Open / closed type analyzer

Abstract

An apparatus and / or system and / or method for analyzing a sample, such as by NIR, is provided. The system (11) includes an open / closed device (13) and a tunable laser (51). The device (13) has a fixed first portion (15) and a second portion (19) that is movable between an open position, for example, for loading a sample, and a closed position, for scanning an absorbance spectrum. By lowering the second portion (19) to the closed position, a sample detection region (12) is formed between an input rod (126) and an output rod (128). In one exemplary arrangement, a reference detection means (69) is located in the first portion (15) and a sample detection means (81) is located in the second portion (19).

Description

Related Applications

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 423,856, filed November 9, 2022, and U.S. Provisional Patent Application No. 63 / 469,502, filed May 29, 2023, both of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Many processes in industries such as chemical, biochemical, pharmaceutical, food, and beverage industries benefit from some type of analysis. Identifying and sometimes quantifying the substances present is crucial. The general approach involves obtaining a sample and detecting one or more components (sometimes called analytes) in the sample.

[0003] Analytes can be characterized using a variety of optical spectroscopic approaches, perhaps the most common of which is absorption spectroscopy. Incident light excites the analyte's electrons from a low-energy ground state to a higher-energy excited state, and the energy can be absorbed by non-bonding n- and π-electrons in the molecular orbitals. Absorption spectroscopy can be performed in the ultraviolet, visible, and / or infrared regions, allowing analytes of various material phases and compositions to be probed with specific wavelengths or wavelength bands of light. The resulting transmitted light can then be used to resolve the absorption spectrum to determine one or more specific properties of the analyte or sample, such as composition, temperature, or pH, for applications in medical diagnostics, drug development, and food and beverage quality control.

[0004] Many existing instruments use light in the visible and / or ultraviolet (UV) regions of the electromagnetic spectrum. For the past decade, UV or visible light methods have been the gold standard for measuring protein and monoclonal antibody (mAb) concentrations, for example. However, UV or visible light instruments can suffer from limited accuracy and reproducibility due to the dynamic range limitations caused by the extremely strong absorption of light by proteins in the UV region (typical maximum absorption is around 3–4 absorbance units (AU)). The development of systems using variable path lengths has provided a partial solution.

[0005] US Patent Application Publication No. 2019 / 0358632A1 by Hassell et al. describes the analysis of culture media using near-infrared (NIR) spectroscopic techniques.

[0006] US Patent Application Publication No. 2020 / 0240902A1 by Hassell et al. describes the application of NIR-based techniques to the measurement of samples in flow cells.

[0007] US Patent Application Publication No. 2021 / 0088433 by Hassell et al. describes a robust, hands-free, non-destructive, real-time NIR method for identifying and / or quantifying components of any process using in-situ probes that can be inserted and / or maintained within a bioreactor.

[0008] Despite the advantages of in-situ monitoring, there are many applications where sample collection and sample analysis are performed separately. For example, there are often laboratory or industrial settings where samples removed from reactors or other sources are analyzed using benchtop instruments. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2019 / 358632 [Patent Document 2] U.S. Patent Application Publication No. 2020 / 240902 [Patent Document 3] U.S. Patent Application Publication No. 2021 / 088433 Summary of the Invention [Problem to be solved by the invention]

[0010] In other words, there is a continuing need for development of analytical instruments that do not necessarily use in-situ probes.

[0011] Additionally, systems and methods are needed that address limitations associated with existing UV or visible spectroscopic analyzers, such as the challenges posed by current variable path length approaches. The limitations of variable path length become most pronounced in subsequent downstream processing steps and often emerge when mAb concentrations become very high (e.g., above 10 mg / mL). Higher mAb concentrations require shorter path lengths to accommodate sensor limitations. However, very short path lengths (e.g., less than 100 micrometers (microns (μm))) make reproducibility difficult to achieve.

[0012] Additionally, given the advantages associated with NIR spectroscopic techniques, there is a demand for the development of instrumentation that can realize these advantages. Systems that facilitate, refine, or even extend the NIR analytical approach into the mid-infrared (MIR) region are of great interest. Particularly desirable are instruments that do not rely on a variable pathlength approach.

[0013] Focusing on a specific field, advances in pharmacokinetics / pharmacodynamics continue to drive the trend toward higher concentrations for many therapeutics. Consequently, many FDA-approved mAb therapies involve formulations with high concentrations (>100 mg / mL). See, for example, SS Wang et al., "US FDA-approved therapeutic antibodies with high-concentration formulation: summaries and perspectives," Antibody Therapeutics, Vol. 4, No. 4, pp. 262-273 (2021) for details. Therefore, the realization of measurement and quantification methods designed to operate in this high concentration range is desirable.

[0014] There is also great interest in simplified approaches that do not require complex transfer stages or dilutions. [Means for solving the problem]

[0015] In summary, the present invention relates to an apparatus and / or system and / or method that overcomes at least some of the problems and / or meets the needs set forth above.

[0016] In one aspect, the invention features a "clamshell" device that opens to accommodate a sample and then closes over the sample to perform an analysis. The device may be comprised of two parts, halves, sections, or portions, with at least one part configured to open and close like a clamshell (i.e., by flipping over). This movement may be achieved by a link connecting the two parts directly or via a support or base.

[0017] In specific embodiments, some components are disposed in a first portion (often the lower or bottom portion) that is fixed to a support. A second portion (often the upper, top, or "lid"-like portion) houses the other components and is configured to move or "flip" between open and closed positions. In operation, the device can be placed in an open position, e.g., to introduce a sample, etc. Closing the second portion (e.g., the top portion) brings the two portions together to form a sample detection region (also referred to herein as a sample "gap"). While the device is in the closed configuration, an absorbance spectrum of a sample analyte present in the sample detection region can be acquired.

