In-line flow cell for monitoring bioprocesses
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-08-13
Smart Images
Figure US2026013840_13082026_PF_FP_ABST
Abstract
Description
Docket: 0376-0034W01IN-LINE FLOW CELL FOR MONITORING BIOPROCESSESRELATED APPLICATIONS[ oooi] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 63 / 755,498, filed on February 7, 2025, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION
[0002] Many processes in the chemical, biochemical, pharmaceutical, food, beverage and in other industries benefit from some type of analysis. Of particular importance is the identification and often the quantification of substances present. One common technique involves obtaining a sample and detecting one or more of its components, sometimes referred to as analytes.
[0003] Analytes can be assessed by various optical spectroscopy approaches. Among these, probably the most common is absorption spectroscopy. Incident light excites electrons of the analyte from a low energy ground state into a high energy, excited state, and the energy can be absorbed by both non-bonding n-electrons and 7t-electrons within a molecular orbital. Absorption spectroscopy can be performed in the ultraviolet, visible, and / or infrared region, with analytes of varying material phases and composition being interrogated by specific wavelengths or wavelength bands of light. The resulting transmitted light is then used to resolve the absorbed spectra, to determine the analyte's or sample’s composition, temperature, pH and / or other intrinsic properties for applications ranging from medical diagnostics, pharmaceutical developments, food and beverage quality control, to list a few.
[0004] Many existing instruments use light in the visible and / or ultraviolet (UV) region of the electromagnetic spectrum. For the past decade, for example, UV-Visible systems have been the gold standard for measuring protein and monoclonal antibodies (mAb) concentrations. However, the accuracy and reproducibility of UV-visible equipment can suffer from dynamic range limitations due to extremely strong absorption by proteins in the UV region, with typical maximum absorptions of about 3 to 4 absorbance units (AU). A partial solution was the development of systems that utilize variable pathlengths.
[0005] In U.S. Patent Application Publication No. 2019 / 0358632A1, Hassell et al. describe analyses of culture media using near infrared (NIR) spectroscopic techniques.Docket: 0376-0034W01
[0006] Applications of NIR-based techniques to measurements of samples in a flow cell are described in U.S. Patent Application Publication No. 2020 / 0240902 Al, to Hassell et al.
[0007] U.S. Patent No. 11,499,903 to Hassell et al. describes robust, hands-free, nondestructive, real-time NIR techniques for identifying and / or quantifying constituents in a given process, using an in-situ probe that can be inserted and / or maintained in a bioreactor.
[0008] Conducting measurements on static samples is described in International Publication No. WO 2024 / 102825, filed on November 8, 2023. A clam shell apparatus for analyzing a static or flowing sample is disclosed in International Patent Application No. PCT / US2024 / 037166, filed on July 9, 2024. Both documents are incorporated herein by this reference in their entirety.SUMMARY OF THE INVENTION
[0009] Even with the advantages obtained with in-situ monitoring, many applications benefit from analyzing samples outside a processing vessel, a reactor, for example.Accordingly, a need exists for developing equipment and techniques in which a sample is extracted from the reaction vessel and transferred to a sample cell, where it can be analyzed with respect to constituents and / or other parameters. A need also exists for in-line designs that allow real-time measurements of samples flowing through loops or other fluidic arrangements. In-line sample (also referred to herein as “flow”) cells that can be detached for disposable, e.g., single-use, or for multi-use sterilization are of particular interest.Fresh approaches to mitigating bubbles are needed as well.
[0010] Turning to specific applications, a need exists for real-time, simultaneous measurements of excipients (stabilizers, solubilizers, buffers, etc.) to ensure the quality and stability of an end-product, during biopharmaceuticals manufacturing, or other processes.
[0011] Desired too are systems and methods that address limitations associated with existing UV-visible spectroscopic analyzers, such as, for instance, problems raised by current variable pathlength approaches. Variable pathlength limitations are particularly pronounced at later stages in downstream processing, often characterized by very high mAb concentrations (e.g., > lOmg / mL). As concentration of the mAb increases, pathlength must decrease to accommodate the sensor saturation limit. However,Docket: 0376-0034W01pathlength reproducibility at very small lengths (less than 100 micrometers or microns (pm), for example) is difficult to achieve.
[0012] Recognizing the advantages associated with NIR spectroscopic techniques, a need for developing instrumentation that realizes these advantages continues to exist. Systems that facilitate or even enhance NIR analytical approaches or expand these approaches into the mid-infrared (MIR) range are of great interest.
[0013] Particularly desired are instruments that do not rely on variable pathlength approaches. Simplified approaches that do not require complex moving stages or dilution continue to be of interest.
[0014] In general, the invention pertains to equipment and / or techniques that address(es) at least some of the needs described above.
[0015] In one aspect, the invention features a flow cell for in-line measurements. In some examples, the flow cell is designed for single use. In others, the flow cell is a multiuse (i.e., two or more) flow cell that can be sterilized as needed (between different runs, for example), by autoclaving or gamma irradiation.
[0016] The flow cell defines a sample flow pathway that can be supported by a channel or a conduit formed in the flow cell body. For conducting the sample analysis, the flow pathway includes a sample detection region, also referred to herein as a “sample gap”. In specific embodiments, the flow pathway forms an angle, e.g., of 90 degrees (°). Together with a preferred sample flow direction, the angled flow cell design can contribute to bubble mitigation. For instance, the sample can enter the flow cell along a first direction which changes to a second direction due to the angle discussed above, also referred to herein as an “elbow”. In one example, the flow along the second direction moves upwards, against the force of gravity. In this arrangement, the sample detection region is located above the elbow. For a 90° angle, the sample gap is located along the vertical leg of the flow pathway.
[0017] In embodiments, the sample gap is formed between rods (a transmission rod and a detection rod) constructed from a material that transmits light in the desired region of the electromagnetic spectrum, e.g., the near infrared (NIR), short-wavelength infrared (SWIR), mid- wavelength infrared (MIR) or long-wavelength infrared (LWIR). SomeDocket: 0376-0034W01approaches employ rods designed and / or oriented to reduce or minimize reflections / etalons.
[0018] In specific implementations, the rods are fixed in position, providing a sample gap that does not vary. While the sample gap remains set during a measurement or a series of measurements, some construction details allow for resetting the pathlength as a new or different sample is analyzed.
[0019] The distance between the rods, corresponding to the sample gap and thus to the path length over which light interacts with the sample, can be set (fixed and maintained) to a value within a range of from about 0.010 millimeters (mm) to about 10 mm. In illustrative implementations, the pathlength has a value within a range of from about 0.010 mm to about 5 mm.
[0020] The flow cell, including the rods defining the sample gap, can be housed in a (flow cell) module that can further include elements for analyzing the sample. For instance, the module can include one or more light detectors (e.g., a reference photodetector and a sample photodetector) and optical elements (such as a beam splitter, polarizer, etc.). In embodiments, the module is configured to separate or de-couple the sample flow path (defined in the body of a detachable flow cell) from components relied upon to analyze the sample, the latter being non-detachable or permanently part of the module.
[0021] The flow cell module can itself be part of a system for sample analysis. For example, the system can include a light source, e.g., a laser such as a tunable laser. In embodiments, the light source is de-coupled from the module. This can result in light being emitted or generated from a light source that is external to the module.
