteins and substrates, proteins and small molecules, lectins and carbohydrates, receptors and hormones, receptors and effectors, complementary strands of nucleic acids, proteins in combination with nucleic acid repressors and inducers, ligands and cell surface receptors, viruses and ligands, and receptors and ligands.

[0128] Non-limiting examples of antibodies, which may also be binding partners or antibodies, include intact (i.e., full-length) polyclonal and monoclonal antibodies, antigen-binding fragments of polyclonal and monoclonal antibodies (e.g., Fab, Fab’, F(ab’)2, or Fv), single-chain (scFv), single-chain variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and modified constructs of immunoglobulin molecules containing the antigen recognition site of the required specificity. Non-limiting examples of antibodies belonging to the last category include glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. In addition, the binding partner may be an antibody of any class, e.g., IgD, IgE, IgG, IgA, or IgM (or a subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and / or IgA2).

[0129] An antigen may be a molecule or a portion of a molecule to which an antibody can be generated. The antigen may be a peptide, a polysaccharide, and / or a lipid. Some antigens may originate in the body (“self-antigens”), and some antigens may originate from the external environment (“non-self antigens”).

[0130] In some embodiments, antibodies suitable for performing chemical and / or biological reactions specifically bind to epitopes on their target molecules. An epitope (which may also be referred to as an antigenic determinant) may be part of an antigen that is recognized (or bound) by an antibody. For example, an epitope may be a specific portion of an antigen to which an antibody binds. The portion of the antibody that binds to the epitope is sometimes referred to as a paratope. An epitope may be a conformational epitope (composed of discontinuous amino acids or sections of an antigen) or a linear epitope (composed of contiguous amino acids). Some proteins may share segments of high sequence homology and / or structural similarity. These similar proteins may have common epitopes (in other words, epitopes on different antigens may be bound by the same antibody). Further, differently processed proteins (such as proteins that have undergone additional enzymatic processes) may share some epitopes, although not all, with their pre-processing forms. Non-limiting examples of different epitopes that may be added or removed during processing include N-terminal signal peptides (such as those found on prepropeptides), and changes seen when an inactive protein (such as a propeptide) is converted to an active form by post-translational modification.

[0131] When an antibody specifically binds to an epitope, it may perform a binding reaction that can distinguish between a target molecule (e.g., an analyte) and non-target molecules (e.g., molecules other than the analyte of interest). For example, the binding partner may specifically bind to the target molecule with an affinity that is at least 2-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater than its affinity for non-target molecules.

[0132] The binding affinity of an antibody may be parameterized by its affinity (K D ). K Dis the ratio of the dissociation constant to the association constant (K D = K d / K a ). In some embodiments, the binding partners described herein have an affinity (K -5 of 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less. An increase in the affinity K D corresponds to a decrease in the dissociation constant K D or an increase in the association constant (K d ). A higher affinity binding of a binding partner (e.g., an antibody) for a first molecule relative to a second molecule can be indicated by a K a for binding to the first target that is higher (or a smaller numerical value of K a or K D and / or K d ) than the K a for binding to the second target (or a numerical value of K D and / or K d ). In such cases, the antibody has relatively higher specificity for the first molecule (e.g., a protein in a first conformation or a mimetic thereof) than for the second molecule (e.g., the same protein in a second conformation or a mimetic thereof, or a second protein). The difference in binding affinity (e.g., specificity) can be 1.5-fold or greater, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 10-fold or greater, 15-fold or greater, 20-fold or greater, 37.5-fold or greater, 50-fold or greater, 70-fold or greater, 80-fold or greater, 90-fold or greater, 100-fold or greater, 500-fold or greater, 1000-fold or greater, 10,000-fold or greater, 10 5 -fold or greater.

[0133] In some embodiments, the reagent may be immobilized on the surface of the probe via a covalent bond. Prior to such immobilization, the surface of the probe may be functionalized to include a plurality of functional groups suitable for forming such covalent bonds. As an example, the surface of the probe may be functionalized by reaction with a bifunctional reagent that includes a siloxane group that facilitates attachment to the probe and a functional group that facilitates the formation of a covalent bond with the reagent to be immobilized on the probe. As another example, the surface of the probe may be exposed to a plasma or other treatment that generates in situ a functional group that facilitates the formation of a covalent bond with the reagent to be immobilized on the probe. Non-limiting examples of suitable types of functional groups that facilitate the formation of a covalent bond with the reagent to be immobilized on the probe include hydroxyl, amine, and carboxyl.

[0134] The probes described herein may be formed from a variety of suitable materials and / or may include a coating formed from a variety of suitable materials. In some embodiments, the probe includes glass and / or a polymer, and / or a coating that includes glass and / or a polymer. Non-limiting examples of suitable glasses include SiO 2 and Ta 2 O 5 Examples of suitable polymers include polystyrene and polyethylene. The probes described herein may include optical fibers having various suitable diameters. In some embodiments, the probe includes an optical fiber having a core with a diameter of 400 microns or more, 500 microns or more, 600 microns or more, 700 microns or more, 800 microns or more, 900 microns or more, 1000 microns or more, 1100 microns or more, 1200 microns or more, 1300 microns or more, 1400 microns or more, 1500 microns or more, 1600 microns or more, 1700 microns or more, 1800 microns or more, or 1900 microns or more. In some embodiments, the probe includes an optical fiber having a core with a diameter of 2000 microns or less, 1900 microns or less, 1800 microns or less, 1700 microns or less, 1600 microns or less, 1500 microns or less, 1400 microns or less, 1300 microns or less, 1200 microns or less, 1100 microns or less, 1000 microns or less, 900 microns or less, 800 microns or less, 700 microns or less, 600 microns or less, or 500 microns or less. Combinations of the above-described ranges are also possible (for example, 400 microns or more and 2000 microns or less). Other ranges are also possible.

[0135] The instruments described herein may employ various suitable detectors. In some embodiments, the instrument includes an optical detector. Non-limiting examples of suitable types of optical detectors include photon counting devices, spectrophotometers, spectrometers (e.g., Raman spectrometers, infrared spectrometers), polarization detectors, photodiodes, CCD / CMOS sensors, and imaging sensors. Such optical detectors may be configured to detect and / or be capable of detecting variations in an optical signal over one or more periods. As an example, such optical detectors may be able to make relatively rapid measurements of an optical signal and / or measure an optical signal over a relatively short period. It is also possible for some optical detectors to be configured to detect and / or be capable of detecting multiple optical signals (e.g., multiple optical signals each related to a fluid and / or a sample of the fluid). In some embodiments, the optical detector is configured to detect and / or be capable of detecting the intensity of light as a function of position (which may thereby enable detection of the intensity of light as a function of the angle at which the light is reflected from the probe), and / or is configured to detect and / or be capable of detecting the intensity of light over a limited angular range. In some embodiments, the instrument includes a mechanical detector such as a quartz crystal microbalance.

[0136] As described above, in some embodiments, the instrument includes a light source. The light source may serve as a source of light that stimulates the emission of an optical signal. In some embodiments, the light source supplies light of multiple wavelengths. As an example, the instrument may include a light source that includes an incandescent bulb. As a further example, the instrument may include a light source that includes a lamp such as a halogen lamp, a xenon lamp, a mercury lamp, an LED, and / or an arc lamp. When the instrument includes a light source and supplies light at multiple wavelengths, the instrument may further include one or more optical filters. Such optical filter(s) may be positioned between the light source and the location of the chemical species that generate the light signal and / or between the light source and the detector. The former may be beneficial when the light emitted by the light source includes at least one wavelength that would stimulate the generation of optical signals other than the desired ones (e.g., in cases where light of the corresponding wavelength would stimulate emissions from various chemical species that would always be immobilized on and / or in contact with the probe and / or the fluid and / or its sample present in the probe). It is also possible for the light source to supply light over a limited wavelength range (e.g., the light source may include a laser or other narrow-band light source).

[0137] The light sources described herein may also supply light of a single polarization and / or multiple polarizations. When the light source supplies light of multiple polarizations, the instrument may further include one or more polarization filters. Such polarization filter(s) may be positioned between the light source and the location of the chemical species that generate the light signal and / or between the light source and the detector. In some embodiments, the light source supplies light at various (e.g., all, or substantially all) angles. It is also possible for the light source to supply light over a limited angular range.

[0138] As described above, in some embodiments, the amount of an analyte in a fluid and / or a sample of the fluid is determined. Details regarding the possible fluids, fluid samples, and analytes are provided below. In some embodiments, the fluid (and / or a sample thereof) may contain an analyte, according to some. Some methods may also include determining whether such a fluid (and / or a sample of the fluid) actually contains an analyte and / or the amount of analyte contained in the fluid (and / or a sample of the fluid). Thus, some fluids and / or samples of fluids may contain an analyte (in various suitable amounts), and some fluids and / or samples of fluids may lack an analyte. Additionally, it should be understood that reference to the concentration of an analyte in a fluid (and / or a sample of the fluid) encompasses concentrations that are zero as well as concentrations greater than zero.

[0139] The fluids described herein may contain various suitable analytes, non-limiting examples of which include proteins (such as Protein A, Protein G, Protein L, host cell proteins, Fc receptors, streptavidin), peptides, antibodies (such as IgG), antigens, small molecules, viruses, capsids, cells (such as Chinese hamster ovary cells), differentiated cell types, polysaccharides, bacteria, nucleic acids (such as DNA, RNA, mRNA), exosomes, extracellular vesicles, and ions (such as nickel ions). In some embodiments, the fluid contains an analyte that is a tagged protein, such as a protein tagged by recombinant modification. Non-limiting examples of tagged proteins include His-tagged proteins and biotin-tagged proteins.

[0140] The analyte may or may not be labeled. An unlabeled analyte may lack a label that facilitates detection (e.g., a label that facilitates optical detection, a fluorescent label, etc.), may have the same chemical composition (e.g., the same chemical formula) as that within the additional instrument to which it is supplied, and / or may have the same chemical composition as that after being purified to form the final product. In some embodiments, advantageously, the method includes determining the concentration of the unlabeled analyte in the fluid, and / or the instrument is configured to determine, and / or is capable of determining, the concentration of the unlabeled analyte in the fluid. This enables facile label-free detection and / or enables the concentration and / or properties of the analyte to be determined without the need for a labeling step and / or without the influence of the applied label.

[0141] The fluids and fluid samples described herein may contain an analyte at various suitable concentrations. In some embodiments, the fluid and / or fluid sample contains the analyte at a concentration of 0.000001 g / L or more, 0.000002 g / L or more, 0.000005 g / L or more, 0.0000075 g / L or more, 0.00001 g / L or more, 0.00002 g / L or more, 0.00005 g / L or more, 0.000075 g / L or more, 0.0001 g / L or more, 0.0002 g / L or more, 0.0005 g / L or more, 0.00075 g / L or more, 0.001 g / L or more, 0.002 g / L or more, 0.005 g / L or more, 0.0075 g / L or more, 0.01 g / L or more, 0.02 g / L or more, 0.05 g / L or more, 0.075 g / L or more, 0.1 g / L or more, 0.2 g / L or more, 0.5 g / L or more, 0.75 g / L or more, 1 g / L or more, 1.5 g / L or more, 2 g / L or more, 2.5 g / L or more, 3 g / L or more, 3.5 g / L or more, 4 g / L or more, 4.5 g / L, 5 g / L or more, 6 g / L or more, 7.5 g / L or more, 10 g / L or more, 15 g / L or more, 20 g / L or more, 30 g / L or more, or 40 g / L or more. In some embodiments, the fluid and / or fluid sample contains the analyte at a concentration of 5 g / L or less, 50 g / L or less, 40 g / L or less, 30 g / L or less, 20 g / L or less, 15 g / L or less, 10 g / L or less, 7.5 g / L or less, 6 g / L or less, 5 g / L or less, 4.5 g / L or less, 4 g / L or less, 3.5 g / L or less, 3 g / L or less, 2.5 g / L or less, 2 g / L or less, 1.5 g / L or less, 1 g / L or less, 0.75 g / L, 0.5 g / L or less, 0.2 g / L or less, 0.1 g / L or less, 0.075 g / L or less, 0.05 g / L or less, 0.02 g / L or less, 0.01 g / L or less, 0.0075 g / L or less, 0.005 g / L or less, 0.002 g / L or less, 0.001 g / L or less, 0.00075 g / L or less, 0.0005 g / L or less, 0.0002 g / L or less, 0.0001 g / L or less, 0.000075 g / L or less, 0.00005 g / L or less, 0.00002 g / L or less, 0.00001 g / L or less, 0.0000075 g / L or less, 0.000005 g / L or less, or 0.000002 g / L or less. Combinations of the above-described ranges are also possible (by way of example, 0.000001 g / L or more and 50 g / L or less).Other ranges are also possible.