[0018] The sample gap can be formed between rods that transmit light in a desired region of the electromagnetic spectrum, e.g., NIR, MIR, etc. Some approaches use rods configured and / or oriented to reduce or suppress reflections / etalons. In specific embodiments, the dimensions of the gap (i.e., also the light path) are fixed. As an example, the gap is set and maintained at a constant path length during any given measurement. Some design details allow for the path length to be reset when analyzing the next or another sample (the path length remains fixed for the duration of the analysis of the next or another sample).

[0019] The path length (gap) can be a value in the range of about 0.010 millimeters (mm) to about 10 mm. In an exemplary embodiment, the path length is a value in the range of about 0.10 mm to about 5 mm.

[0020] In many arrangements, the first part (e.g., the bottom part) houses elements that direct light (typically from a light source such as a laser) into the sample gap, while the second part (e.g., the lid part) houses elements that detect light that has traversed the sample gap.

[0021] In some embodiments, the device is part of a system that also includes a light source, such as a tunable laser. Thus, in another aspect, the invention features a system including a gated device and a tunable laser that generates a swept wavelength signal. A first section of the device includes components that direct light from the laser to a sample detection region, and a second section includes a light detection means that detects the swept wavelength signal after transmission through the sample detection region. In a specific embodiment, the first section further includes a detection means that detects the swept wavelength signal before transmission through a sample.

[0022] Fiber optic technology allows light from a light source, such as a tunable laser, to be transmitted to the first part of the device.

[0023] In yet another aspect, the invention features a method for analyzing a sample. The method includes generating a swept-wavelength signal, transmitting the swept-wavelength signal through a first portion of an open-closed device to and through a sample detection region, detecting the swept-wavelength signal after transmission through the sample detection region with a second portion of the open-closed device, and resolving an absorbance spectrum of the sample. These steps are performed with the device in a closed configuration. In some embodiments, the method further includes opening the open-closed device to load the sample to be analyzed and / or closing the device to perform analysis of the sample.

[0024] The present invention may further comprise the steps of detecting the swept wavelength signal before transmission through the sample detection region, and resolving the absorption spectrum of the sample using the swept wavelength signal before and after transmission through the sample detection region as a reference.

[0025] The methods and devices described herein may operate within any desired wavelength range, for example, from the near infrared to the mid-infrared. In specific examples, the wavelength may be within the range of about 1350 to about 1800 nanometers (nm), or within the range of about 2050 to about 2400 nm. For MIR analysis, the wavelength may be within the range of about 3.5 to about 10 microns. Multiple light sources emitting different wavelength signals of electromagnetic energy may also be used.

[0026] In one application, the devices and / or systems and / or methods described herein are used in protein production, particularly downstream processing steps where proteins are highly concentrated. In such situations, the approaches described herein offer significant advantages over UV or visible light measurements. At mAb concentrations of 10 mg / mL or greater, typical UV or visible light procedures require dilution. In contrast, the devices, systems, or methods described herein provide accurate, linear results without the complex procedures, the need for translation stages, variable path lengths, or the need for dilutions. In many cases, a sample volume of 15 μL is sufficient to perform the analysis.

[0027] Implementation of the embodiments described herein can provide improved accuracy over existing approaches. When the principles of the present invention are applied to mAb measurement, an error of 1% can be achieved at concentrations between 0.1 and 1000 mg / mL. Yet another advantage is its high versatility. In addition to measuring mAb concentrations between 0.1 and 1000 mg / mL, it is possible to simultaneously measure excipients such as histidine, arginine, methionine, polysorbate, and sucrose. Both measurements can be performed using a single system, typically in a single scan. For example, in addition to measuring mAbs, it is possible to measure histidine at concentrations between 1 and 100 mg / mL and polysorbate at concentrations between 1 and 2 mg / mL.

[0028] In some embodiments, such as mAb measurements, the spectra are consistent and unique regardless of background, a significant improvement over UV or visible techniques that rely on analysis and curve fitting to extinction coefficients for measurement, which can be adversely affected by the presence of excipients in the background.

[0029] Implementation of the embodiments described herein allows for the measurement of a range of amino acids present in a buffer, in contrast to the performance of many existing approaches.

[0030] The robust and reliable instruments and methods of the present invention provide reproducible and easily verified results. Measurements are extremely fast (seconds), in some cases 50 times faster than competing technologies on the market. Scan times are often as short as 5 seconds.

[0031] The instrumentation described herein is easy to use, allowing for a simple workflow with minimal or no setup or calibration. The analysis is entirely uncomplicated and can be performed without expert guidance. As a result, implementation of embodiments of the present invention allows for competitive pricing of capital and services.

[0032] In some embodiments, the rod-mounted configuration replaces the typical cuvettes in existing instruments, reducing the need for consumables. Cleaning and reusing glassware adds labor. Also, the actual cuvette, and even the same cuvette, can have different thicknesses at different scan locations, potentially reducing the quality of the analysis. Furthermore, the cuvette's parallel or near-parallel surface configuration can introduce significant etalons / reflections.

[0033] The need for other consumables (e.g., reagents) and the need for outsourced excipient testing may be reduced, further increasing cost-effectiveness.

[0034] Techniques such as those described herein also improve the quality of the analysis. For example, the embodiments described herein improve, and in some cases even maximize, the signal-to-noise ratio (SNR). This is achieved by launching a light beam from a fiber and / or free-space link through a sample gap and directing the transmitted light to a photodetector. Running the detector cable to the spectrometer (instead of using a fiber optic cable back to a photodiode) eliminates a noise source.