[0022] The system can further include conduits for guiding the light from the light source to the module. In many implementations, the system employs fiber optic technology to transmit light to the module. Connectors and / or electrical cables for transmitting an electrical signal from the reference and / or sample detector to an analyzer also can be part of the system.
[0023] Inside the module, the light traces a light pathway that, in many configurations, is orthogonal to the sample flow pathway, e.g., orthogonal to the channel supporting the sample flow through the flow cell.Docket: 0376-0034W01
[0024] In embodiments, the system also includes conduits, tubing, connectors, or other elements supporting the flow of the sample to or from the flow cell. The module can be inserted into existing conduits (tubing, pipes, etc.) used to introduce or withdraw materials from a vessel; as such, these conduits provide a sterile environment. In one example, the module is inserted in a conduit configured for flowing a sample from one vessel to another, in a chemical or biochemical process.
[0025] The system can further include a controller for controlling the sample flow, laser operation, scanning parameters, spectral analyses and / or other functions.
[0026] The sample in the sample gap of the flow cell is analyzed by absorption spectroscopy in a desired wavelength region of the electromagnetic spectrum. For instance, the interrogation light beam employed can be in the infrared (e.g., near- or mid-IR), visible or ultraviolet (UV) region.
[0027] In another aspect, the invention features a method for analyzing a sample. The method comprises flowing a sample through a sample detection region; generating a swept wavelength signal; transmitting the swept wavelength signal to and though the sample detection region; detecting the swept wavelength signal after transmission through the sample detection region; and resolving an absorption spectrum of the sample. The swept wavelength signal can intersect the sample detection region orthogonally to the sample flow pathway. In some approaches, the swept wavelength signal is detected prior to transmission through the sample detection region. An absorption spectrum of the sample is resolved with reference to the swept wavelength signal before and after transmission through the sample detection region. In some implementations, the method also includes transmitting the swept wavelength signal from an external light source to a module in which the sample detection region is formed in a detachable flow cell.
[0028] In one illustrative example, light generated from a laser external to the module enters the module at a fiber port. Inside the module, the light passes through a polarizer and is then split into a reference beam, that is directed to a reference detector that can be located inside the module, and a sample beam (also referred to herein as an “interrogation” beam) that is directed to and through the sample detection region (sample gap). After interacting with the sample in the detection region, the transmitted light is detected by a sample detector that, in many implementations, is also located within the module. SignalsDocket: 0376-0034W01from the sample and reference detectors can be analyzed, e.g., by a controller, to obtain absorption spectra of species of interest.
[0029] The method can further include flowing the sample along a flow pathway and / or in a direction that results in bubble mitigation in the sample detection region. In some implementations, the sample flow changes direction before entering the sample detection region. For instance, the sample can pass through an elbow first.
[0030] Additional steps can include inserting a detachable flow cell, removing (detaching) the detachable flow cell that supports flowing the sample through the sample detection region from an in-line module; sterilizing the detachable flow cell; and / or reinserting the sterilized flow cell into the in-line module.
[0031] The sample can pass through the flow cell and thus through the sample detection region in a continuous or intermittent flow pattern. In one example, the sample is withdrawn from a vessel such as a bioreactor, delivered to and through the flow cell where it is analyzed, then returned to the original vessel or flown to a different vessel. If desired, the flow can be stopped and the sample can be maintained in a stationary mode, e.g., for a time sufficient to scan or analyze it. In other approaches, the sample is scanned or analyzed as a flowing sample, with the sample being continuously “refreshed’ as it flows through the sample gap.
[0032] Embodiments of the invention have many applications and can be practiced in various upstream or downstream bioprocessing operations. In many cases, practicing techniques described herein can lead to increased accuracy relative to existing approaches. Applying principles of the invention to mAb measurements, for instance, can result in a 1% error for concentrations of 0.1 to 1000 mg / mL.
[0033] Aspects of the invention are highly versatile and can find application in any number of processes or equipment.
[0034] Thus, in some embodiments, the system, module, and / or detachable flow cell described herein are used in upstream bioprocessing operations by placement in a recirculation loop associated with a bioreactor or other cell-culture vessel. In one example, cell-culture fluid is withdrawn from the vessel, directed through the detachable flow cell for in-line spectroscopic measurement, and returned to the vessel in a closed, sterile flow path. In such configurations, the resolved spectra and / or multivariate models derivedDocket: 0376-0034W01therefrom can be used to determine concentrations of active pharmaceutical ingredients (API) or target molecules of interest, nutrients and metabolites, including glucose and lactate, and / or to determine cell-related parameters such as optical density, cell density, and / or viable cell density, while maintaining the sterile boundary of the process. For instance, in a bioreactor producing an antibody, it is possible to measure glucose, lactate, protein titer and cell density.
[0035] In further embodiments, the system is integrated into downstream unit operations including tangential flow filtration (TFF) and associated ultrafiltration / diafiltration (UF / DF), chromatography, and sterile filtration. In examples, the detachable flow cell is installed not only in a retentate or recirculation line of a filtration system, but also in a permeate line downstream of a TFF membrane, to monitor permeate composition and / or membrane performance in real time. In chromatography applications, the flow cell can be positioned to interrogate column effluent during loading, washing, elution, and / or fraction collection for different chromatography types (including Protein A and ion exchange) and different modalities (including proteins and viral vectors such as AAV). In sterile filtration applications, the flow cell can be positioned upstream and / or downstream of a sterilizing-grade filter to monitor feed and / or filtrate and support process endpoint determinations and / or filter performance assessments.
[0036] Advantageously, for mAb applications, in addition to measuring protein concentrations of 0.1 to 1000 mg / mL, it is possible to simultaneously measure excipients such as histidine, arginine, methionine, polysorbate, sucrose, to name a few. To illustrate, in addition to measuring mAb, histidine can be measured at 1 to 100 mg / mL concentrations, while polysorbate can be measured at 1 to 2 mg / mL concentrations.
[0037] Practicing aspects of the invention in In Vitro Transcription (IVT) and / or tangential Flow Filtration (TFF) ultra filtration (UF) unit operations of concentration and buffer exchange and diafiltration (DF) applications, can provide real-time “fingerprinting” of excipients, offering numerous advantages that can reduce the time to reach the DF endpoint and monitor for the desired product concentration.
[0038] While the invention can be practiced with UV, visible or IR electromagnetic radiation, certain applications benefit from using NIR (or other IR-based spectroscopic approaches, MIR, for instance). To illustrate, at mAb concentration of 10 mg / mL and higher, typical UV-visible protocols require dilutions. In contrast, NIR, MIR, etc. canDocket: 0376-0034W01typically yield accurate, linear results, without need for complex protocols, moving stages, variable pathlengths and / or the need for dilutions.
[0039] Also, whereas UV-visible techniques typically rely on analytics or curve fitting to an extinction coefficient for measurement, approaches that may be adversely affected by the presence of excipients in the background, mAb spectra in the NIR region are found to be consistent and unique regardless of background.
[0040] Importantly, all measurements can be carried out using one system and, typically, a single scan. Durable and highly reliable, equipment and techniques according to the invention can provide repeatable results that are easy to validate. Measurements can be very rapid (seconds), often 50 times faster than those available with competing technologies. In many cases, the scan time is about 5 seconds.
[0041] Easy to use instrumentation involves a simple workflow, with minimal or no setup or calibrations. The analysis too is far from complicated and can be conducted without input from highly skilled personnel. As a result, the equipment and methods described herein can offer competitive pricing for capital and service.