[0142] In some embodiments, the fluid and / or sample of the fluid, in addition to possibly containing an analyte, also contains additional species. As an example, as described above, in some embodiments, the fluid and / or sample of the fluid is supplied by an additional instrument. In such embodiments, the fluid and / or sample of the fluid may further contain one or more species that facilitate the use of the additional instrument and / or that are present during the process performed in the additional instrument. As an example, in some embodiments, the fluid to be analyzed is a crude sample and / or the sample of the fluid to be analyzed is a crude sample. As a further example (e.g., in the case where the additional instrument is a chromatography system), the fluid and / or sample of the fluid contains a buffer. As a third example (e.g., when the additional instrument is a bioreactor), the fluid and / or sample of the fluid contains one or more components of a cell medium, non-limiting examples of which include glucose, lactate, amino acids (e.g., one or more types), salts (e.g., one or more types), proteins (e.g., one or more types such as Protein A, Protein G, Protein L, host cell proteins, etc.), peptides, one or more types of nucleic acids, and / or one or more types of cells. It is also possible for the fluid and / or sample of the fluid to have one or more characteristics that are useful and / or necessary for the operation of the additional instrument, such as being sterilized.

[0143] Some fluids and / or samples of fluids may include body fluids and / or biological materials. In some embodiments, the fluid and / or sample of fluid includes an analyte that is a biological material (e.g., located in a biological fluid, located in a buffer). As an example, in some embodiments, the fluid and / or sample of fluid includes cells (e.g., living cells) and / or reagents (e.g., biomolecules) as an analyte and / or as species other than the analyte. The fluid and / or sample of fluid may include some or all of the reagents described elsewhere herein for reagents that may be immobilized on the surface of a probe, and / or may include reagents other than those so described. Such reagents may be the analyte to be detected, or may be present in the fluid and / or sample of fluid that is said to include such an analyte. Non-limiting examples of such reagents include proteins, glycoproteins, peptides, ligands, antibodies, antigens, hormones, nucleic acids (e.g., DNA, RNA), polysaccharides, carbohydrates, small molecules, inorganic compounds, isolated compounds, viruses, extracellular vesicles, exosomes, capsids, cells, differentiated cell types, and bacteria.

[0144] Measurements can also be performed on a reference fluid. As an example, in some embodiments, the measurements are performed on a positive reference and / or a negative reference. A positive reference may be configured to always produce a signal (e.g., an optical signal) and / or always produce a known signal (e.g., an optical signal) when the instrument is operating correctly. Some positive references include a known concentration of an analyte in the fluid. A negative reference may be configured to always produce a signal (e.g., an optical signal) indicating that the analyte is not immobilized when the instrument is operating correctly. Some negative references lack any analyte. Performing the method on a positive reference and / or a negative reference may be useful for calibrating the results obtained from the instrument and / or for verifying that the instrument is operating correctly. Some embodiments may include contacting a probe with a plurality of fluids including the fluid and / or sample of fluid to be analyzed (and optionally lacking a reference) and contacting a second probe with a plurality of fluids including a reference (and optionally lacking the fluid and / or sample of fluid to be analyzed). An embodiment may also include contacting two or more references, such as both a positive and a negative reference and / or two or more positive references with different concentrations of analyte (e.g., in a way that two or more probes each contact a single reference, in a way that one probe contacts both references, in a way that both probes contact both references).

[0145] In some embodiments, the fluid and / or a sample of the fluid is supplied from a source of fluid in fluid communication with the apparatus described herein by a relatively short tube. The tube may have a length of 0.1 cm or more, 0.2 cm or more, 0.5 cm or more, 0.75 cm or more, 1 cm or more, 2 cm or more, 5 cm or more, 7.5 cm or more, 10 cm or more, 12.5 cm or more, 15 cm or more, 17.5 cm or more, 20 cm or more, 22.5 cm or more, 25 cm or more, 27.5 cm or more, 30 cm or more, 32.5 cm or more, 35 cm or more, 37.5 cm or more, 40 cm or more, 42.5 cm or more, 45 cm or more, 47.5 cm or more, 50 cm or more, 55 cm or more, 60 cm or more, 75 cm or more, 1 m or more, 2 m or more, 5 m or more, or 7.5 m or more. The tube may also have a length of 10 m or less, 7.5 m or less, 5 m or less, 2 m or less, 1 m or less, 75 cm or less, 60 cm or less, 55 cm or less, 50 cm or less, 47.5 cm or less, 45 cm or less, 42.5 cm or less, 40 cm or less, 37.5 cm or less, 35 cm or less, 32.5 cm or less, 30 cm or less, 27.5 cm or less, 25 cm or less, 22.5 cm or less, 20 cm or less, 17.5 cm or less, 15 cm or less, 12.5 cm or less, 10 cm or less, 7.5 cm or less, 5 cm or less, 2 cm or less, 1 cm or less, 0.75 cm or less, 0.5 cm or less, or 0.2 cm or less. Combinations of the above-described ranges are also possible (by way of example, 0.1 cm or more and 10 m or less, 5 cm or more and 2 m or less, 5 cm or more and 50 cm or less, or 10 cm or more and 50 cm or less). Other ranges are also possible.

[0146] In some embodiments, a fluid and / or a sample of the fluid is supplied from a source of fluid in fluid communication with the instrument described herein by a tube having a relatively small in-line volume. The tube may have an in-line volume of 10 microliters or more, 20 microliters or more, 30 microliters or more, 40 microliters or more, 50 microliters or more, 80 microliters or more, 100 microliters or more, 200 microliters or more, 500 microliters or more, 800 microliters or more, 1 mL or more, 2 mL or more, 5 mL or more, 8 mL or more, 10 mL or more, 20 mL or more, 30 mL or more, 40 mL or more, 50 mL or more, 80 mL or more, 100 mL or more, 125 mL or more, 150 mL or more, or 175 mL or more. The tube may also have an in-line volume of 200 mL or less, 175 mL or less, 150 mL or less, 125 mL or less, 100 mL or less, 80 mL or less, 50 mL or less, 40 mL or less, 30 mL or less, 20 mL or less, 10 mL or less, 8 mL or less, 5 mL or less, 2 mL or less, 1 mL or less, 800 microliters or less, 500 microliters or less, 200 microliters or less, 100 microliters or less, 80 microliters or less, 50 microliters or less, 40 microliters or less, 30 microliters or less, or 20 microliters or less. Combinations of the ranges described above are also possible (by way of example, 10 microliters or more and 200 mL or less, 40 microliters or more and 40 mL or less, or 80 microliters or more and 10 mL or less). Other ranges are also possible.

[0147] As described above, some instruments include a source of regeneration fluid and some methods include contacting a probe with the regeneration fluid. Further details regarding some suitable regeneration fluids are provided below.

[0148] In some embodiments, the regeneration fluid includes a buffer. The regeneration fluid can also include salts (e.g., acetates such as sodium acetate; citrates, phosphates, Tris buffer salts, sodium hydroxide), and / or organic molecules (e.g., glycine, biotin, histidine). The salts may also assist in buffering the regeneration fluid. In some embodiments, the regeneration fluid includes species such as small molecules or salts that are capable of binding to an analyte, such as an analyte immobilized on a probe, and / or are configured to bind. As an example, the regeneration fluid may include biotin to regenerate a probe employed to determine the concentration of a streptavidin analyte. As another example, the regeneration fluid may include histidine to regenerate a probe employed to determine the concentration of a nickel ion analyte. It is also possible for a species present in the regeneration fluid to bind to a reagent immobilized on the probe, thereby displacing the analyte immobilized thereon (e.g., biotin in the regeneration fluid may bind to a streptavidin reagent immobilized on the probe, thereby displacing the analyte therefrom).

[0149] The regeneration fluid described herein may have various suitable pH values. In some embodiments, the regeneration fluid is acidic. An acidic regeneration fluid may be particularly suitable for probes employed with fluids and / or samples of fluids that contain, according to some, analytes that are antibodies, proteins (e.g., protein A, protein G, protein L), and / or nucleic acids (e.g., DNA, RNA). The regeneration fluid may have a pH of 1 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, 3 or greater, 3.5 or greater, 4 or greater, or 4.5 or greater. The regeneration fluid may have a pH of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, or 1.5 or less. Combinations of the ranges described above are also possible (e.g., 1 or greater and 5 or less). Other ranges are also possible. As an example, it is also possible for the regeneration fluid to have a neutral or basic pH.

[0150] As described above, some devices include a source of neutralizing fluid, and some methods include contacting a probe with a neutralizing fluid. Further details regarding some suitable neutralizing fluids are provided below. In some embodiments, the neutralizing fluid includes a buffer. Non-limiting examples of suitable buffers include phosphate buffered saline and Tris buffer.

[0151] The neutralizing fluids described herein may have various suitable pHs. In some embodiments, the neutralizing fluid has a neutral or near-neutral pH. The neutralizing fluid may have a pH of 6 or greater, 6.5 or greater, 7 or greater, 7.5 or greater, 8 or greater, 8.5 or greater, or 9 or greater. The neutralizing fluid may also have a pH of 9 or less, 8.5 or less, 8 or less, 7.5 or less, 7 or less, or 6.5 or less. Combinations of the ranges described above are also possible (by way of example, 6 or greater and 9 or less, or 6 or greater and 8 or less). Other ranges are also possible.

[0152] As described above, in some embodiments, the instrument includes a source of fluid other than a source of sample, a source of regeneration fluid, and / or a source of neutralization fluid. Similarly, in some embodiments, the instrument includes a valve that includes one or more locations in fluid communication with a source of fluid other than a source of sample, a source of regeneration fluid, and / or a source of neutralization fluid. Further non-limiting examples of sources of fluid include a source of fluid containing a primary antibody, a source of fluid containing a secondary antibody, a source of wash buffer, and a source of fluid containing a substrate for the secondary antibody. Without wishing to be bound by theory, these sources of fluid may be beneficial when the instrument is employed to form an assay. As an example, a primary antibody present in a liquid contacting a probe may become immobilized on the probe. Such a primary antibody may be suitable for immobilizing an analyte present in a fluid sample. As another example, a secondary antibody present in a fluid contacting a probe may become immobilized on an analyte immobilized on the probe. The secondary antibody may facilitate detection of the analyte. Similarly, a fluid containing a substrate for the secondary antibody may facilitate detection of the analyte by interaction with the secondary antibody. As a third example, a wash buffer may be suitable for removing one or more components from the probe.

[0153] A further example of a source of fluid that may be present within the apparatus described herein is a source of diluent. The source of diluent may be positioned such that it is configured to supply diluent for mixing with one or more fluids supplied by a source of fluid (e.g., a sample of fluid). The source of diluent may be positioned upstream of a valve that is capable of, configured to, and / or may supply a diluent fluid to a probe. The apparatus may be configured to mix the diluent with the relevant fluid. In some embodiments, this mixing may occur upstream of a valve that is capable of, configured to, and / or supplies a diluent fluid to a probe. A mixed fluid containing both the diluent and the fluid supplied from the source of fluid may then flow through the valve and contact the probe.

[0154] As described elsewhere herein, the apparatus described herein may include microfluidic channels. The microfluidic channels described herein may have various suitable dimensions perpendicular to the flow of fluid. Such dimensions may be referred to elsewhere herein as "widths" even if they are oriented in the vertical direction. In some embodiments, one or more, or each, microfluidic channel within the apparatus independently has one or more widths within the following ranges: 0.5 mm or greater, 0.75 mm or greater, 1 mm or greater, 2 mm or greater, 5 mm or greater, or 7.5 mm or greater. In some embodiments, each microfluidic channel within the apparatus independently has a width of 10 mm or less, 7.5 mm or less, 5 mm or less, 2 mm or less, 1 mm or less, or 0.75 mm or less. Combinations of the above-described ranges are also possible (e.g., 0.5 mm or greater and 10 mm or less). Other ranges are also possible.

[0155] As described above, in some embodiments, the probe is contacted with a fluid and / or a sample of the fluid. It is also possible for the probe to be contacted with a plurality of fluids (such as a plurality of fluids including a plurality of fluids containing an analyte and / or a plurality of samples of a plurality of fluids) in a repeating cycle. Further details regarding such contact are provided below. The fluid and the sample of the fluid may be contacted with the probe for various suitable periods. In some embodiments, contacting the probe with the fluid (and / or its sample) includes contacting the probe with the fluid (and / or its sample) for a period of 1 second or more, 2 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, 40 seconds or more, 1 minute or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hours or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 3.5 hours or more, or 4 hours or more. In some embodiments, contacting the probe with the fluid (and / or its sample) includes contacting the probe with the fluid (and / or its sample) for a period of 5 hours or less, 4 hours or less, 3.5 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 5 minutes or less, 2 minutes or less, 1 minute or less, 40 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, or 2 seconds or less. Combinations of the ranges described above are also possible (by way of example, 1 second or more and 5 hours or less, 1 second or more and 1 hour or less, or 10 seconds or more and 30 seconds or less). Other ranges are also possible.

[0156] The repeated cycles in which a plurality of fluids that may contain an analyte, and / or samples of a plurality of fluids, are brought into contact with a probe may be carried out over various suitable cycle times. In some embodiments, the time to complete a cycle is 30 seconds or more, 40 seconds or more, 50 seconds or more, 60 seconds or more, 70 seconds or more, or 80 seconds or more. In some embodiments, the time to complete a cycle is 90 seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds or less, 50 seconds or less, or 40 seconds or less. Combinations of the above-described ranges are also possible (by way of example, 30 seconds or more and 90 seconds or less). Other ranges are also possible.