[0035] These and other features of the present invention, including various novel details of construction and combination of parts and other advantages, will be more fully described hereinafter with reference to the accompanying drawings and pointed out in the claims. It will be understood that the specific methods and apparatus embodying the invention are merely illustrative and not limiting. The principles and features of the invention can be applied to numerous and varied embodiments without departing from the scope of the invention.

[0036] In the accompanying drawings, reference characters refer to the same structures / components throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [Brief explanation of the drawings]

[0037] [Figure 1] 1 is a perspective view of the top of an open-close device supported on a cabinet-like base of the present invention; FIG. [Figure 2] FIG. 1 is a cross-sectional view of a system with an open-close device in an open position. [Figure 3A] FIG. 1 is a cross-sectional view of a system with an open-close device in a closed position. [Figure 3B] FIG. 10 is another cross-sectional view of the system with the open-close device in the closed position. [Figure 3C] FIG. 10 is a plan view showing the layout of another reference detection means. [Figure 4A-4B] Figure 4A is a side perspective view showing the rods that make up the sample detection region, and Figure 4B is a side perspective cross-sectional view of the rods of Figure 4A rotated 90° relative to each other about their longitudinal axes. [Figure 5] FIG. 1 is a cross-sectional view of a portion of an open / close device showing the specific geometry and orientation of the rods that make up the sample detection region. [Figure 6A] 4C is a schematic diagram showing how a light beam travels through each rod of the structure and orientation shown in FIGS. 4A and 4B. FIG. [Figure 6B] 4C is a graph illustrating the lack of overlap between the main beam and secondary beams obtained with each rod of the configuration and orientation shown in FIGS. 4A and 4B. [Figure 6C] The plot shows the effective etalon intensity upon reflection in water on the vertical axis and the angle θ between the beams on the horizontal axis. [Figure 7] Data showing multiple analytes (mAb, histidine and polysorbate) in a single scan. [Figure 8] 1 is a graph comparing UV or visible light absorbance versus mAb concentration and absorbance measurements obtained by practicing an embodiment of the present invention. [Figure 9] This graph shows the observed spectral signature of a mAb product, which is identical to the NIST mAb standard (A) and varies proportionally with dilution (B, C, and D). [Figure 10] 1 is a graph comparing the error observed in UV analysis with the error seen with the techniques described herein at various dilutions. [Figure 11A] 1 is a graph showing the identification of components in a sample containing sucrose, histidine, and histidine hydrochloride by spectral deconvolution. [Figure 11B] 1 is a graph showing the identification of components in a sample containing arginine and histidine hydrochloride by spectral deconvolution. [Figure 12A] 1 is a spectrum of one component of a buffer that can be quantified using the techniques and / or instruments described herein. [Figure 12B] 10 is a spectrum of other components of the buffer that can be quantified using the techniques and / or instruments described herein. [Figure 12C] 10 is a spectrum of additional components of the buffer that can be quantified using the techniques and / or instruments described herein. [Figure 12D] 10 is a spectrum of additional components of the buffer that can be quantified using the techniques and / or instruments described herein. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, which depict exemplary embodiments of the invention. However, the present invention may be embodied in many other 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.

[0039] As used herein, the terms "and / or" and "and / or," when used in conjunction with one or more items, are intended to encompass any and all combinations of those items. Furthermore, the singular and articles "a," "an," and "the" are intended to encompass the plural, unless expressly stated otherwise. Furthermore, as used herein, the terms "having," "including," "comprising," and / or "having" are intended to identify the presence of stated features and / or entities and / or steps and / or processes and / or elements and / or components, but not to exclude the presence or addition of one or more other features and / or entities and / or steps and / or processes and / or elements and / or components and / or combinations thereof. Furthermore, when an element, such as a component or subsystem, is referred to and / or shown as being connected or coupled to another element, it is understood that one may be directly connected or coupled to the other element, as well as that there may be elements therebetween.

[0040] Unless otherwise defined, the meanings of all terms (including technical and scientific terms) used in this specification are the same as those commonly understood by those skilled in the art to which this invention belongs. Furthermore, even for terms defined in commonly used dictionaries, the meanings should be interpreted according to the meaning in the context of the relevant field, and unless clearly defined in this specification, they should not be interpreted in an idealized or overly formal way.

[0041] The present invention generally relates to an approach for detecting and, in some cases, quantifying compounds (analytes) present in a sample. Substances that can be tested include, but are not limited to, components in culture media, nutrients, metabolites, enzymes, hormones, cytokines, proteins, and the like. By way of example, the application of the present invention is directed to downstream bioprocessing of mAbs. The sample under investigation may contain multiple target analytes.

[0042] The techniques described herein use a spectroscopic approach to determine the spectral response of sample analytes, with particular focus on infrared spectroscopy, which typically covers the near-infrared (0.75-1.4 μm; NIR), short-wavelength infrared (1.4-3 μm; SWIR), mid-wavelength infrared (3-8 μm; NWIR), long-wavelength infrared (8-15 μm; LWIR), and far-infrared (15-1000 μm; FIR) regions of the spectrum.

[0043] NIR or SWIR spectroscopy covers the region of the electromagnetic spectrum between 780 nanometers (nm) and 2500 nm by examining the overtone and combination vibrations of molecules. In simple terms, this region from 780 nm to 2500 nm can be shortened to the abbreviation "NIR." For an overview of NIR spectroscopy, see, for example, "An Introduction to Near Infrared (NIR) Spectroscopy," by A.M.C. Davies. http: / / www.impublications.com / content / introduction-near-infrared-nir-spectroscopySee also Cervera, A.E., Petersen, N., Lantz, A.E., Larsen, A., and Gernaey, 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.