[0042] In many of its aspects, the invention provides a sterile flow pathway from the vessel under investigation, for example, to and through the sample cell.
[0043] De-coupling the light source from the flow cell and / or the module helps to ensure measurement repeatability and maintain a sterile environment. Furthermore, arrangements utilizing a light source that is external to the module minimize the module footprint.
[0044] Conveniently, approaches described herein decouple a detachable flow-cell from a module that can remain in its in-line placement, simplifying mounting and dismounting efforts. In addition, the detachable flow cell can be separated from other elements present in the module, safeguarding elements such as detectors, polarizers, beam splitters, etc. from being autoclaved together with the flow cell.
[0045] Since single use equipment is typically pre-sterilized and flow cells developed for repeat use are autoclavable, either approach can ensure a sterile sample flow pathway. Moreover, with a detachable design, the flow cell can be inserted into an already sterile flow conduit arrangement.Docket: 0376-0034W01
[0046] In some implementations, aligning the sample detection region in the light pathway is as simple as securing the flow cell to the module, using screws, for instance. This obviates the need for kinematic mounts or other more complicated alignment devices.
[0047] Techniques such as the ones described herein also improve the quality of the analysis. For example, embodiments described herein can provide improved or even maximum signal to noise ratios (SNR). This is accomplished by launching a light beam straight out of a fiber and / or a free space link, through a sample gap, with the transmitted light impinging onto a photodetector, typically located within the flow cell module.Having the detector cables running back to the spectrometer (rather than using a return fiber optic cable leading to a photodiode) removes a source of noise.
[0048] The rod arrangement relied upon in some implementations addresses reflections / etalons concerns and eliminates the need for cuvettes. This is particularly advantageous considering that even protocols that call for glassware wash and reuse involve extra effort. Also, since cuvettes can display changes in thickness, depending on the particular cuvette, and even depending on where the scan is performed in the same cuvette, quality of the analysis can suffer. Moreover, cuvette designs rely on parallel or nearly parallel surfaces that can cause massive etalons / reflections.
[0049] The fiber tapping techniques described herein ensure stable and consistent performance by extracting a small portion of the optical signal for monitoring purposes without significantly disrupting the main signal.
[0050] The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.BRIEF DESCRIPTION OF THE DRAWINGS
[0051] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:Docket: 0376-0034W01
[0052] FIGS. 1 A through ID are perspective views of the flow cell according to one embodiment of the invention;
[0053] FIG. IE is a cross-sectional view of the flow cell of FIGS. 1A through ID, showing the flow path through the flow cell;
[0054] FIG. IF is another cross-sectional view of the flow cell of FIGS. 1 A through ID, showing optical elements supporting light transmission and defining a sample detection region;
[0055] FIG. 2A is a side perspective view showing rods forming a sample detection region;
[0056] FIG. 2B is a side perspective view of a cross-section showing the rods of FIG.4A rotated from each other by an angle of 90degrees (°) around the longitudinal axis;
[0057] FIG. 3 A is a schematic diagram of a light beam traveling through rods constructed and orientated as shown in FIGS. 4 A and 4B;
[0058] FIG. 3B is a graph showing the absence of overlap between the main beam and a secondary beam obtained using the rods constructed and oriented as shown in FIGS. 4 A and 4B;
[0059] FIG. 3C is a plot of the effective etalon strength with reflection through water as a function of the angle between beams 9;
[0060] FIG. 4A is a perspective view of a module including a flow cell, according to one embodiment of the invention;
[0061] FIG. 4B is a perspective view of a module in which the flow cell has been removed from the module body;
[0062] FIG. 5A is a schematic diagram of a system including the module of FIGS. 4A and 4B, according to one embodiment of the invention;
[0063] FIG. 5B is a schematic diagram of the system of FIG. 5A, showing module components according to one embodiment of the invention;
[0064] FIG. 6 is a series of plots of real-time diafiltration monitoring of the concentration of several substances measured simultaneously according to embodiments of the invention;Docket: 0376-0034W01
[0065] FIG. 7 A presents real-time measurements of BSA during ultrafiltration (UF);
[0066] FIG. 7B illustrates the sensitivity of the method in measuring release of membrane bound BSA once the UF was completed.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0067] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
[0068] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and / or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and / or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.
[0069] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0070] The invention generally relates to approaches for detecting and often quantifying compounds (analytes) present in a sample. The sample can be obtained from a vessel, a bioreactor, or a mixing tank, for example. Materials that can be investigated include but are not limited to components in culture media, nutrients, metabolites,Docket: 0376-0034W01enzymes, hormones, cytokines, proteins, and so forth. In embodiments, the apparatus and method described herein find applications in protein, e.g., mAb, manufacturing.
[0071] Many of the techniques described herein rely on spectroscopic approaches for determining the spectral response of sample analytes. In embodiments, samples are analyzed by absorption spectroscopy in the ultraviolet (UV), visible, or infrared (IR) region of the electromagnetic spectrum.
[0072] Of particular interest is spectroscopy, and particularly absorption spectroscopy, that covers the near infrared (0.75-1.4 pm, NIR), short-wavelength infrared (1.4-3 pm, SWIR), mid- wavelength infrared (3-8 pm, MWIR), long-wavelength infrared (8-15 pm, LWIR), and the far infrared (15-1000 pm, FIR) of the spectrum. In examples, the method and system described herein are operated in the NIR through MIR region, at a wavelength that is within a range of from about 1350 to about 1800 nanometers (nm) or within a range from about 2050 to about 2400 nm. For MIR analyses the wavelength can be within a range from about 3.5 to about 10 microns.
[0073] Probing molecular overtone and combination vibrations, NIR-SWIR spectroscopy covers a region of from 780 nanometer (nm) to 2500 nm of the electromagnetic spectrum. In a shorthand approach, this region between 780 nm to 2500 nm can be simply referred to by the abbreviation “NIR”. An overview of NIR spectroscopy can be found, for example, in an article by A.M.C. Davies in “An Introduction to Near Infrared (NIR) Spectroscopy”, (website www.impublications.com / content / introduction-near-infirared-nir-spectroscopy). See also, Cervera, A. E., Petersen, N., Lantz, A. E., Larsen, A. & Gemaey, K. V. “Application of near-infrared spectroscopy for monitoring and control of cell culture and fermentation”, Biotechnol. Prog. 25, 1561-1581 (2009); and Roggo Y, et al., “A review of near infrared spectroscopy and chemometrics in pharmaceutical technologies”, Journal of Pharmaceutical and Biomedical Analysis, Volume 44, Issue 3, 2007.
[0074] For many applications, the light employed to carry out the sample analysis is generated by a tunable laser, which can be part of a laser spectrometer. Other technologies are used in other examples such as dispersive systems and / or Fourier transform infrared (FTIR) systems. One example employs a widely tunable quantum cascade laser (QLC).
[0075] Tunable laser spectrometers will typically have a wavelength reference and a power reference. The wavelength reference allows the device to track its wavelengthDocket: 0376-0034W01sweep through the tunable laser’s spectral scan band. The power reference detects the instantaneous power during the sweep so that any variance in the power can be compensated to accurately resolve the absorption spectra of the material of interest.