[0157] The repeated cycles in which a plurality of fluids that may contain an analyte, and / or samples of a plurality of fluids, are brought into contact with a probe may be carried out at various suitable intervals. In some embodiments, the interval is 30 seconds or more, 1 minute or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hours or more, 2 hours or more, 3 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, or 16 hours or more. In some embodiments, the interval is 24 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 5 minutes or less, 2 minutes or less, or 1 minute or less. Combinations of the above-described ranges are also possible (by way of example, 30 seconds or more and 24 hours or less). Other ranges are also possible.

[0158] The repeated cycles in which a plurality of fluids, and / or samples of a plurality of fluids, are contacted with the probe, depending on the analyte, may be carried out for various suitable total times. In some embodiments, the repeated cycles are carried out for a period of 0.5 minutes or more, 1 minute or more, 2 minutes or more, 5 minutes or more, 7.5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 9 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 1 day or more, 1.5 days or more, 2 days or more, 5 days or more, 1 week or more, 2 weeks or more, or 1 month or more (by way of example, continuously, at a pre-set interval). In some embodiments, the repeated cycles are carried out for a period of 3 months or less, 1 month or less, 2 weeks or less, 1 week or less, 5 days or less, 2 days or less, 1.5 days or less, 1 day or less, 18 hours or less, 15 hours or less, 12 hours or less, 9 hours or less, 6 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 7.5 minutes or less, 5 minutes or less, 2 minutes or less, or 1 minute or less. Combinations of the ranges described above are also possible (by way of example, 0.5 minutes or more and 3 months or less). Other ranges are also possible.

[0159] In some embodiments, the methods described herein are carried out with the aid of a computer and / or a processor, such as via a computer-implemented control system. In addition, some of the systems and devices described herein include a computer and / or a processor. Such methods, systems, and devices are not limited to introduction into any particular computer system described herein, and many other different machines may be used.

[0160] The computer-implemented control system described herein is configured and / or programmed to control and adjust the operating parameters of an instrument and / or system and / or to analyze, calculate, and / or determine a value (e.g., concentration), and can be part of the instrument and / or system or be coupled thereto in conjunction therewith. In some embodiments, the computer-implemented control system can transmit and receive a reference signal to set and / or control the operating parameters of the instrument and / or system, such as when a defined value or threshold is reached. In some embodiments, the computer-implemented control system can be separated from the instrument and / or be located remotely from the instrument and / or receive data from one or more remote sample instruments via indirect and / or portable means such as via a portable electronic data storage device such as a magnetic disk or via communication by a computer network such as the Internet or a local intranet.

[0161] The computer-implemented control system may include components and circuits such as a processing unit (i.e., a processor), a memory system, input / output devices and interfaces (e.g., an interconnection mechanism), and / or a transport circuit (e.g., one or more buses), a video and audio data input / output (I / O) subsystem, other components such as dedicated hardware, and / or other components and circuits, as described in more detail below. Further, the computer-implemented control system may be a multiprocessor computer system and / or may include a plurality of computers connected by a computer network.

[0162] A computer-implemented control system may include a commercially available processor such as, for example, one of a series of x86, Celeron, and Pentium processors available from Intel, similar devices available from AMD and Cyrix, 680X0 series microprocessors available from Motorola, PowerPC microprocessors available from IBM, and ARM processors. Many other processors are available and the computer system is not limited to a particular processor. In some embodiments, the processor executes a program called an operating system, examples of which are Windows NT, Windows 95 or 98, Windows 7, Windows 8, UNIX®, Linux®, DOS, VMS, MacOS, and OSX®, and iOS, controls the execution of other computer programs, and / or provides scheduling, debugging, input / output control, accounting, compilation, storage allocation, data management and / or memory management, communication control and / or other related services. The processor and the operating system may together define a computer platform on which application programs are written in a high-level programming language. The computer-implemented control system is not limited to a particular computer platform.

[0163] In some embodiments, the processor is in electronic communication with, capable of being in electronic communication with, and / or configured to be in electronic communication with one or more other components present within the system described herein. By way of example, the processor may be in electronic communication with a detector. In some embodiments, the processor is programmed to perform one or more of the methods described herein and / or one or more values are determined for use by the processor. By way of example, in some embodiments, the processor is programmed to determine the concentration of an analyte (e.g., a first concentration of a first analyte, a second concentration of the first analyte, a second concentration of a second analyte) in a fluid (e.g., a first fluid, a second fluid). This determination may be made based on variations in a signal (e.g., a signal detected by a detector such as an optical signal detected by an optical detector) over a period of time. As another example, the processor may be programmed to determine whether a threshold has been reached based on variations in a signal (e.g., a signal detected by a detector such as an optical signal detected by an optical detector) over a period of time. The threshold may indicate a particular value of a first derivative of the signal, such as a value of the first derivative of the signal that is the limit of what the instrument is capable of detecting and / or is configured to detect.

[0164] The computer-implemented control system may include a memory system, which may include a computer-readable and writable non-volatile recording medium, examples of which are magnetic disks, optical disks, flash memories, and tapes. Such recording media may be removable, such as floppy disks, read / write CDs, memory sticks, etc., or may be permanent, such as hard drives, for example. Such recording media may typically store signals in binary form (i.e., a form interpreted as a sequence of 1s and 0s). A disk (e.g., magnetic or optical) may have a number of tracks on which such signals are typically stored in binary form (i.e., a form interpreted as a sequence of 1s and 0s). Such signals may define a software program (e.g., an application program) executed by a microprocessor, or information processed by an application program.

[0165] The memory system of a computer-implemented control system may also include integrated circuit memory elements that are typically volatile, random access memories, such as dynamic random access memory (DRAM) or static random access memory (SRAM). In operation, the processor may cause the program and data to be read from the non-volatile recording medium into the integrated circuit memory element, which may enable the processor to access the program instructions and data faster than the non-volatile recording medium can. The processor may also handle data within the integrated circuit memory element according to the program instructions. Then, after the processing is complete, the processed data may be copied to the non-volatile recording medium. The computer-implemented control system is not limited to a particular memory system.

[0166] At least a portion of such a memory system described above may be used to store one or more of the data structures (such as a lookup table) or equations described above. For example, at least a portion of the non-volatile recording medium may store at least a portion of a database that includes one or more of such data structures. Such a database may be, for example, a file system that includes one or more flat file data structures in which data is organized into data units separated by delimiter characters, a relational database in which data is organized into data units stored in tables, an object-oriented database in which data is organized into data units stored as objects, another type of database, or any combination thereof, any of various types of databases.

[0167] A computer-implemented control system may include a video and audio data I / O subsystem. The audio portion of the subsystem may include an analog-to-digital (A / D) converter that receives analog audio information and converts it to digital information. The digital information may be compressed using a known compression system for storage on a hard disk for use at another time. The video portion of the I / O subsystem may include a compression / decompression program for video images. Such a compression / decompression program may convert analog video information to compressed digital information or vice versa. The compressed digital information may be stored on a hard disk for use at another time.

[0168] A computer-implemented control system may include one or more output devices. Examples of output devices include cathode ray tube (CRT) displays, liquid crystal displays (LCDs), and other video output devices, printers, communication devices such as modems or network interfaces, storage devices such as disks or tapes, and audio output devices such as speakers. Such output devices may include an output interface that is capable of and / or configured to output information to an operator, instrument, component of the system, and / or components of different systems that are capable of receiving and / or configured to receive such information. In some embodiments, the information may take the form of a signal, such as an electronic signal encoding such information, a visual signal notifying an operator of such information, and / or an electronic signal encoding an instruction (e.g., an instruction supplied to an additional instrument such as a bioprocessing system based on determination of the concentration of an analyte in a fluid). As an example, in some embodiments, the output interface is capable of and / or configured to indicate the concentration of an analyte in a fluid.

[0169] In some embodiments, the output interface includes a display interface. The display interface may display information to an operator. In some embodiments, displaying information includes providing a numerical indication of the information on the display interface. By way of example, the display interface may indicate the concentration of an analyte in a fluid by providing a numerical indication on the display interface. The display interface may additionally or alternatively display other information such as the state of the instrument, a signal (e.g., an optical signal indicative of a binding such as a binding signal), and / or a first derivative of the signal (e.g., a first derivative of an optical signal indicative of a binding such as a binding signal). Such a display interface may display information obtained simultaneously (e.g., the values of signals obtained simultaneously and / or their first derivatives).

[0170] The computer-implemented control system may also include one or more input devices. Examples of input devices include keyboards, keypads, trackballs, mice, pens and tablets, communication devices as described above, and data input devices such as audio and video capture devices and sensors. Such input devices may include an input interface that can receive information from an operator, the instrument, components of the system, and / or components of different systems that are capable of providing and / or are configured to provide such information. In some embodiments, the information may be in the form of a signal such as a signal whose analyte concentration in a fluid may be determined. The information may be received over a network or may be input directly (e.g., mechanically) into the input interface.

[0171] It should be understood that one or more of any type of computer-implemented control system may be used to perform the various aspects described herein. Aspects of the invention may be implemented in software, hardware, firmware, or any combination thereof. A computer-implemented control system may include specially programmed application-specific hardware (e.g., an application-specific integrated circuit (ASIC)). Such application-specific hardware may be configured to perform one or more of the methods, steps, simulations, algorithms, systems, and system elements described above as part of the computer-implemented control system described above, or as a separate component.

[0172] The computer-implemented control system and its components may be programmable using any of a variety of suitable computer programming languages. Such languages may include procedural programming languages (e.g., C, Pascal, Fortran, and BASIC), object-oriented languages (e.g., C++, Java, and Eiffel), and other languages such as scripting languages or assembly languages. The methods, steps, simulations, algorithms, systems, and system elements may be performed using any of a variety of suitable programming languages, including procedural programming languages, object-oriented programming languages, other languages, and combinations thereof, that may be executed by such a computer system. Such methods, steps, simulations, algorithms, systems, and system elements may be performed as separate modules of a computer program or separately as separate computer programs. Such modules and programs may be executed on separate computers.

[0173] Such methods, steps, simulations, algorithms, systems, and system elements may be implemented as a computer program product tangibly embodied as a computer-readable signal on a computer-readable recording medium (e.g., a non-volatile recording medium, an integrated circuit memory element, or a combination thereof). For each such method, step, simulation, algorithm, system, or system element, such computer program product includes a computer-readable signal tangibly embodied on a computer-readable recording medium, the signal defining, for example, instructions (encoded therein) that, as a result of execution by a computer as part of one or more programs, cause the computer to perform the method, step, simulation, algorithm, system, or system element.

[0174] Paragraph 1: In some aspects, a method is provided. The method includes contacting a probe with a fluid over a first period, where the fluid is supplied by a bioprocessing system, the fluid flows over the probe, an analyte is present in the fluid at a first concentration, and at least a portion of the analyte is immobilized on the probe; detecting a variation in a signal over the first period; determining the first concentration based on the variation in the signal over the first period; and sending an instruction to the bioprocessing system based on the determination of the first concentration.

[0175] Paragraph 2: In some embodiments, a system is provided. The system includes a first instrument including a probe and a detector configured to detect signal variations over a first period; and a bioprocessing system, where the system is configured to supply fluid from the bioprocessing system to the first instrument, the first instrument is configured to determine a first concentration of an analyte in the fluid as the fluid contacts and flows over the probe based on the signal variations over the first period, and the system is configured to send instructions to the bioprocessing system based on the determination of the first concentration.

[0176] Paragraph 3: In some embodiments, the method includes contacting a probe with a fluid over a first period, where the fluid flows over the probe, the analyte is present in the fluid at a first concentration, and at least a portion of the analyte is immobilized on the probe; detecting variations in an optical signal over the first period; and determining the first concentration based on the variations in the optical signal over the first period, where the optical signal includes light reflected from an interface inside the probe and light reflected from an end of the probe.

[0177] Paragraph 4: In some embodiments, a first instrument is provided. The first instrument includes a probe and an optical detector configured to detect variations in an optical signal over a first period, where the first instrument is configured to determine a first concentration of an analyte in a fluid that contacts and flows over the probe based on the variations in the optical signal over the first period, and the optical signal includes light reflected from an interface inside the probe and light reflected from an end of the probe.

[0178] Paragraph 5: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 4, the fluid is output by a bioprocessing system when contacted with the probe. Paragraph 6: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-5, the fluid is supplied to the first apparatus as an output from the bioprocessing system. Paragraph 7: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-6, the fluid is supplied to the first apparatus in an automated manner.

[0179] Paragraph 8: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-7, the instructions include modifying one or more characteristics of the fluid within the bioprocessing system. Paragraph 9: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-8, the system includes a second bioprocessing system. Paragraph 10: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-9, the second bioprocessing system is configured to supply a second fluid output from the second bioprocessing system to the first apparatus. Paragraph 11: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-10, the detector is configured to detect fluctuations in a second signal over a second period.

[0180] Paragraph 12: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-11, the first apparatus is configured to determine a second concentration of a second analyte in the second fluid while the second fluid contacts and flows over the probe based on fluctuations in the second signal over a second period. Paragraph 13: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-12, the system is configured to send a second instruction to the bioprocessing system based on the determination of the second concentration. Paragraph 14: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-13, the second instruction includes modifying one or more properties of a fluid within the bioprocessing system, continuing to supply a second fluid to the probe, supplying the second fluid to a different location, and / or doing nothing. Paragraph 15: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-14, the bioprocessing system includes a chromatography system.