[0044] In many aspects, the present invention features a system that includes a tunable laser, which may be part of a tunable laser spectrometer, and a gated analyzer that performs the analysis. The system may further incorporate additional elements, such as a controller.

[0045] Typically, a tunable laser spectrometer is equipped with a wavelength reference and a power reference. The wavelength reference allows the instrument to track the wavelength sweep within the spectral scan band of the tunable laser. The power reference detects the instantaneous power during the sweep, allowing any changes in power to be compensated for. This allows for accurate resolution of the absorption spectrum of the substance of interest.

[0046] In some embodiments, the laser produces narrowband emission, such as a full width at half maximum (FWHM) of less than 20 nm, preferably less than 10 nm, less than 5 nm, or even less than 2 nm. The narrowband emission is wavelength swept or tuned within a range of about 1350 to about 1800 nm, such as about 1350 nm to about 1400 nm, about 1350 nm to about 1500 nm, about 1350 nm to about 1600 nm, about 1350 nm to about 1700 nm, about 1400 nm to about 1500 nm, about 1400 nm to about 1600 nm, about 1400 nm to about 1700 nm, about 1400 nm to about 1800 nm, about 1500 nm to about 1600 nm, about 1500 nm to about 1700 nm, about 1500 nm to about 1800 nm, about 1600 nm to about 1700 nm, about 1600 nm to about 1800 nm, and about 1700 nm to about 1800 nm. In another embodiment, a wavelength within the range of about 2050 nm to about 2400 nm is used, such as about 2050 nm to about 2100 nm, about 2050 nm to about 2200 nm, about 2050 nm to about 2300 nm, about 2100 nm to about 2200 nm, about 2100 nm to about 2300 nm, about 2100 nm to about 2400 nm, about 2200 nm to about 2300 nm, about 2200 nm to about 2400 nm, and about 2300 to about 2400 nm.

[0047] In the MIR region, the wavelengths are from about 3.5 microns to about 4 microns, from about 3.5 microns to about 5 microns, from about 3.5 microns to about 6 microns, from about 3.5 microns to about 7 microns, from about 3.5 microns to about 8 microns, from about 3.5 microns to about 9 microns, from about 4 microns to about 5 microns, from about 4 microns to about 6 microns, from about 4 microns to about 7 microns, from about 4 microns to about 8 microns, from about 4 microns to about 9 microns, from about 4 microns to about 10 microns, from about 5 microns to about 6 microns, from about 5 microns to about 7 microns, and from about 5 microns to about 8 microns. The wavelength sweep of the laser is targeted in a range of about 3.5 microns to about 10 microns, such as about 10 microns to about 8 microns, about 5 microns to about 9 microns, about 5 microns to about 10 microns, about 6 microns to about 7 microns, about 6 microns to about 8 microns, about 6 microns to about 9 microns, about 6 microns to about 10 microns, about 7 microns to about 8 microns, about 7 microns to about 9 microns, about 7 microns to about 10 microns, about 8 microns to about 9 microns, about 8 microns to about 10 microns, and about 9 to about 10 microns.

[0048] The instantaneous narrowband emission from the laser in the MIR region is typically less than 500 nm wide at FWHM, often less than 250 nm wide, but can be less than 100 nm wide, even less than 50 nm, less than 25 nm, or less than 10 nm.

[0049] In some applications, the tunable laser is optimized for a specific wavelength range that contains the chemical information of interest, such as the key chemical information of proteins (e.g., CH, OH, etc.). In one embodiment, the tunable laser sweeps wavelengths within a spectral band that includes wavelengths of 2.3 micrometers and over wavelengths of greater than 100 nanometers. In one embodiment, the laser sweeps over a spectral band that spans wavelengths on the order of 2.2-2.4 micrometers.

[0050] High-performance operation requires perfect compensation for all power variations. One source of power variations is the highly polarized nature of diode lasers. Therefore, even small changes in polarization state, combined with polarization-dependent losses (PDL) in various components such as lenses, beam splitters, and detectors, can lead to off-track power variations that degrade the accuracy of the extinction spectrum.

[0051] To ensure the polarization stability necessary to manage PDL, polarization-maintaining fibers, such as single-mode polarization-maintaining optical (PANDA) fibers, are sometimes used with tunable laser spectrometers. However, the use of polarization-maintaining fibers does not completely solve the problem. Some power is usually present in the non-desired polarization, which beats with the power of the desired polarization, resulting in power fluctuations. This also leads to the phenomenon of fiber polarization beating and PANDA ripple. In summary, when two waves with different linear polarizations propagate through a birefringent polarization-maintaining (PM) fiber, their phases evolve differently. The differential phase delay is proportional to the fiber length.

[0052] As described below, specific embodiments address at least some of these issues, as further described in U.S. Patent Application No. 18 / 483,882, filed October 10, 2023, and International Application No. PCT / US2023 / 075442, filed September 28, 2023, both of which are incorporated by reference in their entireties.

[0053] Figures 1 to 3B show the device in an open / close configuration. Specifically, Figure 1 is a perspective view of a support holding the top of the open / close device, while Figure 2 is a cross-sectional view of the device in the open position, and Figures 3A and 3B are cross-sectional views of the device in the closed position.