[0076] In some implementations, the wavelength of the light generated by the laser is within a region of from about 1350 to about 1800 nm, such as from about 1350 nm to about: 1400 nm, 1500 nm, 1600 nm, 1700 nm; or from about 1400 nm to about: 1500 nm, 1600 nm, 1700 nm or 1800 nm; or from about 1500 nm to about: 1600 nm, 1700 nm, 1800 nm; or from about 1600 nm to about: 1700 nm, 1800 nm; or from about 1700 nm to about 1800 nm. In other implementations, the wavelength employed is within a range of from about 2050 nm to about 2400 nm, such as from about 2050 nm to about: 2100 nm, 2200nm, 2300 nm; or from about 2100 nm to about: 2200 nm, 2300 nm, 2400 nm; or from about 2200 nm to about: 2300 nm, 2400 nm; or from about 2300 to about 2400 nm.
[0077] The instantaneous narrow band emission from the laser is typically less than 100 nm wide, Full Width at Half Max (FWHM). More often it is less than 50 nm wide and can be less than 25 nm wide or even less than 15 nm.
[0078] In the MIR region, the wavelength of the laser light can be within a range of from about 3.5 microns to about 10 microns, such as from about 3.5 microns to about: 4, 5, 6, 7, 8, 9 microns; or from about 4 microns to about 5, 6, 7, 8, 9, 10 microns; or from about 5 microns to about: 6, 7, 8, 9 10 microns; or from about 6 microns to about: 7, 8, 9, 10 microns; or from about 7 microns to about: 8, 9, 10 microns; or from about 8 microns to about: 9, 10 microns; or from about 9 to about 10 microns.
[0079] For some applications the tunable laser is optimized for a specific wavelength range which contains relevant, e.g., protein-critical chemical information (C-H, O-H, etc.). In one embodiment the tunable laser sweeps its wavelength in a spectral band including 2.3 micrometers in wavelength and sweeps through greater than 100 nanometers in wavelength. In one implementation, the laser sweeps through a spectral band extending from about 2.2 to 2.4 micrometers in wavelength.
[0080] Currently, the tunable laser has a semiconductor gain chip for amplification. The laser’s amplification is provided by a GaSb gain chip, in one example. Nevertheless, other material systems can be selected for the gain chip according to other examples.Common material systems are based on III-V semiconductor materials, including binary materials, such as GaN, GaAs, InP, GaSb, InAs, as well as ternary, quaternary, andDocket: 0376-0034W01pentenary alloys, such as GaAlAs, InGaN, InAlGaN, InGaP, AlGaAs, InGaAs, GalnNAs, GalnNAsSb, AlInGaAs, InGaAsP, AlGaAsSb, AlGalnAsSb, AlAsSb, InGaSb, InAsSb, and InGaAsSb. Collectively, these material systems support operating wavelengths from about 400 nanometers (nm) to 2500 nm, including longer wavelength ranges extending into multiple micrometer wavelengths. Semiconductor quantum well, quantum cascade and quantum dot gain regions are typically used to obtain especially wide gain and spectral emission bandwidths, and support operation up to 250 pm in wavelength. Quantum well layers may be purposely strained or unstrained depending on the exact materials and the desired wavelength coverage.
[0081] For high performance operations, all power variability must be fully compensated. And one source of power variability arises from the highly polarized nature of diode lasers. As a result, small changes in the polarization in conjunction with polarization dependent loss (PDL) in the different components such as lenses, beam splitters and detectors will result in an untracked power variability that will degrade the accuracy of the absorption spectra.
[0082] To ensure the polarization stability that is required to manage PDL, polarization maintaining fiber, such as polarization maintaining single mode optical (PANDA) fiber can be employed in conjunction with the tunable laser spectrometer. The use of polarization maintaining fiber, however, does not entirely solve the problem. The phenomenon of fiber polarization beat or PANDA ripple also arises because there is usually some power in the non-preferred polarization and this will beat with the power in the preferred polarization, causing power fluctuations. More generally, when two waves with different linear polarization states propagate in the birefringent polarization maintaining (PM) fiber, their phases will evolve differently. The difference in phase delay will be proportional to the fiber length.
[0083] Specific embodiments address at least some of these issues, as further described below. Details also can be found in US Patent Publication No. 2024 / 0117293 and International Publication WO 2024 / 076868, both published on April 11. 2024, both being incorporated herein by this reference.
[0084] In some of its aspects, the invention features an arrangement that separates or de-couples the fluidics (conduits, channels, etc., that are used to direct a sample to andDocket: 0376-0034W01through a sample detection region) from elements (lenses, polarizer, beam splitter, detectors, fiber optics, etc.) used to analyze the sample.
[0085] One aspect of the invention involves an in-line flow cell, a flow cell that can be integrated, e.g., inserted, into a flow arrangement in which a sample is extracted from a vessel (a bioreactor, mixing vessel, etc.), directed to the flow cell where it is analyzed, then returned to the original vessel or passed on to a different vessel.
[0086] The flow cell can be made from a metal, stainless steel, for instance, or a suitable plastic material. A detachable design ensures that the flow cell can be inserted into a flowing arrangement and / or can be removed for disposal or for cleaning.
[0087] For repeated use, detaching the flow cell for autoclaving (an approach particularly well suited for stainless steel construction) or gamma irradiation (for plastic flow cells) ensures a sterile sample flow pathway. A detachable design is also compatible with disposable, e.g., single use flow cells, which can be pre-sterilized (for a sterile sample flow pathway) and discarded at the end of a given experiment.
[0088] Shown in FIGS. 1A, IB, 1C, ID, IE and IF is flow cell 11 having base 13 and flow cell body 15. Base 13 can be mounted into a module (further described below), using screws and respective holes 17 or other suitable attachments.
[0089] Flow cell body 15 defines a flow path supported, for instance by conduit or channel 19. This channel can be formed by a technique that can be selected by considering the flow cell material, flow cell dimensions and / or other criteria. For example, a channel can be formed in a plastic flow cell by constructing the flow cell using 3D printing. In a metal flow cell body, channel 19 can be formed by laser or plasma machining or other suitable techniques. More than one technique can be employed to produce different sections of the channel.
[0090] As seen in FIG. IE, channel 19 includes segments 21 and 23, angled (e.g., by 90°) with respect to each other at elbow 25. Openings 27 and 29, optionally provided with fittings, are configured to receive connectors for the in-line mounting of the flow cell into a flowing arrangement. In one implementation, connectors 31 and 33 are barbed connectors.
[0091] The angled geometry of channel 19 can be combined with a preferred flow direction to mitigate bubbles. Thus, in embodiments, the sample enters at opening 27, asDocket: 0376-0034W01indicated by the arrow (FIG. 1 A), proceeds through segment 21 and exits via segment 23 (at opening 29).
[0092] If desired, the flow cell can be provided with one or more devices for measuring sample parameters (pH, temperature, conductivity, flow rates, etc.), as illustrated by device 30 in FIGS. 1C and IE. In examples, device 30 is or includes a thermistor, a pH sensor, a conductivity sensor and / or a flow meter. Such a device can be placed at other suitable points along segments 21 or 23, e.g., before or after elbow 25, and can be within or outside the flow cell body 15.
[0093] Opening 35 (FIG. 1C) allows a light beam to enter the flow cell along a light pathway that is orthogonal to the flow path of the sample. An opening opposite opening 35 allows the light beam to exit the flow cell. Both entry and exit openings are fitted, respectively, with input rod 126 and output rod 128 (FIG. IF), the two rods being made of a material transparent in the spectroscopic region of interest. In specific implementations, rods 126 and 128 are made from sapphire or quartz.