[0181] Paragraph 16: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-15, the system further includes a second bioprocessing system, and the second bioprocessing system includes a bioreactor. Paragraph 17: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-16, the bioprocessing system includes a bioreactor. Paragraph 18: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-17, the bioreactor is a batch-fed bioreactor. Paragraph 19: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-18, the bioreactor is a fed-batch bioreactor. Paragraph 20: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-19, the bioreactor is a perfusion bioreactor. Paragraph 21: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-20, the bioprocessing system includes a filtration system.

[0182] Paragraph 22: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-21, the filtration system is a tangential flow filtration system and / or an ultrafiltration / diafiltration system. Paragraph 23: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-22, the bioreactor includes a centrifugation system and / or a centrifuge. Paragraph 24: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-23, the signal is an optical signal. Paragraph 25: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-24, the optical signal includes light reflected from an interface inside the probe and light reflected from the end of the probe. Paragraph 26: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-25, the optical signal includes light reflected from the surface of the probe over a limited range of angles.

[0183] Paragraph 27: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-26, the signal is a mechanical signal. Paragraph 28: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-27, the first device includes a light source. Paragraph 29: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-28, the light source supplies light over a limited range of wavelengths. Paragraph 30: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-29, the light source includes an LED. Paragraph 31: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-30, the analyte is unlabeled. Paragraph 32: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-31, the fluid is supplied from a column within a chromatography system.

[0184] Paragraph 33: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-32, the different locations are different columns and / or chromatographic media within the chromatography system. Paragraph 34: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-33, the fluid is supplied from a probe to a waste container Paragraph 35: In some embodiments, the method described in any one of paragraphs 1-34 further includes washing the column to which the fluid is supplied, and / or the system and / or first apparatus described in any one of paragraphs 1-34 are further configured to wash the column to which the fluid is supplied.

[0185] Paragraph 36: In some embodiments, the method described in any one of paragraphs 1-35 further includes eluting the column to which the fluid is supplied, and / or the system and / or first apparatus described in any one of paragraphs 1-35 are further configured to elute the column to which the fluid is supplied. Paragraph 37: In some embodiments, the method described in any one of paragraphs 1-36 further includes regenerating the column to which the fluid is supplied, and / or the system and / or first apparatus described in any one of paragraphs 1-36 are further configured to regenerate the column to which the fluid is supplied. Paragraph 38: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-37, the system includes a processor in electronic communication with a detector, and wherein the processor is programmed to determine a first concentration based on fluctuations in the signal over a first period.

[0186] Paragraph 39: In some embodiments, a computer for performing the method described in any one of paragraphs 1-38 is provided, and / or the system and / or the first instrument described in any one of paragraphs 1-38 includes a computer. The computer includes an input interface configured to receive a signal, at least one processor programmed to determine a first concentration based on the variation of the signal over a first period, and an output interface configured to send instructions to a bioprocessing system. Paragraph 40: In some embodiments, in the computer, system, or first instrument described in any one of paragraphs 1-39, the input interface is configured to receive a signal via at least one network. Paragraph 41: In some embodiments, in the computer, system, or first instrument described in any one of paragraphs 1-40, the output interface is configured to indicate a first concentration of an analyte in a fluid. Paragraph 42: In some embodiments, in the computer, system, or first instrument described in any one of paragraphs 1-41, the output interface includes a display interface, and indicating a first concentration of an analyte in a fluid here includes providing a numerical indication of the first concentration on the display interface.

[0187] Paragraph 43: In some embodiments, in the computer, system, or first instrument described in any one of paragraphs 1-42, the signal is received using the input interface, and the first concentration is determined using at least one processor. Paragraph 44: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-43, a computer-readable recording medium is encoded with a plurality of instructions that, when executed by a computer, implement the method described in any one of paragraphs 1-43. Paragraph 45: In some embodiments, in the method described in any one of paragraphs 1-44, the fluid contacted with the probe is a first sample supplied by a sample source, and by switching a valve positioned upstream of the probe to remove the sample source from fluid communication with the probe and place a regeneration fluid source in fluid communication with the probe; and further contacting the probe with the regeneration fluid. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1-44 are configured to perform this method.

[0188] Paragraph 46: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-45, the system and / or the first instrument further includes a valve positioned upstream of the probe; and a regeneration fluid source positioned upstream of the valve, where: the valve is switchable between a plurality of positions, each position in the plurality of positions places the probe in fluid communication with a source within a plurality of sources, the plurality of sources includes a regeneration fluid source, and the plurality of sources includes a sample source. Paragraph 47: In some embodiments, in the method described in any one of paragraphs 1-46, the fluid contacted with the probe is a first sample supplied by a sample source, and the method further includes closing a first valve to remove the sample source from fluid communication with the probe; opening a second valve to place a regeneration fluid source in fluid communication with the probe; and further contacting the probe with the regeneration fluid, where the sample is directly supplied to the probe by opening the first valve and / or the regeneration fluid is directly supplied to the probe by opening the second valve. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1-46 are configured to perform this method.

[0189] Paragraph 48: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-47, the system and / or the first instrument further includes a plurality of valves positioned upstream of the probe, where: the plurality of valves includes a first valve positioned between the sample source and the probe and a second valve positioned between the regeneration fluid source and the probe, and by opening the first valve, the sample is directly supplied to the probe and / or by opening the second valve, the regeneration fluid is directly supplied to the probe.

[0190] Paragraph 49: In some embodiments, in the method described in any one of paragraphs 1-48, the method is implemented in a first instrument, the fluid is a first sample, the probe is a first probe, and the method in the first instrument is as follows: Contacting the first probe with a first sequence of fluids, where the first sequence of fluids includes the first sample and the regeneration fluid, Contacting a second probe with a second sequence of fluids, where the second sequence of fluids includes a second sample and the regeneration fluid, and After contacting the first probe with the regeneration fluid, contacting the first probe with a third sample. The method further includes performing the steps. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1-48 is configured to perform this method.

[0191] Paragraph 50: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-49, the system and / or the first instrument further includes a plurality of probes, where the plurality of probes includes probes; a plurality of inlets; and a regeneration fluid source, where: each inlet is in fluid communication with a probe among the plurality of probes, each inlet is configured to be reversibly in fluid communication with the regeneration fluid source and / or the sample source, and the first instrument is configured to alternately contact each probe with a sample among the plurality of samples supplied from the sample source and the regeneration fluid. Paragraph 51: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-50, the signal or optical signal is related to the sample. Paragraph 52: In some embodiments, in the method described in any one of paragraphs 1-51, the method further includes comparing the signal to a model signal profile. In some embodiments, the system and / or first instrument described in any one of paragraphs 1-51 are configured to perform this method.

[0192] Paragraph 53: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-52, in the method described in claim 51, the model signal profile is related to the first instrument functioning properly. Paragraph 54: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-53, the model signal profile is related to the first instrument malfunctioning. Paragraph 55: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-54, the model signal profile is related to the presence of bubbles in the fluid in contact with the probe. Paragraph 56: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-55, the variation of the signal is its first derivative.

[0193] Paragraph 57: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-56, the fluid is the first sample among a plurality of samples, and the analyte is present in the first sample at a first concentration. Paragraph 58: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-57, the variation of the signal indicates the binding constant between the analyte immobilized on the probe and the reagent. Paragraph 59: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 58, the signal variation indicates the dissociation constant between the analyte immobilized on the probe and the reagent. Paragraph 60: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 59, the signal variation indicates the affinity between the analyte immobilized on the probe and the reagent. Paragraph 61: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 60, the signal includes fluorescence.

[0194] Paragraph 62: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 61, the regeneration fluid is configured to cause at least partial detachment of the analyte immobilized on the probe. Paragraph 63: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 62, the regeneration fluid includes a buffer. Paragraph 64: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 63, the regeneration fluid buffer has a pH of 1 or more and 5 or less.

[0195] Paragraph 65: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 64, the regeneration fluid buffer includes glycine. Paragraph 66: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 65, the regeneration fluid buffer includes sodium acetate. Paragraph 67: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 66, the regeneration fluid buffer includes citrate. Paragraph 68: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 67, the regeneration fluid buffer includes phosphate, Tris buffer, and / or sodium hydroxide. Paragraph 69: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 68, the regeneration fluid contains biotin. Paragraph 70: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 69, the regeneration fluid contains histidine.

[0196] Paragraph 71: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 70, the regeneration fluid contains nickel ions. Paragraph 72: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 71, the first instrument includes a source of neutralization fluid. Paragraph 73: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 72, the neutralization fluid contains a buffer. Paragraph 74: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 73, the neutralization fluid buffer has a pH of 6 or more and 8 or less. Paragraph 75: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 74, the neutralization fluid has a pH of 9 or more. Paragraph 76: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 75, the neutralization fluid buffer is phosphate buffered saline.

[0197] Paragraph 77: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 76, the neutralization fluid buffer is a Tris buffer. Paragraph 78: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 77, the first instrument includes a valve configured to switch between a source of fluid, a source of regeneration fluid, and a source of neutralization fluid. Paragraph 79: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-78, the valve is positioned upstream of the inlet. Paragraph 80: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-79, the plurality of supply sources includes a supply source of neutralizing fluid. Paragraph 81: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-80, the plurality of supply sources includes a supply source of fluid containing a primary antibody. Paragraph 82: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-81, the plurality of supply sources includes a supply source of fluid containing a secondary antibody.

[0198] Paragraph 83: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-82, the plurality of supply sources includes a supply source of wash buffer. Paragraph 84: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-83, the plurality of supply sources includes a supply source of fluid containing a substrate for the secondary antibody. Paragraph 85: In some embodiments, the method described in any one of paragraphs 1-84 further includes switching a valve to remove a supply source of regeneration fluid from fluid communication with the probe and place a supply source of neutralizing fluid in fluid communication with the probe. In some embodiments, the system and / or first instrument described in any one of paragraphs 1-84 is configured to perform this method. Paragraph 86: In some embodiments, the method described in any one of paragraphs 1-85 further includes contacting the probe with the neutralizing fluid. In some embodiments, the system and / or first instrument described in any one of paragraphs 1-85 is configured to perform this method.

[0199] Paragraph 87: In some embodiments, the method described in any one of paragraphs 1-86 further includes switching a valve to remove the source of neutralization from fluid communication with the probe and place the source of the sample in fluid communication with the probe. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1-86 are configured to perform this method. Paragraph 88: In some embodiments, the method described in any one of paragraphs 1-87 further includes contacting a second sample supplied by the source of the sample with the probe. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1-87 are configured to perform this method. Paragraph 89: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-88, the first instrument includes a waste container. Paragraph 90: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-89, the waste container is positioned downstream from the outlet.

[0200] Paragraph 91: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-90, the first instrument further includes a purification filter. Paragraph 92: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-91, the purification filter is positioned between the source of the sample and the probe. Paragraph 93: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-92, the first instrument further includes a degassing filter. Paragraph 94: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-93, the degassing filter is positioned between the source of the sample and the probe. Paragraph 95: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-94, the first instrument further includes a vacuum degassing device.

[0201] Paragraph 96: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 95, the first instrument further includes an ultrasonic degassing device. Paragraph 97: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 96, the first instrument further includes a heater or a cooler configured to perform degassing. Paragraph 98: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 97, the first instrument includes a manifold. Paragraph 99: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 98, the manifold supplies fluid from a fluid source to a probe. Paragraph 100: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 99, the probe is in fluid communication with a microfluidic channel positioned within the manifold.

[0202] Paragraph 101: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 100, the microfluidic channel includes a bend. Paragraph 102: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 101, the microfluidic channel includes a step. Paragraph 103: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 102, the first instrument further includes a temperature control system. Paragraph 104: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 103, the temperature control system is associated with the probe. Paragraph 105: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-104, the temperature control system is associated with a tube that fluidly connects the probe to the valve. Paragraph 106: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-105, the temperature control system is associated with a manifold.

[0203] Paragraph 107: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-106, the first instrument further includes a source of diluent. Paragraph 108: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-107, the first instrument is configured to mix a plurality of samples upstream of the valve with the diluent. Paragraph 109: In some embodiments, the method described in any one of paragraphs 1-108 further includes contacting the probe with a plurality of fluids in repeated cycles. In some embodiments, the system and / or first instrument described in any one of paragraphs 1-108 are configured to perform this method. Paragraph 110: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-109, the second probe is contacted with a second sample while the probe is contacted with a regeneration fluid. Paragraph 111: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1-110, the second probe is contacted with a second sample while the probe is contacted with a neutralizing fluid.

[0204] Paragraph 112: In some embodiments, the method described in any one of paragraphs 1-111 further includes contacting two or more probes with a common sample simultaneously. In some embodiments, the system and / or first instrument described in any one of paragraphs 1-111 are configured to perform this method. Paragraph 113: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 112, the first instrument further includes at least one probe that does not contact the sample while two or more probes contact a common sample. Paragraph 114: In some embodiments, the method described in any one of paragraphs 1 - 113 further includes detecting signals generated from each probe that contacts a common sample. In some embodiments, the system and / or first instrument described in any one of paragraphs 1 - 113 are configured to perform this method. Paragraph 115: In some embodiments, the method described in any one of paragraphs 1 - 114 further includes comparing signals generated from probes that contact a common sample. In some embodiments, the system and / or first instrument described in any one of paragraphs 1 - 114 are configured to perform this method.