[0054] Device 13, a component of system 11, has a first and a second part. First part 15 is fixed and secured to a base or support 27, which in this embodiment constitutes the lower or bottom part of the open / close device. In this configuration, first part 15 is suspended from support 27 and does not move or tip over. Rather, the opening and closing action is performed by second part 19, which in this embodiment constitutes a top, apex, or lid. Other arrangements and orientations of these two parts are possible.

[0055] More specifically, FIG. 1 depicts top 19 supported by an enclosure or housing, such as cabinet 21. Linkage 23 provides a clamshell or "flip-over" configuration for opening and closing the device. Various mechanisms can be used to move the top between open and closed configurations. For example, linkage 23 can be or include one or more elements known in the art, such as a jar, hinge, soft-close arrangement, rod, or lever. In the perspective view of FIG. 1, knobs, rods, and soft-close structures 25 raise and lower top 19 relative to bottom 15 (hidden within cabinet 21 in the perspective view of FIG. 1). In some approaches, a lip allows linkage 19 to be raised or lowered, for example, with a hinge mechanism. In general, linkage 23 can be constructed using elements and techniques known in the art.

[0056] In some embodiments, the two sections are directly connected by a connecting portion 23. For example, the connecting portion 23 can be supported by the fixed first portion 15 and can include a mechanism that allows the second portion to be attached and moved between the open and closed configurations. In other embodiments, other components provide support for the connecting portion 23 or its components. For example, a support 27 can be used to position the top portion 19 against a structure (e.g., the top of the cabinet 21 in FIG. 1 , or a pedestal, table, or other configuration) to ensure precise butting of the top and bottom portions in the closed configuration. Other types of fastening means, such as bolts 29, can also be used. In the illustrated example, the base 27 incorporates or supports a hinge 31, an example element of the connecting portion 23.

[0057] The support 27 further comprises a sample pedestal 35 which includes a sample receiving area 37. In operation, the section 19 is opened and a sample, for example in the form of a droplet, is placed in the receiving area 37 and then closed like a lid, allowing the sample to be analysed.

[0058] Light from the tunable laser may be transmitted to device 13 via fiber optics. Electrical signals from one or more optical detection means used may be collected from device 13 and transmitted to the tunable laser spectrometer by a cable, such as one or more electrical harnesses. The tunable laser may be part of the tunable laser spectrometer together with a controller. Some or all of these components (e.g., fiber optics and / or electrical cables external to the open / close device 13), the tunable laser, the controller, and unit 15 or parts of unit 15 may be housed in cabinet 21.

[0059] An exemplary embodiment of the system and apparatus of the present invention will now be described in detail with reference to FIG. 2 (open configuration of the open-closed apparatus 13) and FIGS. 3A and 3B (closed configuration of the apparatus 13).

[0060] The light beam generated by tunable laser 51 is transmitted through an optical fiber arrangement, such as an optical fiber patch cable 53 (e.g., as shown in FIG. 3A ). The light then enters section 15 of device 13 at fiber port 55. In some embodiments, fiber patch cable 53 is a PANDA fiber. Fiber port 55 can be configured as a collimator for light exiting the optical fiber patch cable and directed to polarizing means 57 (which can be used to filter out orthogonally polarized light). Rotation mount 59 allows polarizing means 57 to rotate in a plane perpendicular to both bench 61 and the optical axis of the beam exiting the fiber. In some embodiments, mount 59 allows fine rotational adjustment of polarizing means 57 to align it with the desired polarization axis of the PANDA fiber.

[0061] The beam splitting means (for tapping a beam, such as the PANDA ripple submission) includes a partially reflective sapphire window 63, such as a wedge-shaped window, held by a pitch-yaw mount 65. The window 63 is fixed to the bench 61 by the pitch-yaw mount 65. The partially reflective sapphire window 63 reflects a portion of the laser beam (referred to herein as the "reference" beam) that enters the gate-type device through the fiber port 55 toward a ripple reference ("tap") optical detection means 69, such as an In-GaAs detector. A focusing lens 71 couples the reference beam to the active area of ​​the ripple reference detection means 69. In addition to holding the sapphire window 63, the pitch-yaw mount 65 adjusts the free-space beam reflected and transmitted by the window 63 so that it propagates and is incident on the active area of ​​the ripple reference detection means 69.

[0062] The ripple detection means 69 is mounted on a head-side printed circuit board (PCB) 73. The head-side printed circuit board 73 includes a transimpedance amplifier that amplifies the electrical response of the ripple reference detection means. The head-side printed circuit board may be provided with a thermistor that enables temperature compensation of the detection means 69 and the transimpedance amplifier. The response of the ripple reference light detection means may then be transmitted as an electrical signal to the tunable laser spectrometer 203, for example, via an electrical connection 91A of an electrical wiring harness arrangement.

[0063] The beam splitting means generates a second beam portion, separate from the reference beam, referred to herein as the "input" or "probe" beam, which propagates from the partially reflective sapphire window 63 toward the sample detection region 12, which is formed by an optical transmission port and an opposing optical detection port. In this example, the optical transmission port is formed by a quartz or sapphire input rod 126, and the optical detection port is formed by a quartz or sapphire output rod 128. The rod waveguide arrangement described herein eliminates the need for a cuvette, a typical component of existing instrumentation.

[0064] In some embodiments, rod 126 is held by rod holder 131 and rod 128 is held by rod holder 133. One or both rod holders may be heated.

[0065] Figure 3C depicts a right-angle optical arrangement in which the fiber port 55 (and therefore the optical entry portion 15) is positioned at an angle (typically 90°) to the straight line of the arrangement shown in Figures 3A and 3B (as opposed to, for example, a straight tap (see Figures 3A, 3B, etc.), where both the fiber port and the detector must be adjusted simultaneously). In this right-angle approach, the interrogation beam 95 is directed by a folding mirror 92 mounted on a kinematic mount 93 fixed to the bench 61.