[0094] The innermost rod ends, namely transmission port 126P and detection port 128P, define sample gap or sample detection region 12. In embodiments, the sample detection region 12 is formed in segment 23 of channel 19 (see FIG. IE, for example), after the sample has passed through elbow 25. This location registers less bubbles due to the bubble mitigation discussed above.
[0095] During operation, a light beam enters input rod 126 at port 126 A, travels through the input rod, enters the sample gap 12 at transmission port 126P, traverses the sample gap, then enters detection rod 128 at detection port 128P. The distance between ports 126P and 128P represents the pathway traversed by the light beam during the optical sample analysis. Setting the sample gap traversed by the light beam can be realized by approaching or distancing the rods relative to each other. In specific implementations, one or both rods are provided with a mechanism that allows the translation of the rod. In many embodiments the light pathway (sample gap) does not vary (remains fixed) during a given spectroscopic measurement or even during successive measurements of a flowing sample.
[0096] The selected pathlength can have a value within a range of from about 0.010 millimeters (mm) to about 5 mm or, in some cases, to about 10 mm, such as, for example, within a range of from about 0.01 mm to about: 0.05 mm, 0.1 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 0.05 mm to about:Docket: 0376-0034W010.1 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 0.1 mm to about: 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 0.5 mm to about: 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 1 mm to about: 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 2 mm to about: 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 3 mm to about: 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 4 mm to about: 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 5 mm to about: 6 mm, 7 mm, 8 mm, 9 mm, 10 mm; or from about 6 mm to about: 7 mm, 8 mm, 9 mm, 10 mm; or from about 7 mm to about: 8 mm, 9 mm, 10 mm; or from about 8 mm to about: 9 mm, 10 mm; or from about 9 mm to about 10 mm.
[0097] One illustrative pathlength is about 5 mm. Another illustrative pathlength is 1 mm. For Mid-IR measurements, the pathlengths can be reduced, to about 10 microns, for instance.
[0098] In specific implementations, the rods have a diameter within a range from about 2 to about 8 mm, e.g., about 4 mm. This arrangement can allow a beam of between 0.5 and 3 mm in diameter, preferably about 1 mm in diameter, to progress through the rods and the sample in the sample gap 12. In many cases, both rods have the same diameter. In others, the rods have different diameters. Other rod dimensions can be selected.
[0099] In many optical arrangements, etalons are formed between parallel reflecting surfaces. For instance, surfaces will reflect due to the refraction index mismatch between air and the bulk material of the input rod 126. An index mismatch between the fluid in the detection sample region 12, the input rod 126 and the output rod 128 can occur as well. Another refraction index mismatch can occur when the light beam exits the output rod 128. Even if surfaces are antireflection coated, residual reflectivity can still be present.
[0100] Some measures that can be taken to avoid or mitigate reflections / etalons are described with reference to FIGS. 2A, 2B and 3A.
[0101] Illustrated in FIG. 2A are input rod 126 and output rod 128, defining sample gap 12 between a transmission port 126P and a detection port 128P. Wedge angles 9 are formed on ports 126 A, 126P of the input rod 126, and, also, on ports 128P and 128 A of the output rod 128, resulting in slanted or sloping wedged surfaces. Typically, the wedge angles 9 are a few tenths of a degree, such as, for instance, withing a range of from aboutDocket: 0376-0034W010.09 to about 0.6 degrees. In one example, angle 9 (FIG. 2A) is 0.125 degrees. In further embodiments, the angles of the input rod 126 and the output rod 128 are rotated (see circular arrow in FIG. 2A) 90 degrees relative to each other, resulting in the orientation of FIG. 2B. In this example, the ports 128A, 128P of the output rod 128 are angled left to right in the plane of the figure. The ports 126 A, 126P of the input rod 126 are angled fore and aft in the plane of the figure.
[0102] A schematic diagram of light traveling though input rod 126 then output rod 128, constructed and oriented as described above with reference to FIGS. 2A and 2B, is shown in FIG. 3 A. When clocked at 90 degrees, none of the reflections overlap. This can be seen in FIG. 3B, showing no superposition (overlap) of the main beam (partly filled circles) and primary and secondary reflections (open circles). FIG. 3C presents the etalon strength with reflection through water. The rods are angled (angle 9) such that the strength of the etalon is below the noise floor, approximately less than about 10'6mAU (milliabsorbance units) or greater than about 9.4 degrees.
[0103] Accordingly, the rods in sample cell 11 can incorporate geometric and orientation details that reduce or minimize reflections / etalons. Specifically, a rod can be angled with opposing wedges, and a rod pair can include rods that are “clocked” 99 degrees relative to each other.
[0104] The flow cell can be fitted in a module. The module design described herein separates the fluidic function provided via flow cell 11 from module components needed to conduct the sample analysis. Thus, a detachable flow cell that receives a sample flow can be removed, sterilized and reinstalled into the module, without disturbing other module components (components that do not support sample flow), such as detectors or optical elements, to name a few. These analysis or scanning elements are not exposed to the sample, do not need to be sterilized and can be kept out of the autoclave.
[0105] Thus, while the flow cell can be removed as often as needed, e.g., for disposal or sterilization, the module can remain in-line, e.g., for repeated measurements or series of measurements of the same or different samples, over any desired time period. Analysis or scanning elements (for propagating a light beam to and through sample detection region 12 (of the flow cell) and / or for detecting the light before and / or after its interaction with the sample in the sample detection region) are part of the module. Although they can beDocket: 0376-0034W01removed for trouble shooting, repair or replacements (e.g., when problems arise), they are not detachable in the sense in which the flow cell is detachable.
[0106] As shown in FIGS. 4A and 4B, module 50 includes a recessed volume 52, configured to receive flow cell 11. In practice, the flow cell can be removed while the sample flow through the module is stopped, at the end point of an operation, for example. A sterilized or a replacement flow cell can be (re)inserted into the module and the sample flow resumed. In some cases, a short interruption in flow permits the attachment of a temporary connector, a piece of tubing, for example, that can be used to replace the flow cell and maintain fluid communication and flow through the module, albeit without measurement capabilities. Another interruption in flow permits the removal of the temporary connector and reinsertion of the flow cell. Once the easily detachable flow cell is removed, to be autoclaved, for example, the operator can connect to the system / process by suitable tubing, thus keeping the detector / tap assembly, further described below, free from the sterilization (e.g., autoclave) operation.
[0107] For carrying out the sample analysis, opening 55 allows light (generated by an external laser, for instance) to enter the module. After interacting with the sample in the flow cell, specifically in sample detection region 12, the light re-enters the module body 54 through opening 56. Electrical signal(s) can exit the module at output connector 91, which, in this configuration, is on the same module face as opening 55. Other arrangements are possible.
[0108] The module can be part of a system which can further include additional components such as, for instance a tunable laser (or another suitable light source), a controller, conduits for transmitting a light beam from an external light source to the module, electrical wires or cables for conveying an electrical signal from a detector to be analyzed, e.g., by the controller. In some implementations, the controller and the tunable laser can be part of a tunable laser spectrometer.