[0205] Paragraph 116: In some embodiments, the method described in any one of paragraphs 1 - 115 further includes determining whether there are any abnormalities associated with one or more probes that contact a common sample based on the comparison of the signals. In some embodiments, the system and / or first instrument described in any one of paragraphs 1 - 115 are configured to perform this method. Paragraph 117: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 116, at least one probe contacts a fluid other than the sample. Paragraph 118: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 117, the plurality of fluids includes a fresh sample of the fluid. Paragraph 119: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 118, the plurality of fluids includes a regenerating fluid.

[0206] Paragraph 120: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 119, the plurality of fluids includes a buffering agent. Paragraph 121: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 120, the plurality of probes includes probes that are different from each other. Paragraph 122: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 121, the plurality of probes includes two or more probes that are not different from each other. Paragraph 123: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 122, any of the plurality of probes is not different from each other.

[0207] Paragraph 124: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 123, the plurality of probes includes probes that are in series with each other. Paragraph 125: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 124, the plurality of probes includes probes that are in parallel with each other. Paragraph 126: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 125, contacting the probe with the fluid includes contacting the probe with the fluid for a period of 1 second or more and 5 hours or less. Paragraph 127: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 126, contacting the probe with the fluid includes contacting the probe with the fluid for a period of 5 seconds or more and 1 minute or less. Paragraph 128: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 127, the repetitive cycle occurs over a period of 30 seconds or more and 90 seconds or less.

[0208] Paragraph 129: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 128, the variation of the signal over time indicates the binding rate of the analyte to the probe. Paragraph 130: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 129, the fluid is a crude sample. Paragraph 131: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 130, the crude sample further contains a buffer. Paragraph 132: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 131, the crude sample further contains one or more components of the cell culture medium. Paragraph 133: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 132, the crude sample further contains glucose.

[0209] Paragraph 134: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 133, the crude sample further contains lactate. Paragraph 135: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 134, the crude sample further contains one or more types of amino acids. Paragraph 136: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 135, the crude sample further contains one or more types of salts. Paragraph 137: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 136, the crude sample further contains one or more types of proteins. Paragraph 138: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 137, the one or more types of proteins include protein A.

[0210] Paragraph 139: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 138, one or more types of proteins include host cell proteins. Paragraph 140: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 139, the crude sample further includes peptides. Paragraph 141: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 140, the crude sample further includes one or more types of nucleic acids. Paragraph 142: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 141, the crude sample further includes one or more types of cells. Paragraph 143: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 142, the first instrument is in fluid communication with an additional instrument. Paragraph 144: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 143, the first instrument is configured to divide the fluid flowing out from the second instrument into a plurality of samples.

[0211] Paragraph 145: In some embodiments, the method described in any one of paragraphs 1 to 144 further includes outputting a signal when the amount of the analyte exceeds a predetermined amount. In some embodiments, the system and / or the first instrument described in any one of paragraphs 1 to 144 are configured to implement this method. Paragraph 146: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 145, the signal is an electrical signal. Paragraph 147: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 146, the signal is transmitted via a standard specified in open platform communication. Paragraph 148: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 147, the signal commands an additional instrument to perform an action. Paragraph 149: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 148, the action is to pause.

[0212] Paragraph 150: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 149, the action is to change the flow of fluid flowing out of an additional instrument. Paragraph 151: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 150, the action is to supply the fluid flowing out of an additional instrument to a different container. Paragraph 152: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 151, the action is to change the flow of fluid flowing within an additional instrument. Paragraph 153: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 152, the fluid is received from an additional instrument in a manner that maintains the sterility of the fluid remaining in the additional instrument.

[0213] Paragraph 154: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 153, the additional instrument is a chromatography system. Paragraph 155: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 154, the additional instrument is a bioreactor. Paragraph 156: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 155, the additional instrument is a filtration device. Paragraph 157: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 156, the additional instrument is a centrifuge. Paragraph 158: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 157, the additional device is a pump. Paragraph 159: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 158, the additional device is a valve.

[0214] Paragraph 160: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 159, the reagent is immobilized on the probe. Paragraph 161: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 160, the reagent is a protein. Paragraph 162: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 161, the protein is Protein A. Paragraph 163: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 162, the protein is Protein G. Paragraph 164: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 163, the protein is Protein L. Paragraph 165: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 164, the reagent is a peptide. Paragraph 166: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 165, the reagent is an antibody.

[0215] Paragraph 167: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 166, the reagent is an antigen. Paragraph 168: In some embodiments, in the method, system, or first device described in any one of paragraphs 1 to 167, the reagent is a small molecule. Paragraph 169: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 168, the reagent is a virus. Paragraph 170: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 169, the reagent is a cell. Paragraph 171: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 170, the reagent is a differentiated cell type. Paragraph 172: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 171, the reagent is a polysaccharide. Paragraph 173: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 172, the reagent is a bacterium.

[0216] Paragraph 174: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 173, the reagent is a nucleic acid. Paragraph 175: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 174, the nucleic acid is DNA. Paragraph 176: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 175, the reagent is streptavidin. Paragraph 177: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 176, the reagent is aminopropylsilane. Paragraph 178: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 177, the reagent is Ni-NTA. Paragraph 179: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 178, the reagent is a lectin. Paragraph 180: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 179, the reagent is glutathione.

[0217] Paragraph 181: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 180, the first instrument includes an optical cable, where the optical cable transmits light to an optical detector. Paragraph 182: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 181, the first instrument includes a plurality of optical detectors. Paragraph 183: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 182, each optical detector among the plurality of optical detectors is associated with a probe. Paragraph 184: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 183, the probe is an optical probe. Paragraph 185: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 184, the probe is an optical fiber probe. Paragraph 186: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 185, the first instrument includes an optical cable.

[0218] Paragraph 187: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 186, the probe transmits light through one or more openings. Paragraph 188: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 187, the one or more openings are positioned on the side of the probe opposite the optical cable. Paragraph 189: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 to 188, the optical cable is configured to transmit light to the probe. Paragraph 190: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 189, the optical cable transmits light from a light source to a probe. Paragraph 191: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 190, the light source supplies light at a plurality of wavelengths.

[0219] Paragraph 192: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 191, the light source is a halogen lamp. Paragraph 193: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 192, the first instrument includes a plurality of light sources, each associated with a different probe. Paragraph 194: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 193, two or more probes are associated with a single light source. Paragraph 195: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 194, the first instrument includes an optical switch configured to switch which probe the light source is associated with. Paragraph 196: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 195, the optical cable is configured to transmit light from the probe.

[0220] Paragraph 197: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 196, the optical cable transmits light from the probe to an optical detector. Paragraph 198: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 197, the optical detector is a spectrometer. Paragraph 199: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 198, the analyte is a protein. Paragraph 200: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-199, the protein is protein A. Paragraph 201: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-200, the protein is a host cell protein. Paragraph 202: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-201, the protein is an Fc receptor.

[0221] Paragraph 203: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-202, the analyte is a peptide. Paragraph 204: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-203, the analyte is an antibody. Paragraph 205: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-204, the antibody is IgG. Paragraph 206: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-205, the analyte is an antigen. Paragraph 207: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-206, the analyte is a small molecule. Paragraph 208: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-207, the analyte is a virus. Paragraph 209: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-208, the analyte is a capsid.

[0222] Paragraph 210: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1-209, the analyte is a cell. Paragraph 211: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-210, the analyte is a differentiated cell type. Paragraph 212: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-211, the analyte is a polysaccharide. Paragraph 213: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-212, the analyte is a bacterium. Paragraph 214: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-213, the analyte is a nucleic acid. Paragraph 215: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-214, the nucleic acid is DNA. Paragraph 216: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-215, the analyte is RNA.

[0223] Paragraph 217: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-216, the analyte is mRNA. Paragraph 218: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-217, the analyte is an exosome. Paragraph 219: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-218, the analyte is an extracellular vesicle. Paragraph 220: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-219, the analyte is a plasmid. Paragraph 221: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-220, the analyte is an antibody fragment. Paragraph 222: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-221, the analyte is a nutrient component. Paragraph 223: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-222, the analyte is a metabolite.

[0224] Paragraph 224: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-223, the analyte is a metabolic byproduct. Paragraph 225: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-224, the analyte is a hormone. Paragraph 226: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-225, the first device further includes a control device. Paragraph 227: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-226, the control device provides commands periodically. Paragraph 228: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-227, the control device provides commands as needed. Paragraph 229: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-228, the control device provides commands, and the commands relate to the flow of fluid.

[0225] Paragraph 230: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-229, the control device provides commands, and the commands relate to the detection of optical signals. Paragraph 231: In some embodiments, in the method, system, or first device described in any one of paragraphs 1-230, the first device is interfaced with an additional device that is performing a bioprocess. Paragraph 232: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 231, the first instrument monitors a biological process. Paragraph 233: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 232, the first instrument reports the results of measurements performed on a fluid undergoing a biological process. Paragraph 234: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 233, the measurement is performed on a sample of the fluid undergoing the biological process.

[0226] Paragraph 235: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 234, the reporting of the measurement results has a time lag that is low enough to enable biological process control. Paragraph 236: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 235, when the fluid flows over the probe, the analyte becomes immobilized on the probe. Paragraph 237: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 236, the instructions include instructions to modify one or more properties of the fluid within the bioprocessing system, instructions to supply the fluid to a different location, instructions to pause, and / or instructions to do nothing. Paragraph 238: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 237, the fluid within the bioprocessing system is different from the fluid at one or more points.

[0227] Paragraph 239: In some embodiments, in the method, system, or first instrument described in any one of paragraphs 1 - 238, the system includes a processor in electronic communication with a detector, and here the processor is programmed to determine whether a threshold has been reached based on the variation of a signal over a first period. Paragraph 240: In some embodiments, in the method, system, or first apparatus described in any one of paragraphs 1 to 239, the output interface includes a display interface, and here the display interface is configured to provide a numerical indication of the signal and / or the first derivative of the signal. Paragraph 241: In some embodiments, the method described in any one of paragraphs 1 to 240 further includes outputting a signal when the variation in the derivative of the optical signal exceeds a predetermined amount. In some embodiments, the system and / or the first apparatus described in any one of paragraphs 1 to 240 are configured to implement this method.

[0228] Example 1 This example presents advantages associated with detecting the temporal variation of an optical signal. FIG. 19 shows simulated optical signals of the equilibrium immobilization of an analyte on a probe at various concentrations of the analyte in the fluid contacting the probe for an analyte having a binding constant K D = 1 nM. As can be seen from FIG. 19, there is a range of analyte concentrations in the fluid that result in an easily distinguishable optical signal (enclosed by the shaded square). At analyte concentrations higher or lower than this value, the optical signals become more difficult to discriminate. This is schematically shown in FIG. 20, with some such concentrations enclosed within the shaded frame.

[0229] However, when the temporal variation of the optical signal is measured instead, the concentration of the analyte in the range shown within the shaded frame may be readily distinguishable. This is shown schematically in FIG. 21. As can be seen in FIG. 21, fluids having different concentrations of analyte but having similar equilibrium levels of immobilization on the probe exhibit analyte immobilization on the probe at different rates. In FIG. 21, concentration C1 is higher than concentration C2, but fluids having both analyte concentrations represent similar equilibrium levels of analyte immobilization. However, the fluid having analyte concentration C1 exhibits analyte immobilization at a faster rate than the fluid having analyte concentration C2. Thus, it may be adopted to measure the variation of the optical signal over time to readily determine the concentration of the analyte in such fluids with relatively high accuracy.

[0230] Example 2 In this example, a method for regenerating the probe is described. Figures 22 - 24 schematically depict a method in which a probe contacts a fluid containing an analyte, and is then regenerated. As shown in these figures, the three-way valve is positioned upstream of the inlet to the housing containing the probe. The three-way valve switches between positions in fluid communication with a fluid source for the probe (in this case, a chromatography column labeled "sample from chromatography column"), a regeneration fluid, and a neutralization fluid (labeled "buffer"). A pump and a waste container (labeled "waste") are located downstream of the probe, and the pump pumps fluid from the housing containing the probe into the waste container. As shown in Figure 22, the first step is to contact the probe by flowing a sample of fluid from the chromatography column over the probe. During this step, fluctuations in the optical signal are detected and employed to determine the concentration of analyte in the fluid sample. Next, as shown in Figure 23, the three-way valve selects the regeneration fluid such that it contacts the probe, the regeneration fluid is supplied, and then it flows over the probe and then into the waste container. Finally, as shown in Figure 24, the three-way valve selects the neutralization fluid such that it contacts the probe, the neutralization fluid is supplied, and then it flows over the probe and into the waste container. After the step shown in Figure 24, the steps shown in Figure 22 (and, optionally, the steps shown in Figures 23 and 24) may be repeated.