[0066] Furthermore, the fiber port 55 is fixed to the part 15 (and therefore to the bench 61) via a goniometer 91, which allows the angle of the PANDA fiber to be adjusted to properly align it with the axis of the polarizing means 57.

[0067] In the embodiment shown in Figures 2, 3A and 3B, the input rod 126 is mounted on the sample seat 35, while the output rod 128 is part of section 19 of the open / closed device 13. In the open position (Figure 2), a sample (or a blank, for calibration purposes) can be introduced onto rod 126 in the sample-receiving area 37 of seat 35. By moving section 19 to the closed position, the two rods are aligned. The distance between these ports determines the path traveled by the input (probe) beam 95 within the sample gap. A precision ball screw 75 extending from one end of section 19 towards the sample seat 35 allows the rods to be offset to set the path to a desired, constant path length.

[0068] The path length value may be in the range of about 0.010 millimeters (mm) to about 5 mm, in some cases up to about 10 mm, e.g., in the range of about 0.01 mm to about 0.05 mm, about 0.1 mm, to about 0.5 mm, to about 1 mm, to about 2 mm, to about 3 mm, to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, e.g., in the range of about 0.05 mm to about 0.1 mm, to about 0.5 mm, to about 1 mm, to about 2 mm, to about 3 mm, to about 4 mm and within the range of about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm, and within the range of about 0.1 mm to about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm, and within the range of about 0.5 mm to about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm. in the range of about 1 mm to about 2 mm, to about 3 mm, to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, in the range of about 2 mm to about 3 mm, to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, in the range of about 3 mm to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, in the range of about 4 mm to about 5 mm mm, up to about 6 mm, up to about 7 mm, up to about 8 mm, up to about 9 mm, or up to about 10 mm, within the range of about 5 mm to about 6 mm, up to about 7 mm, up to about 8 mm, up to about 9 mm, or up to about 10 mm, within the range of about 6 mm to about 7 mm, up to about 8 mm, up to about 9 mm, or up to about 10 mm, within the range of about 7 mm to about 8 mm, up to about 9 mm, or up to about 10 mm, within the range of about 8 mm to about 9 mm, or up to about 10 mm, within the range of about 9 mm to about 10 mm, etc.

[0069] An example path length is about 5 mm. Another example path length is 1 mm. For MIR systems, an example path length can be reduced to, for example, about 100 microns.

[0070] In a detailed embodiment, the diameter of each rod is in the range of about 2 to about 8 millimeters (mm), e.g., about 4 mm. This configuration allows for a droplet of about 15 microliters (mL) to be held by surface tension while allowing a beam 95 of 0.5 to 3 mm, preferably about 1 mm, diameter to travel through the rod and the sample in the sample gap 12. In many cases, both rods have the same diameter. In other cases, the rods have different diameters. In one example, the base window (rod) is enlarged to allow for easier cleaning.

[0071] The output rod 128 is optically coupled to a sample light detector 81 equipped with a lens 83. The electrical signal registered by the light detector 81 can be transmitted to the tunable laser spectrometer 203, for example, by electrical connection 91B of a wire harness arrangement. In one example, the light detector is a TO-46 In-GaAs detector with a dome lens. The detector can be electrically connected to a sample detector printed circuit board (PCB) 85. The detector PCB can itself include a transimpedance amplifier that amplifies the detector response and transmits it to the tunable laser spectrometer 203. The detector PCB can include a thermistor to detect temperature and allow for temperature offset.

[0072] In operation, swept wavelength light from the tunable laser 51 is coupled into the fiber optic patch cable 53 toward the gate-and-gate device 13 section 15. The PANDA fiber optic patch cable operates to eliminate modes and minimize ripple. The short length of the fiber patch cable reduces or minimizes losses. Additionally, the polarization means 57 eliminates modes and random polarization fluctuations and accounts for polarization-dependent losses in optical components. A portion of the polarized light is detected by the ripple reference light detection means 69, which can improve operation by accounting for random power attenuation. Polarizing the light eliminates the risk of polarization-dependent losses in optical components. This ripple reference signal is transmitted back to the tunable laser spectrometer 203, for example, via the spectrometer electrical wiring harness.

[0073] The remaining light travels towards input rod 126, couples into input rod 126 and exits through the light transmission port. It then propagates through sample detection region 12 and is altered by the analyte present in the sample. Preferably, the analyte will absorb some wavelengths more than others. It exits sample detection region 12 through the light detection port and enters output rod 128. It propagates through output rod 128 and is detected by sample light detection means 81.

[0074] In many cases, the light beam entering the open / closed device travels in free space (from one optical element to the next) and not just through an optical fiber, see the arrows (FIG. 3A) tracing the light path inside the open / closed device 13.

[0075] A controller 200, which may be part of the tunable laser spectrometer 203, monitors the responses of the sample light detector 81 and the ripple reference detector 65. That is, the controller can resolve the sample absorption spectrum by monitoring the spectral scan across the scan band of the tunable laser 51 against the time response of the sample light detector 81. Any noise associated with sources such as ripple from the optical fiber is compensated for by the response from the ripple reference detector 69. Typically, the tunable laser or tunable laser system sweeps its emission across a narrow band or portion of the electromagnetic spectrum, such as the NIR and / or SWIR and / or MIR regions.

[0076] In some embodiments, the control unit 200 uses the temperature detected by the thermistor on the PCB on which the sample detection means 81 is provided and the temperature detected by the thermistor on the reference detection means 69 to compensate for changes in the response of the ripple reference light detection means 69 and the sample light detection means 81, as well as changes in the gain used in the transimpedance amplifier on each PCB.