[0109] Construction details characterizing an illustrative system such as system 70 in FIGS. 5 A and 5B as well as an illustrative module such as module 50 can be described by tracing the pathway of a light beam generated by an external light source, e.g., tunable laser 51. As shown in FIGS. 5 A and 5B, an optical fiber arrangement such as fiber optics 53, which can be a PANDA fiber, guides light from an external light source (e.g., tunable laser 51, FTIRs, dispersive (grating) systems, broadband systems with tunable filters, etc.)Docket: 0376-0034W01to the module, specifically to fiberport 55. Fiberport 55 can be configured as a collimator for the light that exits the optical fiber patch cable. From the fiberport, the light pathway continues (as shown in FIG. 5B) to polarizer 57 (for filtering and removing the light in the orthogonal polarization). A rotational mount 59 allows for the rotation of polarizer 57 in a plane that is orthogonal to bench 61 and orthogonal to the optical axis of the beam exiting from the fiber. In some implementations, mount 59 provides fine rotational adjustment of the polarizer 57 so that it can be aligned to the preferred polarization axis of the PANDA fiber.
[0110] Abeam splitter (see, e.g., US 2024 / 0117293 or WO 2024 / 076868, both being incorporated herein by this reference) includes a partially reflecting sapphire window 63, such as a wedge window, held on a pitch yaw mount 65 that secures the window 63 to the bench 61. The partially reflecting sapphire window 63 reflects a portion of the beam, referred to herein as a “reference” beam, to a “tap” photodetector (e.g., an integrated optical device that combines an optical tap (coupler) and a photodetector in one package) and lens arrangement. The light tapping approach described herein extracts a small portion of the optical signal entering the module for monitoring the optical power levels continuously and providing feedback for stabilization purposes, without a significant disruption of the main signal.
[0111] In more detail, the arrangement shown in FIG. 5B includes a ripple reference photodetector 69, such as an In-GaAs (indium gallium arsenide) detector. Focusing lens 71 couples the beam onto the active area of the ripple reference detector 69. In addition to holding window 63, the pitch yaw mount 65 allows the adjustments of the free space beam reflected and transmitted through the sapphire window 63 so that the beam will propagate to strike the active area of the ripple reference detector 69.
[0112] The ripple detector is mounted onto a head printed circuit board (PCB) 73, which includes a transimpedance amplifier to amplify the electrical response of the ripple reference detector. A thermistor can be provided on the head printed circuit board to allow for temperature compensation of the detector 69 and transimpedance amplifier. The response of the ripple reference photodetector 69 can then be transmitted as an electrical signal to tunable laser spectrometer 203 (which can include controller 200 and tunable laser 51, as seen, e.g., in FIG. 5B) via electrical connection 91A, in an electrical wiring harness arrangement, for example.Docket: 0376-0034W01
[0113] In addition to the reference beam, the beam splitter generates a second beam, referred to herein as an “input”, “sample”, “main” or “interrogation” beam. From partially reflecting sapphire window 63, the main beam pathway progresses towards flow cell 11 and passes through the sample detection region 12, defined, as described above (FIGS. IE, IF. 2A, 2B, 3A), by optical transmission port 126P and an opposed optical detection port 128P.
[0114] In embodiments, the module is configured to align the flow cell with other elements within the module to ensure that light intersects the sample in the sample detection region 12. In a simple approach, this can be accomplished by using screws 17 (FIGS. 1 A, 1C, for example) to secure flow cell 11 to the module base 58, in recessed volume 52 (FIG. 4B). The screws can hold the flow cell in position, while some tolerance can be provided by how the rods defining the sample gap 12 are aligned with the light beam. For increased precision, convenience, and / or insertion / removal ease, base 58 can be provided with a kinematic platform configured to receive and / or secure base 13 of flow cell 11. The platform can use a commercial or customized magnetic design to replace screws 17, for example. An example of a suitable kinematic base that can be used is a Newport Kinematic Base, Magnetic, e.g., 25 mm Square, Metric Model: M-BK-1 A. This device includes a bottom plate that is secured to the base 58. The top plate is secured to the underside of the base 13 of flow cell 11. The kinematic base enables mounting, removal and / or replacement of the flow cell with high repeatability. The base includes three polished steel balls in the bottom plate to register with a hardened cone, groove, and flat in the top plate so the two plates relocate precisely.
[0115] Output rod 128 optically couples to sample (also referred to herein as “main”) photodetector 81, provided with lens 83 (FIG. 5B). The electrical signal registered by the photodetector 81 can be transmitted to tunable laser spectrometer 203 via electrical connection 91B, e.g., in a wire harness arrangement. In one example, the photodetector is a dome lens TO-46 In-GaAs detector which can be electrically connected to a sample PCB 85. The detector PCB can include its own transimpedance amplifier for amplifying the detector response and transmitting that response to tunable laser spectrometer 203. A thermistor can be included on the detector PCB to detect the temperature, allowing temperature offsets.Docket: 0376-0034W01
[0116] Another aspect of the invention relates to a method for analyzing a sample. One illustrative embodiment is described below.
[0117] During operation, the tunable laser or tunable laser system sweeps its narrow band emission over some region of the electromagnetic spectrum such as the NIR and / or SWIR and / or MIR regions, or portions thereof. The swept wavelength light from the tunable laser 51 is coupled into the optical fiber patch cable 53, e.g., a PANDA optical fiber, to fiberport 55 of module 50 (FIG. 5A).
[0118] In many cases, once it has entered the module, the light beam does not need to be confined to an optical fiber but can travel (from one element to another) in free space. Thus, in some implementations, light beams inside the module propagate without being guided by fiber optics. Other implementations employ a combination of fiber optics and free space light propagation, while in further implementations, light beams inside the module are guided by fiber optics.
[0119] The PANDA fiber optical fiber patch cable operates to reject modes, minimizing ripple. Short fiber patch cable lengths reduce or minimize losses. Polarizer 57 further removes modes and random polarization fluctuations and addresses polarization dependent loss in the optical components. The ripple reference detector can improve operations by addressing random power attenuation. Polarizing the light removes the risk of polarization dependent loss in the optical components. A portion of the polarized light is detected by the ripple reference photodetector 69. This ripple reference signal is transmitted back to the tunable laser spectrometer 203 (connection 91 A in FIG. 5B) via a spectrometer electrical wiring harness, for example.
[0120] The remaining light travels toward and is coupled into input rod 126, exiting out at the optical transmission port 126P (FIGS. IF, 2A, 2B). It then propagates through the sample detection region 12. After interacting with and being modulated by analytes in the sample in the sample detection region 12, the transmitted light enters output rod 128 through optical detection port 128P, propagates through the output rod 128 (FIGS. IF, 2A, 2B), and exits at port 128A. It is detected by the sample photodetector 81. In the sample detection region, the analytes would have preferentially absorbed some wavelengths relative to others.
[0121] Controller 200, which can be part of the tunable laser spectrometer 203, monitors the response of the sample photodetector 81 as well as the ripple referenceDocket: 0376-0034W01photodetector 65. The controller resolves the absorption spectra of the sample by monitoring the spectral scanning of the tunable laser 51 over its scan band relative to the time-response of the sample photodetector 81. Any noise associated with ripple or other sources from the optical fiber is compensated by the response from the ripple reference photodetector 69.