[0231] Figure 25 shows the amount of analyte bound to the probe during each of the steps described above when a chromatography column is employed in a system that supplies a fluid containing the analyte, which is a monoclonal antibody, and the probe is functionalized with Protein A. As can be seen in Figure 25, while the probe is in contact with the fluid containing the analyte (method step No. 0), both the optical signal and its first derivative (smoothed by a numerical filter) increase, and the latter increases more rapidly than the former. Then, when the probe is in contact with the regeneration fluid (method step No. 1), the optical signal drops to a value close to zero, and its first derivative becomes zero. This is thought to be due to a decrease in the pH of the fluid that contacted the probe during regeneration, which causes a decrease in the affinity of the monoclonal antibody for Protein A, thereby causing the monoclonal antibody to detach from the probe. The optical signal and its first derivative remain relatively constant also during the subsequent exposure of the probe to the neutralization fluid (method step No. 2). An increase in the pH of the fluid that contacted the probe during neutralization was thought to enable Protein A to bind during the subsequent contact of the fluid sample with the probe.

[0232] Example 3 The method described in Example 2 may also be implemented in an instrument that includes one or more microfluidic channels instead of tubes. Figure 26 depicts one non-limiting example of such an instrument. As shown in Figure 26, the instrument may include a housing that contains a microfluidic channel, a probe in fluid communication with the microfluidic channel, and a switchable valve positioned upstream of the probe. The switchable valve positioned upstream of the probe may be the following: A position that places the probe in fluid communication with the column, A position that places the probe in fluid communication with a buffer supply, and A position that places the probe in fluid communication with a regeneration fluid supply It may also include three positions. The switchable valve may also switch between these three positions to fluidly connect the probe to these three fluid sources (in the order of, according to the situation, the column, then the regenerating fluid source, and then the buffer source). The valve may also be switched in an iterative manner between these three positions to alternately contact the probe with the fluid sample supplied by the column and to regenerate the probe. While the probe is in contact with the fluid sample, the amount of analyte in the fluid sample may be detected. This process may be repeated for a preset period, until the analyte exceeds a preset amount, and / or until the operator pauses the operation.

[0233] Example 4 In this example, an instrument including a plurality of probes will be described. Except for possibly including a plurality of valves and a plurality of probes, an instrument similar to the instrument including microfluidic channels described in Example 2 may be provided. Such an instrument is schematically shown in FIG. 27. The plurality of valves may include an uppermost switchable valve including a plurality of positions, and each of the positions fluidly connects a column to one of a plurality of downstream switchable selector valves.

[0234] The uppermost switchable valve may also be switched between different positions to fluidly connect different downstream selector valves to the column. Each downstream switchable selector valve is associated with one of the plurality of probes and includes the following: A position that fluidly connects the associated probe to the uppermost switchable valve, A position that fluidly connects the probe to the buffer source, and A position that fluidly connects the probe to the regenerating fluid source It may also include three positions.

[0235] The downstream switchable selector valve and the uppermost upstream switchable valve may be switched together, thereby fluidly connecting each probe to these three fluid sources (according to which, in the order of the column, then the source of the regeneration fluid, and then the source of the buffer). This switching may also continuously provide a fluid sample from the column to the instrument, causing at least one of the plurality of probes to always be in fluid communication with the column.

[0236] Example 5 In this example, an instrument including a plurality of different types of probes is described. FIG. 28 shows one non-limiting example of an instrument including a plurality of different types of probes. The instrument shown in FIG. 28 is similar to that described in Example 2 including microfluidic channels, except that it includes three different types of probes. The three different types of probes are arranged continuously downstream of the valve. The fluid flowing through the instrument may flow continuously over each of these types of probes. Such an instrument is useful, for example, when the fluid sample contains one or more analytes whose concentration is desired to be detected. Different types of probes may be configured to detect different types of analytes, and thus may enable the detection of multiple types of analytes within a single instrument and / or a single flowing fluid.

[0237] Example 6 In this example, an instrument including two filters, a source of diluent, and a temperature control system is described. Figure 29 shows one non-limiting example of an instrument that includes a purification filter, a degassing filter, a source of diluent, and a temperature control system. The instrument shown in Figure 29 is similar to that described in Example 2 that includes microfluidic channels, except that it includes these additional components. The purification filter may be placed upstream of the source of diluent, and the degassing filter may be placed downstream of a switchable valve. The temperature control system may be suitable for controlling the temperature of the switchable valve, the degassing filter, and / or the housing.

[0238] Example 7 In this example, an instrument that includes a switchable valve having seven positions is described. Figure 30 shows one non-limiting example of an instrument that includes a switchable valve having seven positions. The instrument shown in Figure 30 is similar to that described in Example 2 that includes microfluidic channels, except that the switchable valve has more positions. The switchable valve shown in Figure 30 can reversibly place the probe in fluid communication with a column, a source of primary antibody, a source of secondary antibody, a wash buffer, a regeneration fluid, and a substrate for the secondary antibody. An instrument such as that shown in Figure 30 may be suitable for performing an assay on a sample of fluid obtained from a column.

[0239] Example 8 In this example, a method for detecting breakthrough of an analyte from a chromatography column is described. In a device employed to perform continuous perfusion culture of Chinese hamster ovary cell lines, monoclonal antibodies were produced. Cross-flow tangential filtration was employed to remove cells from the perfusion permeate, which was collected in a surge vessel. The surge vessel served as a supply solution for affinity chromatography carried out in a continuous simulated moving bed chromatography system. The continuous simulated moving bed chromatography system was in fluid communication with an instrument containing a probe functionalized with Protein A as a reagent. The instrument was operated in the same manner as the instrument described in Example 2. Here, the continuous simulated moving bed chromatography system containing an in-built chromatography column served as the chromatography column, the regeneration fluid was 10 mM glycine buffer having a pH of 2, and the neutralization fluid was phosphate buffered saline having a pH of 7.4. The cycle during which these three fluids contacted the probe occurred for more than 1 minute.

[0240] Figure 31 shows the first derivative of the optical signal (labeled "gradient" therein) measured over a period of 27 cycles. During the first 800 seconds, the optical signal had a constant and low first derivative value of less than 0.4 nm / min. This supports the claim that no significant breakthrough of the monoclonal antibody occurred during this period. Subsequently, the first derivative of the optical signal began to increase, indicating a breakthrough of the monoclonal antibody. Finally, the first derivative of the optical signal increased above the previously defined threshold of 1 nm / min (shown as a dashed line in Figure 31), which triggered a column switch, redirecting the fluid flowing out of the column in the continuous simulated moving bed chromatography system and contacting the probe to flow instead to another column therein. At this point, the supply solution from the surge tank was redirected to another column according to the process of simulated moving bed chromatography.

[0241] An offline measurement was carried out on the tortuous flow, and when the threshold was reached, the concentration of the monoclonal antibody in the fluid was 0.02 g / L, indicating that the loss of the monoclonal antibody was minimal. After the column switch was triggered, the flow from the other column was not supplied to the probe for 108 minutes, which was a shorter period than the shortest possible breakthrough time for the other column. Subsequently, the flow from the other column was supplied to the probe, the optical signal related to the probe was measured, the instrument was operated again as described above, and the time to column breakthrough was recorded. The process described in the previous paragraph was repeated for three days, and the measured time to column breakthrough for each operation of the instrument is shown in Figure 32. The overall yield for the monoclonal antibody (i.e., 100% multiplied by the ratio of the monoclonal antibody in the final eluate to all the monoclonal antibody removed from the continuous simulated moving bed chromatography system) was 96.9%, and the concentration of the monoclonal antibody in the final eluate was 15.1 g / L (the approximate concentration of the monoclonal antibody in the feed solution was 0.7 g / L). In addition, DNA and HCP impurities were highly removed (i.e., 2.1 log removal to 30 ppm and 3.6 log removal to 38 ppm, respectively).

[0242] Example 9 Summary Biopharmaceuticals such as monoclonal antibodies (mAbs) are used in the treatment of numerous severe diseases such as cancer, infectious diseases, autoimmune diseases, and inflammatory diseases. Due to their high specificity, activity, and low side effects compared to conventional drugs, the market for mAbs has been continuously rising. To improve the economics and adaptability of manufacturing and obtain more consistent product quality, continuous or semi - continuous biomanufacturing for process intensification would be desirable. However, due to the high process complexity, the difficulty in (digital) process integration, and the need to address process variations, there are still numerous challenges to be overcome.

[0243] This study presents fully integrated and continuous upstream and capture steps with an overall control strategy for mAb production. An advanced control strategy was achieved by integrating comprehensive custom software components, associated equipment, and novel process analytical technologies. Using these custom software components, the operation of specific equipment was triggered and controlled based on the monitored process data. Starting from a perfusion process with an alternating tangential flow module for cell retention, mAb was captured from the permeate by simulated moving bed (SMB) affinity chromatography. Changes and variations in setpoints throughout the upstream process were addressed by dynamic flow control. Additionally, to detect mAb breakthrough within the flow-through, dynamic loading of the SMB was enabled by a newly developed sensor based on continuous biolayer interferometry. This novel approach offers several advantages such as high specificity and low background signal, resulting in achieving a desirable (e.g., high) resin utilization rate while reducing (and in some cases, avoiding) product loss. Supported by an overall control strategy, a robust continuous process was operated for several days to obtain high yields and purity of mAb. The results of this study and the newly adopted analytical approach present great potential for establishing adaptable continuous manufacturing of biopharmaceuticals.

[0244] Introduction Monoclonal antibodies (mAbs) are used in the treatment of numerous severe diseases such as cancer, infectious diseases, autoimmune diseases, and inflammatory diseases. Due to their high specificity, activity, and low side effects compared to conventional drugs, the market for mAbs has been continuously rising, representing over 60% of the total revenue of the global biopharmaceutical industry. To reduce manufacturing costs and enable broader patient access, continuous or semi - continuous biomanufacturing for process intensification would be desirable. For continuous upstream process (USP) cultivation, improvements would be desirable. Among USP cultivations, some, unfortunately, require continuous addition of unused medium, removal of spent medium containing mAb by permeate recovery, and retention of cells within the bioreactor. Thus, the introduction of USP in the highly regulated biomanufacturing industry remains difficult due to the complexity of process development and the required automation control strategies. Therefore, it is not yet widely used in the industry for mAb production.

[0245] To avoid bottlenecks and maximize the achievable benefits of such continuous USP, an enhanced downstream process (DSP) would be beneficial. Among DSP processes, some, unfortunately, exhibit limitations in loading capacity and high costs. Reducing such disadvantages by cycling them several times can have an adverse effect on throughput reduction, prolonged processing time, and the associated potential impact on product quality. Applying simulated moving bed (SMB) chromatography as an approach for DSP enhancement presents increased productivity, involving reducing buffer consumption and production facility footprint simultaneously while maintaining product yield and purity at laboratory and pilot scales. Introduction of SMB technology enables resin particles to remain within the packed bed, while "transport" of the resin is achieved using column inlet and outlet switching. Utilization of the multi-column chromatography (MCC) approach loads the breakthrough of the product of one column onto another column to avoid product loss, while simultaneously improving resin utilization and achieving beyond the dynamic binding capacity. This enables a significant improvement as large batch chromatography columns may only be loaded to 65% of their static binding capacity. Once fully loaded, column switching is applied and the column is washed, eluted and regenerated before entering the next loading cycle. Using a countercurrent sequence further improves process efficiency due to enhanced driving force for mass transfer throughout the entire contact zone. As a result, mass transfer can exceed the thermodynamic limits of batch or simultaneous chromatography processes.

[0246] For continuous SMB processes, it may be undesirable to rely on a predefined recipe with respect to loading amount and flow rate, and consequently column switching time. This requires several assumptions, such as the product concentration in the feed solution being constant and known, all columns having the same binding capacity, and the binding capacity remaining constant throughout the process. Deviations from these assumptions can result in either an inefficient process with low productivity or low yield due to product loss. Dynamic loading can be obtained by detecting the breakthrough of mAb in the flow-through of the last column during the loading cycle, which serves as a trigger for column switching. This presents several advantages, such as the ability to correct for variable mAb concentrations in the feed solution (e.g., due to variations in cell culture) and reducing product loss due to column capacity fluctuations.

[0247] (Substantially) real-time process monitoring and control can be implemented using several process analytical technology (PAT) tools for rapid mAb quantification, such as IR or Raman spectroscopy. UV spectroscopy is an example of another technique that can be used for mAb breakthrough detection. One approach is to use UV absorption at 280 nm to calculate the differential signal of two detectors placed at the column inlet and outlet. Another approach is to employ partial least squares regression modeling on the UV / Vis absorption spectrum instead of single wavelength measurements. However, these methods present several disadvantages, such as the low specificity of the UV signal (where mAb and impurities contribute to absorption at 280 nm) and the extremely high background signal due to media components in the feed solution. This poses the challenge of detecting extremely low mAb concentrations within the flow-through to avoid product loss.

[0248] A promising method to overcome these limitations would be biolayer interferometry (BLI). This label-free technology is based on the interference pattern obtained from the combination of white light reflected from an internal reference surface and a biolayer (Figure 33). Due to the high affinity of the analyte to the molecule immobilized on the biocompatible surface and subsequent binding, the thickness at the surface of the biosensor increases, which is then measured in sequence as a concentration-dependent signal by the change in optical interference (Figure 33). For the quantification of mAb, a specific biosensor with protein A immobilized on its surface can be used. Applications to process control strategies such as dynamic loading in continuous SMB chromatography would benefit from online or at-line measurements. In this study, a novel BLI-based prototype enabling continuous at-line measurement of mAbs for breakthrough detection in a continuous SMB chromatography process is presented. This approach was implemented in a comprehensive control strategy of dynamic loading as well as dynamic flow control. The continuous mAb perfusion culture was combined with a continuous capture step and was fully autonomously controlled by an established comprehensive control strategy and run for over 3 days.