[0077] In many optical arrangements, an etalon is formed between parallel reflective surfaces. In the apparatus 13, reflections occur at each surface due to a refractive index mismatch between the air in the beam's free-space path and the bulk material of the input rod 126. A refractive index mismatch typically also exists between the fluid in the sample region 12 and the input and output rods 126 and 128. The beam also propagates in free space between the output rod 128 and the detection means 81. Even if surfaces are anti-reflective coated, residual reflectivity may still be present.

[0078] Some of the measures that can be taken to prevent or mitigate reflections / etalons are described with reference to Figures 4A, 4B, 5, and 6A-6C. In certain embodiments, the rods 126, 128 are configured so that the light input and output surfaces of each rod are not parallel flat surfaces. For example, the faces of each rod may be angled, as described below.

[0079] FIG. 4A shows the input rod 126 and output rod 128 forming the sample gap 12 between the transmission port 126P and the detection port 128P. The ports 126A, 126P of the input rod 126 and the ports 128P, 128A of the output rod 128 are formed with a wedge angle θ, resulting in an inclined or slanted wedge-shaped surface. Typically, the wedge angle is a few tenths of a degree, e.g., in the range of about 0.09 to about 0.6°. In one example, the angle θ (FIG. 4A) is 0.125°. In a further embodiment, the input rod 126 and the output rod 128 are rotated 90° relative to each other (see the circular arrows in FIG. 4A) and oriented as shown in FIG. 4B. In this example, ports 128A and 128P (forming the light exit surface from the rod 128) of the output rod 128 are angled from left to right on the page, and ports 126A and 126P (forming the light entrance surface to the rod 126) of the input rod 126 are angled from front to back on the page.

[0080] Figure 5 shows a cross-sectional view of a portion of device 13. The depicted portion may incorporate geometric and orientation details that reduce or suppress reflections / etalons. Specifically, Figure 5 illustrates how the rods are wedged and angled on both sides, with each rod in a pair being 90° "advanced" from the other to offset the reflected waves.

[0081] Figure 6A is a schematic diagram showing light traveling through input and output rods 126 and 128 configured and oriented as described above. Due to the 90° time advance, none of the reflected waves overlap. This can be seen in Figure 6B, which shows the lack of overlap between the main beam (dark circle) and the primary and secondary reflected waves (light circle). Figure 6C shows the etalon intensity upon reflection in water. Each rod (angle θ) is measured at a point where the etalon intensity is below the noise floor (approximately 10 -6 The tilt angle is adjusted to be less than 100 mAU or greater than about 0.4°.

[0082] The workflow of the present invention typically involves a few simple steps: With the open device in the open position, a blank or sample (e.g., 15 μL) is pipetted onto rod 126 in sample receiving area 37 of pedestal 35. Portion 19 is then lowered toward portion 15 to close the open device, forming the sample detection area containing the sample (or blank). A scan (often taking about 5 seconds at a time) can then be performed, after which the results can be read out.

[0083] In many embodiments, the results are automatically analyzed and displayed in a suitable viewer, such as that shown in Figure 7, showing the scaled absorbance of the sample components mAb (A), histidine (B), and polysorbate (C), a comparison of the sample with a reference library complex (D), and the scaled absorbance of the residual (E). The various components can be measured simultaneously, presenting data for the mAb and excipients in a single scan.

[0084] The following non-limiting examples further illustrate embodiments of the present invention. [Example]

[0085] Example 1

[0086] As mentioned, an application of great interest relates to protein measurement. The techniques described herein can operate within a wavelength range where mAb absorbance is two orders of magnitude lower than in the UV region, resulting in a detection limit two orders of magnitude higher than UV measurements (maximum absorbance of approximately 5 AU). This is illustrated in Figure 8 (which shows a comparison of UV / visible results (1 mm, 0.1 mm) with results from embodiments of the present invention). The excellent SNR and sensitivity allow detection limits around 0.01 mg / mL to be achieved, providing a significantly wider dynamic range than other current optical methods.

[0087] The data described below were obtained using a tunable laser developed for a specific wavelength range containing key protein chemical information of interest (e.g., CH, OH) and a NISTmAb reference standard. Application of embodiments of the present invention confirmed spectral identity and qualified sample concentration with a high correlation coefficient (>0.99) against the reference standard.

[0088] Figure 9 shows the observed spectral signature of a 100 mg / mL mAb product (A), which is identical to the NISTmAb (10.03 mg / mL) and varies proportionally with dilution (B, C, and D). More specifically, the user's sample was compared to the NISTmAb reference material. Instantaneous statistics were performed to qualify the measurements. The measured (and actual) concentrations for samples A through D were 99.39 (100) mg / mL, 49.9 (50) mg / mL, 25.6 (25) mg / mL, and 12.7 (12.5) mg / mL, respectively.

[0089] For measurements of mAb in the presence of excipients, mAb was applied in a histidine and poly80 buffer (5 mg / mL histidine + 1 mg / mL poly80). Figure 10 shows that the UV analysis had an error of approximately 20%, whereas the method described here only had an error of approximately 1% at any dilution.

[0090] Example 2

[0091] Using the open-close system described above, various amino acids present in buffer were measured. The basic steps were: 1) pipette 15 μL of deionized (DI) water onto the pedestal and perform a scan, 2) wipe with a Kimwipe®, 3) pipette 15 μL of sample (of known concentration) and perform a scan, and 4) select the analyte to be quantified, run the analysis, and obtain the results.