[0122] In some implementations, controller 200 uses the temperatures detected by the thermistor on the PCB 85 (associated with the sample detector 81) and registered by the thermistor associated with the reference detector 69, on PCB 73, to compensate for change in the response of the ripple reference photodetector 69 and the sample photodetector 81 and changes in the gain of the transimpedance amplifiers on the PCBs employed.
[0123] The results can be automatically analyzed and displayed on a suitable viewer.
[0124] Controller 200 can also monitor and / or adjust the sample flow through flow cell 11 (measured, for instance, by a flow meter (e.g., device 30 in FIGS. 1C, IE). For example, monitoring a protein signal spectroscopically can be combined with monitoring the sample flow. The measurements (spectroscopic signal and flow rate) can be integrated to provide “total” protein information, a parameter not typically available using current techniques or equipment.
[0125] If actuators are employed, they can be controlled by controller 200 or independently.
[0126] In some cases, conductivity data, gathered e.g., by a conductivity sensor such as device 30 in FIGS. 1C and IE, could be used to accurately subtract (for chemometrics) the salt gradient commonly used in Ion Exchange Chromatography.
[0127] Other suitable approaches can be employed, however. If integrated into or inline to a larger fluidic system, in a protein manufacturing process, for example, the sample flow can be monitored and / or controlled by the same equipment employed to move fluid through the overall fluidic system, e.g., from one vessel to another.
[0128] The flow cell, module, system, or method described herein can be used in various applications. For example, the module can be integrated into protein downstream operations. To illustrate, embodiments of the invention can be integrated into an In Vitro Transcription (IVT) system or a tangential flow filtration (TFF) system that can be used for protein sieving in perfusion. In one example, embodiments of the invention are employedDocket: 0376-0034W01in a system such as described in the Nirrin Technologies Provisional Patent Application No. 63 / 751,353, filed on January 30, 2025, and the International Patent Application No. PCT / US26 / 12497, filed on January 26, 2026, both with the title Filtration system control based on spectroscopic analysis, and both being incorporated herein in their entirety by this reference.
[0129] In embodiments in which module 50 is integrated into tangential flow filtration (TFF) systems, the detachable flow cell 11 can be placed in one or more locations, including, for example, a feed line, a retentate line, a retentate recirculation loop, and / or a permeate line downstream of the filtration membrane. Placement in the permeate line (post- TFF) allows in-line monitoring of permeate composition to assess passage of one or more species through the membrane. Such monitoring can be used, for example, to track clearance of small molecules during diafiltration, to detect undesired passage of larger species (e.g., product loss or breakthrough), and / or to provide a real-time indication of membrane integrity and performance. In some implementations, multiple modules 50 (or multiple detachable flow cells) are deployed at different locations (e.g., retentate and permeate) to provide a mass balance and / or improved endpoint determination.
[0130] Module 50 also can be integrated into downstream chromatography operations. For example, the detachable flow cell 11 can be positioned in a line receiving effluent from a chromatography column, such as at or downstream of a column outlet and upstream of a fraction collector, to enable real-time measurement of one or more species in the effluent stream. Chromatography operations can include, by way of non-limiting example, affinity chromatography (including Protein A), ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, size exclusion chromatography, and / or combinations thereof. Such chromatography techniques can be applied to different product modalities including monoclonal antibodies and other proteins, as well as viral vectors (including adeno-associated virus (AAV)) and other biological or biochemical products. Controller 200 can use resolved spectra to support decisions and / or control actions including column loading endpoints (e.g., breakthrough detection), wash and elution transitions, gradient formation (including salt gradients), fraction collection timing, and pooling of desired fractions. In some implementations, conductivity data gathered by a conductivity sensor such as device 30 is combined with the spectroscopic data, e.g., toDocket: 0376-0034W01account for or subtract effects of salt gradients (such as in ion exchange chromatography) in chemometric models.
[0131] Deployment in upstream bioreactor recirculation arrangements is also possible. For instance, module 50 can be integrated into a recirculation arrangement in which a portion of cell-culture fluid (e.g., culture media containing cells, nutrients, metabolites, and product) is withdrawn from a bioreactor, passed to and through sample detection region 12 of detachable flow cell 11 for in-line measurement, and returned to the bioreactor. The sample can be analyzed continuously, semi-continuously, or intermittently (including stopflow measurement modes) while maintaining a closed sterile flow pathway. The resolved absorption spectra and / or chemometric analyses based on the spectra can be used to determine one or more upstream process parameters, including (by way of non-limiting example) glucose concentration, lactate concentration, concentrations of other nutrients and metabolites, and / or cell-related parameters such as optical density, cell density, and / or viable cell density. In some implementations, controller 200 uses the measured parameters to provide feedback for upstream control actions such as adjusting feed rates, perfusion rates, bleed rates, dilution rates, or other process settings.
[0132] In some processes, module 50 is integrated into sterile filtration operations. In one example, detachable flow cell 11 is located upstream of a sterile filter to monitor a feed stream and / or downstream of the filter to monitor a filtrate stream. In-line measurement of the filtrate can be used to verify concentration and composition targets, detect process transitions, confirm completion of a filtration step, detect filter fouling or breakthrough, and / or provide an indication of filter performance, while maintaining a closed sterile flow path and without requiring off-line sampling.
[0133] Thus, embodiments described herein present significant versatility and can be adapted or adopted in any number of situations, replacing reliance on off-line measurements. In TFF ultifiltration (UF) operations of concentration and buffer exchange and diafiltration (DF) units, for example, practicing embodiments of the invention can replace existing off-line approaches. One or more modules such as described above can be integrated into the loop of a filtration system allowing real-time measurements of buffer / excipient and product concentrations. Excipients (e.g., stabilizers, solubilizers and / or other auxiliary substances) often are used to ensure the quality and stability of theDocket: 0376-0034W01final product; the exact measurement and control of solution constituents is critical in determining the end point of the operation.
[0134] Additional UF / DF and / or other implementations include monitoring different product modalities and buffer systems, and the examples herein are intended to be illustrative rather than limiting with respect to analyte identity, concentration ranges, or unit-operation configurations.
[0135] The invention is further described in the following non-limiting example.
[0136] Example
[0137] 1-mg / mL BSA was prepared in PBS and exchanged into a 5% sucrose + 22mM histidine buffer. Measurements were performed using a system such as described above which allowed monitoring protein and excipients simultaneously and in flow, providing a real-time look into the DF process.
[0138] Real-time data is presented in FIG. 6, with the plot reaching the highest concentration at 4 diavolumes (represented by a broken line) corresponding to sucrose; the plot reaching the lowest concentration at 4 diavolumes (represented by a light solid line) corresponding to BSA; and the plot reaching an intermediate concentration at 4 diavolumes (represented by a dark solid line) corresponding to histidine.
[0139] FIG. 7 A presents real-time measurement of BSA concentration during ultrafiltration. The sensitivity of the system allowed one to capture the release of membrane-bound BSA once UF was complete (FIG. 7B).
[0140] The theoretical DF exchange endpoint without real-time monitoring was 7 diavolumes before concentrating the protein. By applying techniques described herein, however, the measured DF endpoint was at 4 diavolumes. This result highlights the reduced amount of filtration time and diafiltration buffer required for a process monitored according to embodiments described herein.