[0249] Furthermore, surface plasmon resonance (SPR) technology may be used to implement one or more of the process control strategies disclosed herein, such as dynamic loading and dynamic flow control of continuous SMB chromatography. Like BLI, SPR is a label-free binding technology that can measure the binding rate of biomolecular interactions in real time. In SPR, a sensor chip is used to immobilize one or more binding partners on its surface. The SPR technology may enable continuous on-line or at-line measurement of mAbs for breakthrough detection in a continuous SMB chromatography process and help avoid product loss.

[0250] The SPR technology also brings benefits for mAb breakthrough detection. Similar to BLI, one of the benefits offered by the SPR technology is improved specificity, which results in a lower background signal, a lower detection limit, and enables early detection of mAb breakthrough. This is particularly important for detecting very low mAb concentrations to avoid product loss.

[0251] Materials and Methods Perfusion Culture From a standard batch seed culture of the Cellca CHO DG44 cell line (Sartorius, Germany) expressing IgG1-type mAb, 0.2x10 6They were seeded at cells / mL. The process was controlled at 36.8 °C, pH 7.1, and DO 60%. After a 3-day batch phase, the pH was shifted to 6.95, and perfusion was initiated at 1 vessel volume per day (VVD) using a proprietary perfusion media formulation. For cell retention, an ATF2 device (Repligen, USA) was connected to the bottom drain of the bioreactor. During the initial cell growth phase, the perfusion rate was increased as needed to maintain a cell-specific perfusion rate (CSPR) of 50 pL / cell / day. The process control strategy is described in Figure 34. Briefly, the perfusion rate was controlled using a gravimetric feed flow controller in combination with the removal of cell-free permeate and maintained at a constant working volume of 2 L. Automatic cell bleed was utilized to maintain the target viable cell concentration (VCC). This was achieved by controlling the bleed pump speed based on in-line biomass measurements (BioPAT(R) ViaMass, Sartorius, Germany) using the PID controller of BioPAT(R) MFCS (Sartorius, Germany).

[0252] Continuous capture The cell-free perfusion permeate was transferred to a 2 L intermediate surge tank and continuously processed there by a BioSMB PD chromatography system (Sartorius, Germany). A 5 mL MabSelect SuRe(TM) prepacked HiTrap column (column height 2.5 cm, column diameter 1.6 cm, Cytiva, USA) was used for affinity mAb capture. The initial recipe for all phases was: loading of perfusion permeate, 3 column volumes (CV) interconnect wash, 8.5 CV wash, 4 CV elution, 5 column CV equilibration, 5 CV cleaning in place (CIP) wash, and 5 CV equilibration. Equilibration and washing (PBS, pH 7.4), elution (50 mM C 2 H 3 NaO 2, pH 3.0), and all buffer chemicals used for CIP (0.1 M NaOH) were purchased from Carl Roth (Germany). The flow rate was kept constant at 1.3 mL / min in all steps except loading.

[0253] For the advanced control strategy, the dynamic flow control and dynamic loading functions of the BioSMB PD system were used via their respective OPC UA interfaces. To allow for changes between phases during operation, the dynamic flow function used three phases based on the same recipe with different flow rates for loading (low = 2.1 mL / min, medium = 2.4 mL / min, and high = 2.7 mL / min), equal transition counts, and relative durations for each step. Based on the dynamic loading function, the loading of the perfusion permeate continued beyond the minimum loading volume of 52 CV until mAb was detected in the flow-through of the last column in the loading cycle.

[0254] Continuous Biolayer Interference mAb breakthrough in the flow-through of the SMB capture process was measured by a continuous BLI prototype (Figure 35). For the specific mAb measurements, a biosensor with immobilized Protein A was used (Figures 35 - 36, Octet ProA biosensor, Sartorius, Germany).

[0255] One measurement cycle consisted of the following steps: Sample application (step number 1; flow-through of the last connected column during the loading step) Regeneration (step number 2; 10 mM glycine adjusted to pH 2.0 with hydrochloric acid), and Neutralization (step number 3; PBS, pH 7.4). One complete cycle lasted for 60 seconds (left of Fig. 37). By using a valve upstream of the biosensor and a pump downstream, different solutions required for each step were provided (left of Fig. 35, Fig. 36). In all steps, the pump speed was 120 rpm, and as a result, the flow rate was 1.09 mL / min. The biosensor was irradiated with a lamp using white light, and the interference between the reflected light from the internal coating in the biosensor and the reflected light from the probe tip was detected by a spectrometer (Fig. 36). According to the principle of BLI, as a result of the immobilization of mAb to the protein A biosensor, the thickness of the probe tip increased, and as a result, the wavelength shift and the binding signal increased (left of Fig. 37). This is proportional to the mAb concentration in the solution in order.

[0256] To obtain a concentration-dependent signal with less time lag, the binding rate at step number 1 corresponding to the first derivative of the binding signal was used. In the left of Fig. 37, an exemplary course of the binding signal and the binding rate among three steps in three consecutive cycles is shown. From this, it became clear that the maximum value of the binding rate was obtained more rapidly compared to the binding signal itself that reaches the maximum plateau only after a long time has passed (left of Fig. 37). The maximum binding rate obtained as a function of the mAb concentration of the purified mAb sample diluted with PBS showed an explicit non-linear correlation (right of Fig. 37).

[0257] Control strategy system The process control components used to organize the instruments incorporated into the continuous bioprocess were implemented using the Node-RED integrated development environment (Version 1.3.4, OpenJS Foundation, CA, USA). All devices were part of the same network infrastructure. The communication nodes for interacting with the experimental setup were "node-red-contrib-opcua" for interacting with the OPC UA interfaces of BioSMB implemented in BioPAT(R) MFCS and the permeate surge tank balance (Cubis II, Sartorius, Germany), and the built-in TCP nodes for interacting with the continuous BLI prototypes (all Sartorius Stedim Biotech GmbH, Gottingen, Germany). Timer-related events were implemented using the "node-red-contrib-looptimer-advanced" node. Additionally, control scripts were established using the built-in core nodes (Figure 38). The software components were introduced into a Raspberry Pi 4 B 4GB single-board computer operating on Raspberry Pi OS (Debian 11; Kernel 5.15, Raspberry Pi Foundation, United Kingdom).

[0258] Analysis For the determination of the mAb concentration, size exclusion chromatography (SEC) was used as high performance liquid chromatography (HPLC) with a Dionex UltiMate 3000 HPLC System (ThermoFisher Scientific, Waltham, USA) and a Yarra 3μm SEC 3000 column (Phenomenex, Torrance, USA) at a flow rate of 1 mL / min. 0.1 M Na 2 SO 4 , 0.05 M NaH 2 PO 4 , and 0.05 M Na 2 HPO 4An SEC buffer (final pH 6.6) containing (All chemicals were purchased from Carl Roth, Karlsruhe, Germany) was used. The SEC method has been previously validated using analytical Protein A HPLC (data not shown) to ensure determination of the appropriate mAb. If necessary, samples were diluted with SEC buffer and filtered through a Minisart RC4 0.2 μm syringe filter (Sartorius, Göttingen, Germany) prior to analysis. The mAb concentration was determined based on the peak area (at 220 nm) of a calibration curve obtained using a known reference mAb material.

[0259] The HCP concentration was determined by CHO HCP-ELISA (Cygnus Technologies, Southport, USA) according to the manufacturer's instructions. Samples were diluted with ELISA buffer (20 mM TRIS, 50 mM NaCl; all chemicals were purchased from Carl Roth, Karlsruhe, Germany) if necessary. Measurements were performed using an Infinite M Nano Plus plate reader (Tecan Trading AG, Switzerland) with a detection limit of 1 ng / mL. The DNA concentration was measured by Quant-iT(TM) PicoGreen(TM) dsDNA Assay Kit (ThermoFisher Scientific, Waltham, USA). Samples were diluted with TE buffer (10 mM TRIS, 1 mM EDTA, 0.1% SDS; all chemicals were purchased from Karl Roth, Karlsruhe, Germany) if necessary. Measurements were performed by an Infinite M Nano Plus plate reader with a detection limit of 31.25 ng / mL. The parts per million (ppm) values of HCP and DNA (ppm imp ) were calculated according to Equation 1, where c imp is the concentration of each impurity, and c mAb is the mAb concentration.

Equation

[0260] Results and Discussion Continuous USP Perfusion Culture To enable continuous production of mAb, a 2L stirred tank bioreactor was operated in perfusion mode. By utilizing a membrane-based cell retention device, the product could be continuously removed from the bioreactor and transferred to an intermediate surge tank prior to further processing using a continuous chromatography system. Figure 34 shows a schematic of the perfusion setup and the main control loop implemented to enable a fully automated and robust process even at high cell concentrations. A gravimetric flow controller was used to maintain a stable addition of fresh perfusion medium. The permeate controller was linked to the weight of the bioreactor to maintain a constant working volume. The working volume is affected not only by the addition of feeds, but also by the current bleeding rate and the addition of corrective agents (for example, for foam and pH control), as a result, the permeate flow rate becomes more unpredictable and is more susceptible to fluctuations. To correct for the resulting fluctuations in the permeate flow rate and enable a robust and integrated process, dynamic flow regulation was performed as part of an extensive DSP control strategy. This control strategy was highly advantageous due to reliable culture conditions and an optimal supply of nutrients.

[0261] Cell growth and viability of the perfusion cell culture are shown in Figure 39. As a result of the culture conditions by the applied control loop, high viability and fast cell growth similar to those previously reported for this cell line were observed during the initial growth phase. The target of 45x10 6On the fifth day, immediately before the VCC of cells / mL was obtained, automated cell bleed was initiated to assist in the smooth transition to a steady-state-like phase of the process (Figure 39). The target VCC had only a significant deviation once on the tenth day and was able to adequately control the remaining perfusion operations. Here, the actual VCC was approximately 30% lower than the target VCC (Figure 39), which is a change in the VCC-capacitance correlation and is thus considered to result from the deviation between the online and offline VCC measurements. After recalibration was performed, the process rapidly stabilized, but this deviation affected the current bleed flow rate and thereby also affected the permeate flow rate due to the implementation of the control strategy described above. However, continuous product capture from the perfusion permeate was performed between days 12 and 16 of the culture. During this period, the average mAb concentration in the permeate was approximately 0.7 g / L.

[0262] Adaptive control strategy for dynamic flow and loading for continuous capture Continuous affinity SMB chromatography was used to directly and continuously capture mAb from the perfusion culture. An extensive advanced control strategy was developed to establish a robust process and be able to respond to fluctuations in USP.

[0263] Cell-free perfusion permeate was collected in a surge tank placed on a scale. The surge tank was directly connected to the loading inlet of the SMB chromatography system. The balance signal was transmitted via the OPC UA interface. To correct for fluctuations in the perfusion flow rate from USP and for dynamic flow control that may also occur, the loading flow rate of the SMB chromatography system was adjusted to maintain a constant surge tank weight of approximately 1 kg. The surge tank weight and the current loading flow rate were constantly obtained by an extensive control strategy. A weight of the surge tank less than 0.9 kg resulted in a switch from a medium loading flow rate to a low loading flow rate, while a weight exceeding 1.1 kg induced a high loading flow rate (Figures 38 and 40).

[0264] To correct for the variability of titer within the USP and the different breakthrough times as a result of different loading flow rates, a dynamic loading function was also implemented in the comprehensive control strategy. Based on the original chromatography recipe, the loading and sequential connection to transfer the flow-through of one column to another column continued until the continuous BLI prototype detected the mAb breakthrough in the subsequent column flow-through (Figure 40). After breakthrough detection, the continuous BLI measurement was in standby mode for 108 minutes. This corresponds to the minimum loading time of SMB chromatography based on the highest expected titer throughout the USP to avoid unnecessary measurements. To obtain synchronization between the SMB chromatography system and the continuous BLI prototype, the software component of the comprehensive control strategy continuously polled the status of the chromatography system. The pause / resume function of the overall control script and the associated waiting time until the measurement of the continuous BLI prototype were implemented to ensure a stable process even in the case of unexpected events such as potential overpressure within the chromatography system (Figure 38).

[0265] Throughout continuous capture, the SMB chromatography system performed a total of 34 loading steps in approximately 3 days (Figure 41). To start the process and for the initial loading of the columns, without the dynamic control strategy, the first three loading steps were performed according to the predefined original recipe. Therefore, the loading times for loading steps 1 - 3 were exactly 108 minutes, which is the loading time of the original recipe and corresponds to 1 g / L of the highest expected mAb titer throughout the USP (Figure 41).