[0092] Figure 11A shows an on-demand excipient analysis for buffer validation of a sample containing sucrose (270 mM), histidine (20 mM), and HCl (used as a pH buffer). Spectral deconvolution confirmed the presence and simultaneous measurement of histidine and histidine hydrochloride (two species representing different protonated forms of histidine with pKa around 6.0). More specifically, the results confirmed a sucrose concentration of 270 mM, along with the detection of 7.2 mM histidine and 13.24 mM histidine hydrochloride.

[0093] FIG. 11B is a graph showing the concentrations of arginine hydrochloride, arginine, histidine hydrochloride, and histidine in a sample containing 10 mM arginine and 10 mM histidine hydrochloride by spectral deconvolution measurements.

[0094] Example 3

[0095] 12A-12D show examples of measurements of complex buffer components, such as surfactant species, chelating compounds, and buffer components such as specific sugars.

[0096] While the present invention has been particularly shown and described with reference to preferred embodiments, those skilled in the art will recognize that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. 1. A system for analyzing a sample, comprising: a tunable laser spectrometer that generates a swept wavelength signal; an open / close device forming a sample detection region, the open / close device including a reference light detection means for detecting the swept wavelength signal from the tunable laser spectrometer and a sample light detection means for detecting the swept wavelength signal after passing through the sample detection region; wherein the open / close device has a fixed first part and a second part configured to move between an open position and a closed position of the open / close device.

2. 2. The system of claim 1, wherein the reference light detecting means is in the fixed first part of the open / closed apparatus and the sample light detecting means is in the second part of the open / closed apparatus.

3. 3. The system of claim 1 or 2, wherein the swept wavelength signal generated by the tunable laser is in the range of about 1350 to about 1800 nm, in the range of about 2050 to about 2400 nm, or in the range of about 3.5 to about 10 microns.

4. 4. The system of claim 1, wherein the sample detection region defines a fixed path length selected from the range of about 0.010 mm to about 5 mm.

5. 5. The system of claim 1, wherein the sample detection region is formed between a transmission port of an input rod and a detection port of an output rod.

6. 6. The system of claim 5, wherein the input and output rods are wedge shaped at an angle θ and rotated 90 degrees relative to each other.

7. 6. The system of claim 5, wherein the distance between the transmission port of the input rod and the detection port of the output rod is adjustable.

8. The system of any one of claims 1 to 7, further comprising: beam splitting means for directing a reference beam to said reference light detection means and an interrogation beam to said sample light detection means; A system comprising:

9. The system of any one of claims 1 to 8, further comprising: Polarizing means, A system comprising:

10. The system of any one of claims 1 to 9, further comprising: a cable for transmitting light from a tunable laser to said device; wherein the cable is a single-mode polarization-maintaining optical fiber.

11. The system of any one of claims 1 to 10, further comprising: a cable for transmitting an electrical signal from the reference light detecting means and an electrical signal from the sample light detecting means to the tunable laser spectrometer; A system comprising:

12. 12. The system of claim 1, further comprising: control unit, A system comprising:

13. 1. A method for analyzing a sample, comprising: generating a swept wavelength signal; transmitting the swept wavelength signal to a first portion of a gate-type device; detecting the swept wavelength signal within the open / closed device prior to transmission through a sample detection region formed in the closed configuration of the open / closed device; detecting the swept wavelength signal after transmission through a sample in the sample detection region; resolving the absorption spectrum of the sample with reference to the swept wavelength signals before and after transmission through the sample detection region; A method comprising:

14. 14. The method of claim 13, wherein the sample detection region defines a fixed path length having a value selected from the range of about 0.010 mm to about 5 mm.

15. 15. The method of claim 13 or 14, wherein the generated wavelength signal is in the range of about 1350 to about 1800 nm, in the range of about 2050 to about 2400 nm, or in the range of about 3.5 to about 10 microns.

16. 16. The method of claim 13, wherein the step of detecting the swept wavelength signal before transmission through the sample detection region is performed at a fixed portion of the open / close device, and the step of detecting the swept wavelength signal after transmission through the sample in the sample detection region is performed at a lid portion of the open / close device.

17. 17. The method of any one of claims 13 to 16, further comprising: a step of opening and closing the open-close type device; A method comprising:

18. 18. The method of any one of claims 13 to 17, wherein the sample detection region is formed between a transmission port in an input rod and a detection port in an output rod.

19. 20. The method of claim 18 further comprising: suppressing reflections when the input rod and the output rod pass through; A method comprising:

20. 20. The method of any one of claims 13 to 19, further comprising: The process of transmitting light through a single-mode polarization-maintaining optical fiber, A method comprising:

21. 21. The method of any one of claims 13 to 20, wherein the swept wavelength signal is passed through polarizing means before being detected as a reference signal.

22. An open / close type device, a first section including a polarizing means, a beam splitting means, and a reference light detecting means; a second part including a sample light detection means; an input rod having a transmission port and an output rod having a detection port; wherein the transmission port and the detection port form a sample detection region in a closed configuration of the device.

23. 23. The openable / closable device of claim 22, wherein the distance between the transmission port and the detection port in the closed configuration is fixed.

24. 24. The open / closed device of claim 23, wherein the distance between the transmission port and the detection port in the closed configuration of the open / closed device is a value selected from the range of about 0.010 mm to about 5 mm.

25. 25. The device of any one of claims 22 to 24, wherein the first part is fixed and the second part is configured to move between an open position and a closed position.

26. 26. The apparatus of any one of claims 22 to 25, further comprising: a sample receiving area; An apparatus comprising:

Images