[0141] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Claims
Docket: 0376-0034W01CLAIMSWhat is claimed is:
1. A system for analyzing a sample, the system comprising:a tunable laser for generating a swept wavelength signal;a module comprising a detachable flow cell for passing a flowing sample to and through a sample detection region, a reference photodetector for detecting the swept wavelength signal from the tunable laser and a sample photodetector for detecting the swept wavelength signal after transmission through the sample detection region,wherein,the tunable laser is external to the module;the flow cell is configured to reduce bubbles in the sample detection region; and / orthe system is configured to direct a sample through an elbow and then through the sample detection region, the sample detection region being located above the elbow.
2. The system of claim 1, further comprising a controller.
3. The system of claim 2, wherein the controller is configured to control at least one unit operation selected from a filtration operation, a chromatography operation, and a sterile filtration operation based on the detected swept wavelength signal after transmission through the sample detection region.
4. The system of claim 3, wherein the controller is configured to control a chromatography fraction collection and / or pooling based on the resolved absorption spectrum.
5. The system of claim 3, wherein the controller is configured to determine a diafiltration endpoint based on the resolved absorption spectrum.
6. The system of claim 1, wherein the flow cell includes a conduit for supporting a sample flow, wherein the conduit includes the elbow, wherein the sample detectionDocket: 0376-0034W01region is above the elbow, and wherein the system is configured for a sample flow direction that passes through the elbow, then through the sample detection region.
7. The system of claim 1, further comprising a beam splitter for directing a reference beam to the reference photodetector and an interrogation beam to the sample photodetector.
8. The system of claim 1, further comprising a polarizer.
9. The system of claim 1, wherein the sample detection region is defined between a transmission port of an input rod and a detection port of an output rod.
10. The system of claim 1, further comprising an arrangement for securing the flow cell to the module.
11. The system of claim 1, wherein the flow cell is part of a fluidic system.
12. The system of claim 11, wherein the fluidic system comprises a recirculation loop coupled to a bioreactor, the recirculation loop being configured to withdraw a portion of cell-culture fluid from the bioreactor, pass the portion of cell-culture fluid through the detachable flow cell, and return the portion of cell-culture fluid to the bioreactor.
13. The system of claim 11, wherein the fluidic system comprises a tangential flow filtration system, and wherein the detachable flow cell is disposed in a permeate line downstream of a tangential flow filtration membrane.
14. The system of claim 11, wherein the fluidic system comprises a chromatography system including a chromatography column, and wherein the detachable flow cell is disposed in a line that receives effluent from the chromatography column.
15. The system of claim 14, wherein the chromatography column performs Protein A affinity chromatography or ion exchange chromatography.Docket: 0376-0034W0116. The system of claim 14, wherein the chromatography column performs at least one of hydrophobic interaction chromatography, mixed-mode chromatography, size exclusion chromatography, or affinity chromatography.
17. The system of claim 14, wherein the effluent comprises a viral vector comprising AAV, or a protein product.
18. The system of claim 11, wherein the fluidic system comprises a sterile filtration system including a sterile filter, and wherein the detachable flow cell is disposed upstream of the sterile filter and / or downstream of the sterile filter.
19. The system of claim 1, wherein the module includes components that do not require sterilization.
20. The system of claim 1, wherein the flow cell is autoclavable.
21. The system of claim 1, wherein the swept wavelength signal generated by the tunable laser is transmitted to the module via a PANDA optical fiber.
22. The system of claim 1, wherein, within the module, the swept wavelength signal propagates in free space.
23. The system of claim 1, wherein the module is configured to de-couple sample flow components from sample analysis components.
24. A filtration arrangement including the system of claim 1.
25. The filtration arrangement of claim 24, wherein the filtration arrangement comprises a tangential flow filtration system, and / or wherein the detachable flow cell is disposed in a retentate line, a retentate recirculation line, and / or a permeate line downstream of a filtration membrane.
26. A method for analyzing a sample, the method comprising:passing a flowing sample through a sample detection region in a detachable flow cell;generating a swept wavelength signal;Docket: 0376-0034W01transmitting the swept wavelength signal to a module that includes the detachable flow cell;detecting the swept wavelength signal in the module prior to transmission through the sample detection region;detecting the swept wavelength signal after transmission through the flowing sample in the sample detection region; andresolving an absorption spectra of the sample with reference to the swept wavelength signal before and after transmission through the sample detection region.
27. The method of claim 26, wherein the swept wavelength signal is generated by a tunable laser and is within a range of from about 1350 to about 1800 nm, or within a range of from about 2050 to about 2400 nm, or within a range of from about 3.5 to about 10 microns.
28. The method of claim 26, wherein the flowing sample changes direction before entering the sample detection region.
29. The method of claim 26, further comprising inserting or removing the flow cell into or from the module.
30. The method of claim 26, wherein an interrogation beam traverses the sample detection region orthogonally to a sample flow pathway.
31. The method of claim 26, further comprising inserting the flow cell into a conduit for directing the sample to or from the flow cell.
32. The method of claim 26, further comprising passing the swept wavelength signal through a polarizer before detecting it as a reference signal.
33. The method of claim 26, further comprising controlling flow parameters of the sample.
34. The method of claim 26, wherein the method measures simultaneously (i) a concentration of a product species and (ii) at least one additional parameter, wherein the product species is a protein, peptide, nucleic acid, or viral vector, andDocket: 0376-0034W01wherein the at least one additional parameter is selected from excipient concentration, nutrient concentration, metabolite concentration, salt concentration, conductivity, pH, temperature, optical density, cell density, and / or viable cell density.
35. The method of claim 34, wherein the nutrient concentration comprises glucose concentration or wherein the metabolite concentration comprises lactate concentration or wherein the product species comprises an active pharmaceutical ingredient or a target molecule of interest.
36. The method of claim 34, wherein the method determines cell density and / or viable cell density from the resolved absorption spectra and / or a multivariate model derived therefrom.
37. The method of claim 33, further comprising supplying the swept wavelength signal to the module through a PANDA optical fiber.
38. The method of claim 33, wherein, within the module, the swept wavelength is propagated in free space.
39. A module, comprising:a section including a polarizer, a beam splitter, a reference photodetector and a sample photodetector;a detachable flow cell for directing a flowing sample to and from a sample detection region, the sample detection region being defined by a transmission port of an input rod and a detection port of an output rod; wherein the detachable flow cell includes an elbow below the sample detection region.
40. The module of claim 39, wherein the flow cell is part of a fluidic system.
41. The module of claim 40, wherein the fluidic system is de-coupled from the section including the polarizer, the beam splitter, the reference photodetector and the sample photodetector.Docket: 0376-0034W0142. A system including the module of claim 39, a source for generating a swept wavelength signal, and a controller.
43. A system for analyzing a sample, the system comprising:a source generating a swept wavelength signal;a module comprising a detachable flow cell for passing a flowing sample to and through a sample detection region, a reference photodetector for detecting the swept wavelength signal from the source generating the swept wavelength signal and a sample photodetector for detecting the swept wavelength signal after transmission through the sample detection region, wherein,the source generating the swept wavelength signal is external to the module; the flow cell is configured to reduce bubbles in the sample detection region; and / orthe system is configured to direct a sample through an elbow and then through the sample detection region, the sample detection region being located above the elbow.
44. A chromatography arrangement comprising a chromatography column and the system of claim 1, wherein the detachable flow cell is disposed in an effluent line of the chromatography column.
45. A sterile filtration arrangement comprising a sterile filter and the system of claim 1, wherein the detachable flow cell is disposed upstream of the sterile filter and / or downstream of the sterile filter.