[0266] The second cycle corresponding to the 4th loading step began, and after 108 minutes of loading time, continuous BLI measurements were automatically initiated by the comprehensive control strategy. During the interconnected loading steps, the flow-through of the last (second in this setup) column in series was monitored for mAb breakthrough. In Figure 41 (top), the binding rate signal from the continuous BLI prototype is exemplarily shown for the 7th loading step. Based on the periodic measurement principle, the binding rate signal was obtained per minute. The binding rate remained at a low baseline level of less than 0.4 nm until reaching a measurement time of 800 seconds, indicating that no significant amount of mAb was present during the flow-through (Figure 41). Thereafter, the value continued to increase, indicating that mAb was gradually breaking through from the column. At the 27th cycle of the continuous BLI measurement (starting after 1560 s), the defined threshold of the binding rate of 1 nm / min was exceeded (Figure 41), as a result of which the measurement stopped and the column of SMB chromatography was switched by the comprehensive control strategy (Figure 38). It was exemplarily determined that the mAb concentration in the flow-through at the end of the loading step was less than 0.02 g / L for the 7th and 27th continuous BLI measurements, indicating that there was no significant product loss due to dynamic loading.

[0267] Throughout the entire process, mAb breakthrough was only observed after 108 minutes (Figure 41, bottom), thus confirming the expected shortest loading time calculated thereby. Due to insufficient column loading during the initial start-up cycle of SMB chromatography, the loading time of the 4th loading step was significantly longer compared to the other loading steps, which was not controlled by the dynamic loading strategy. In the second half of the process, due to the decrease in the mAb titer in the permeate (Table 1), and because the loading flow rate was partially reduced by dynamic flow control in this time range (Figure 42), a slower mAb breakthrough was brought about (Figure 41, bottom).

[0268] After the initial filling of the surge tank at the start of the process, the first cycle corresponding to the first three loading steps was carried out based on a predefined recipe at a medium loading flow rate (Figure 42). In the next cycle starting from a 5.4 h process time, the dynamic flow control strategy became effective, and as a result, after exceeding the 1.1 kg threshold after approximately 9 h, it switched to a high flow rate (Figure 42). However, due to a slightly increased VCC at the start of the continuous capture implementation (Figure 39) and the resulting higher permeate flow rate, the level of the surge tank further increased to 1.2 kg. However, after the VCC was readjusted back to the target value of approximately 45x10 6 cells / mL and the cell-specific perfusion rate was maintained, the high-speed loading flow rate was sufficient to return the surge tank weight to the desired range. After approximately 45 h of process time, the weight dropped below 1 kg, and as a result, it switched to a medium flow rate, after which the surge tank level increased (Figure 42). Throughout the subsequent process time, the surge tank weight fluctuated between 1 kg and 1.1 kg, which was within the desired range according to the control strategy (Figure 42).

[0269]

Table 1

[0270] Throughout the entire process, only a small amount of mAb (0.013 g / L, the detection limit of SEC used for quantification) was detected in the flow-through, emphasizing again that there was no significant product loss due to the control strategy. Although the pooled waste fractions also did not exhibit a significant mAb concentration, a small amount of product was detected in the wash solution fraction, probably because the wash step of the interconnected columns was too short. However, overall in the process, a high mAb yield of 96.9% was obtained with respect to the total mass of mAb recovered at all outlets. At the same time, throughout the process, process-related impurities were significantly removed. DNA was removed to an average amount of 30.3 ppm, representing a 2.1 log reduction (Figure 43). Moreover, a 3.6 log reduction was obtained for HCP, and as a result, the average amount in the eluate was 37.5 ppm (Figure 43).

[0271] Discussion and conclusions Due to implementing the USP control strategy for perfusion feed, cell bleed, and perfusion permeate, culture conditions with sufficient nutrient supply were obtained, resulting in a robust and well-controlled process that was able to maintain a high cell density with high viability simultaneously. As a result, variations in mAb concentration, as well as potential changes or differences in column binding capacity, were addressed by the comprehensive control strategy, enabling the continuation of such enhanced USP advantages into DSP. Due to dynamic loading control, the column loading was automatically adjusted, achieving good resin utilization without significant product loss. In addition, throughout the entire process, dynamic flow control to maintain the permeate surge tank within the desired range provided a robust continuous process. The resulting changes in loading flow rate changed the breakthrough point during the loading step, which was also addressed by dynamic loading. This further emphasizes the interaction and mutual advantages of the two DSP control strategies applied.

[0272] The advantages of this study's results become even more evident when compared to a similar process without adaptive control. An increase of more than 100% in the mAb concentration in the eluate was obtained by good resin utilization with dynamic loading while leaving the impurity levels equal, compared to a conservative pre-defined recipe. This leads to an improvement in productivity in subsequent DSP unit operations.

[0273] The novel continuous BLI prototype used in this study was able to reliably detect mAb breakthrough at all loading steps. Only a very low background signal was obtained, which probably represents a small amount of mAb leached during loading. Throughout the process, more than 1000 measurement cycles were performed, yet no threshold adjustment or biosensor replacement was necessary. In summary, the novel prototype for continuous BLI presents various advantages. SPR technology may also be used for advanced control of bioprocess unit operations such as mAb capture. Additionally, using different biosensors with other immobilized ligands on the surface presents great potential for the monitoring and process control of several biopharmaceuticals such as recombinant proteins, viruses, exosomes, etc. Moreover, quality attributes such as HCP content, glycosylation pattern, or binding rate can be investigated at-line using appropriate biosensors and system setups.

[0274] Although some aspects of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or advantages thereof. Each such variation and / or modification is to be regarded as within the scope of the invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application(s) for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention provided such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0275] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of defined terms. As used in the specification and claims of this application, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated to the contrary.

[0276] As used herein, the phrase "and / or" as used in the specification and claims is to be understood to mean "either or both" of the elements so connected, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed by "and / or" are to be construed in the same fashion, i.e., as "one or more" of the elements so connected. Other elements may optionally be present whether or not they are related to those specifically identified, apart from the elements specifically identified by the "and / or" clause. Therefore, by way of non-limiting example, saying "A and / or B" when used in conjunction with open-ended words such as "comprising" can mean, in one aspect, only A (optionally including elements other than B), in another aspect only B (optionally including elements other than A), and in yet another aspect both A and B (optionally including other elements).

[0277] As used herein, the "or" as used in the specification and claims is to be understood to have the same meaning as the "and / or" defined above. For example, when separating listed items, "or" or "and / or" is inclusive, i.e., construed as including at least one of a number of elements or a listing of elements, but also including more than one and optionally additional unlisted items. Only terms expressly commanded to the contrary, such as "only one of" or "exactly one of" or "consisting of" when used in a claim, will refer to the inclusion of exactly one of a number of elements or a listing of elements. Generally, as used herein, the term "or" is to be construed to mandate exclusive alternatives (i.e., "not both, one or the other") only when preceded by exclusive terms such as "either", "one of", "only one of", or "exactly one of". "Consisting essentially of" shall have its ordinary meaning as used in the field of patent law when used in a claim.

[0278] As used herein, in the specification and claims, the phrase "at least one," when used in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically listed in the list of elements, and does not exclude any combinations of elements from the list of elements. This definition also allows for the possibility that elements other than those specifically identified from the list of elements referred to by the phrase "at least one" may optionally be present, whether or not related to those specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B," or equivalently "at least one of A and / or B") can, in one aspect, be at least one A where B is absent (and optionally includes elements other than B), including more than one A optionally; in another aspect, be at least one B where A is absent (and optionally includes elements other than A), including more than one B optionally; and in yet another aspect, be at least one A including more than one A optionally and at least one B including more than one B optionally (and optionally includes other elements).

[0279] It should also be understood that, unless expressly ordered to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0280] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like should be understood to be open, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are to be considered closed or semi-closed transitional phrases, respectively, as defined in U.S. Patent Office Patent Examination Manual Section 2111.03.

Claims

1. The system is as follows: A first instrument comprising a probe and an optical detector configured to detect optical signal fluctuations over a first period of time; and Including a bioprocessing system, Here, the system is configured to supply fluid from the bioprocessing system to the first instrument. Here, the first instrument is configured to determine, based on optical signal fluctuations over a first period, the rate at which the analyte is fixed onto the probe while the fluid is in contact with and flowing over the probe. Here, the optical signal includes light reflected from the inner interface of the probe and light reflected from the ends of the probe, and the system is configured to send commands to the bioprocessing system based on the determination of the rate at which the analyte is fixed on the probe. The aforementioned system.

2. The method is as follows: The probe is brought into contact with a fluid for a first period, where the fluid is supplied by a bioprocessing system, the fluid flows over the probe, the analyte is present in the fluid at a first concentration, and at least a portion of the analyte becomes immobilized on the probe; To detect variations in the optical signal over a first period; and This includes determining the rate at which the analyte is fixed onto the probe based on variations in the optical signal over a first period, Here, the optical signal includes light reflected from the inner interface of the probe and light reflected from the ends of the probe. Based on the determination of the rate at which the analyte is fixed onto the probe, a command is sent to the bioprocessing system. The aforementioned method.

3. The first device is as follows: probe; and Includes an optical detector configured to detect changes in an optical signal over a first period of time, Here, the first instrument is configured to contact a probe and determine a first concentration of the analyte in the fluid flowing over the probe, based on the variation of the optical signal over a first period of time. Here, the optical signal includes light reflected from the inner interface of the probe and light reflected from the ends of the probe. The aforementioned device.

4. The system according to claim 1, wherein the fluid is supplied to the first instrument as output from a bioprocessing system, or the fluid is supplied to the first instrument in an automated manner.

5. The system includes a second bioprocessing system, and The system according to claim 1, wherein the second bioprocessing system is configured to supply a second fluid output from the second bioprocessing system to the first instrument.

6. The system according to claim 1, wherein the bioprocessing system includes a chromatography system, a filtration system, and / or a bioreactor.

7. The system according to claim 6, further comprising a second bioprocessing system, wherein the second bioprocessing system comprises a bioreactor.

8. The method according to claim 2, wherein the bioprocessing system comprises a chromatography system, and the method further comprises washing a fluid-supplied column, eluting a fluid-supplied column, and / or regenerating a fluid-supplied column.

9. The method according to claim 2, wherein the fluid in contact with the probe is a first sample supplied by a sample supply source, the following: Close the first valve to remove the sample source from fluid communication with the probe; Open the second valve to bring the regenerating fluid source into fluid communication with the probe; and Bringing the probe into contact with the regenerating fluid, It further includes, Here, by opening the first valve, the sample is supplied directly to the probe, and / or by opening the second valve, the regenerating fluid is supplied directly to the probe. The aforementioned method.

10. The system according to claim 1, wherein: Multiple valves located upstream of the probe, This further includes, here The multiple valves include a first valve positioned between the sample supply source and the probe, and a second valve positioned between the regeneration fluid supply source and the probe, and By opening the first valve, the sample is supplied directly to the probe, and / or by opening the second valve, the regeneration fluid is supplied directly to the probe. The aforementioned system.

11. The method according to claim 2, wherein the method is carried out in a first instrument, the fluid is a first sample, and the probe is a first probe, the following: In the first apparatus, the following: The first probe is brought into contact with a first fluid sequence, where the first fluid sequence includes a first sample and a regenerating fluid. The second probe is brought into contact with a second fluid sequence, where the second fluid sequence includes a second sample and a regenerating fluid, and After bringing the first probe into contact with the regenerating fluid, bring the first probe into contact with the third sample. To carry out the following steps, The method further includes the above.

12. The system according to claim 1, further comprising a valve located upstream of the probe, the valve configured to switch between a fluid source, a regenerating fluid source, and a neutralizing fluid source.

13. The method according to claim 2, wherein the first instrument includes a valve positioned upstream of the probe, the valve is configured to switch between a fluid source, a regenerating fluid source, and a neutralizing fluid source, and the method further includes switching the valve to remove the regenerating fluid source from fluid communication with the probe and to fluid communication with the neutralizing fluid source.

14. The system according to claim 12, wherein the first instrument further includes a source of diluent, and the first instrument is configured to mix a plurality of samples upstream of a valve with the diluent.

15. The system according to claim 1, wherein the first instrument includes a plurality of light sources, each associated with a different probe.

16. The system according to claim 1, wherein the first instrument includes one or more light sources, and two or more probes are associated with a single light source.

17. The system according to claim 1, wherein the first instrument is interfaced with an additional instrument that is carrying out a bioprocess, and the first instrument monitors the bioprocess.

18. The system according to claim 1, wherein the commands include commands to modify one or more properties of a fluid in a bioprocessing system, commands to supply the fluid to a different location, commands to pause, and / or commands to do nothing.

19. The method according to claim 2, for the purpose of determining the binding capacity of a column to an analyte.

20. The method according to claim 2, wherein the thickness of the end of the probe is increased by fixing the analyte on the probe, resulting in increased wavelength shift and coupling signal.

21. The method according to claim 2, further comprising determining a first concentration of the analyte, determining the binding constant of the analyte, and / or determining the dissociation constant of the analyte based on the rate at which the analyte is fixed onto the probe.

22. The method is as follows: The probe is brought into contact with a fluid over a first period of time, the fluid being supplied by a first bioprocessing system, the fluid flowing over the probe, the analyte being present in the fluid at a first concentration, and at least a portion of the analyte being immobilized on the probe; To detect fluctuations in the optical signal over a first period; Determining a first concentration based on the variation of the optical signal over a first period, where the optical signal includes light reflected from the inner interface of the probe and light reflected from the ends of the probe; and Based on the determination of the first concentration, an instruction is sent to the second bioprocessing system. The method, including the method described above.

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