Native fluorescence detection for protein analysis in capillary electrophoresis

Abstract

A method and system for determining the concentration of a target protein in a sample using a capillary electrophoresis (CE) system is disclosed. In a particular embodiment, the method can include flowing a sample through a capillary tube of a CE system and utilizing a light source to generate radiation comprising at least one excitation wavelength suitable for exciting at least one natural fluorophore of at least one target protein in the sample. An excitation beam comprising at least one excitation wavelength can be directed into the transparent portion of the capillary tube to excite the at least one natural fluorophore of the target protein passing through the lumen of the transparent portion to cause the at least one natural fluorophore to generate fluorescence radiation, and at least a portion of the fluorescence radiation emitted by the excited target protein can be detected.

Description

[Technical field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 174,897, filed April 14, 2021, and U.S. Provisional Application No. 63 / 292,277, filed December 21, 2021, the contents of which are incorporated herein in their entireties.

[0002] (Technical field) The present disclosure relates generally to systems and methods for quantification of one or more target proteins in a sample, and in particular to such quantification systems and methods that can provide high sensitivity and selectivity for target samples contained within capillary tubes of a capillary electrophoresis (CE) system. [Background technology]

[0003] Over the past two decades, capillary electrophoresis (CE) has developed into a mature separation technique for biopharmaceutical and biomolecule analysis. In the biopharmaceutical industry, CE-based techniques (including capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and capillary isoelectric focusing (cIEF)) are increasingly replacing gel-based electrophoresis techniques to aid in the analytical characterization and quality control of therapeutic monoclonal antibodies (mAbs).

[0004] In CE, the use of thin capillary tubes in which high electric field strengths can be applied provides an environment that allows for highly efficient separations using minimal sample volumes. However, small capillary inner diameters can pose detection challenges in that they exhibit short optical path lengths and therefore require large sample loading to achieve sufficient sensitivity for many analytes, especially those present in low concentrations.

[0005] UV absorbance detection is the most widely used technique in CE and has been shown to achieve a linear dynamic range of 2-3 orders of magnitude, however, its concentration sensitivity can be lower than required for many applications.

[0006] Laser-induced fluorescence (LIF) detection is often used in an attempt to improve concentration sensitivity and extend the linear dynamic range. However, commercially available excitation sources generally emit in the visible wavelength range, where proteins are not inherently fluorescent. To circumvent this challenge of LIF detection, fluorophore derivatization has been used, but can cause further problems.

[0007] For example, labeling sites on proteins have different reactivities, and labeling reactions invariably generate mixtures of differentially labeled proteins, resulting in peak broadening or splitting as well as quantification challenges. Furthermore, labeling proteins with fluorescent dyes can lead to changes in the net charge of the protein, thereby altering the isoelectric point (pI) of the protein and resulting in an overall change in the properties of the protein compared to its native state.

[0008] In an effort to circumvent the challenges posed by UV and LIF detection modalities, researchers have used deep UV lasers as excitation sources to exploit the intrinsic fluorescence emission from specific amino acids to facilitate protein detection and quantification. Native fluorescence detection (NFD) of proteins was first reported using 257 nm excitation radiation generated by frequency doubling of 514 nm argon-ion laser-generated radiation. Data were acquired using a 50 μm inner diameter capillary, achieving a limit of detection (LOD) of 14 nM for concalbumin.

[0009] The detection sensitivity of the same protein was improved using a 275.4 nm laser line isolated from a water-cooled argon-ion laser. Pulsed lasers, including ND:YAG (266 nm), KrF (248 nm), and He-Ag (224 nm) lasers, were also evaluated as excitation sources for NFD. These lasers are relatively cost-effective, but the concentration sensitivity that can be achieved using such lasers is lower than that achievable using UV argon-ion lasers due to unfavorable excitation wavelengths and pulse-to-pulse variations.

[0010] Lamps such as xenon and deuterium lamps provide a continuous spectrum in the deep UV region, thus allowing flexibility in excitation wavelengths for NFD. However, conventional lamps emit divergent light of low radiant power, presenting a challenge to deliver sufficient energy for fluorescence detection in CE. To improve detection, lamp-based fluorescence detection systems have been introduced, in which light from a mercury-xenon lamp is filtered and subsequently directed through an optical fiber into a capillary to be focused into a detection cell using a ball lens. The emitted fluorescence is guided by total internal reflection along the capillary to a photomultiplier tube (PMT) placed at the end of the cell. This detection system has been successfully adapted to commercial CE instruments for the analysis of various compounds. Results showed that the LOD for the analysis of tryptophan was 6.7 nM, 20 times greater than the sensitivity obtained via absorbance detection at 214 nm. For protein analysis, the LOD was in the range of 10-20 nM, 25 times more sensitive than absorbance detection at 280 nm and comparable to absorbance detection at 214 nm. Replacing the PMT in this system with a charge-coupled device can turn this system into a wavelength-resolved fluorescence detector, which can probe conformational changes in proteins.

[0011] There remains a need for quantification of protein samples using NFDs, which offer greater sensitivity and flexible excitation wavelengths compared to conventional approaches. Summary of the Invention [Means for solving the problem]

[0012] In various embodiments of the present teachings, a system for determining the concentration of a target protein in a sample is provided, the system including a light source for generating radiation and an optical system for directing the radiation onto a capillary tube of a capillary electrophoresis (CE) system through which a sample of interest flows, to illuminate at least the target protein if present in the sample. The radiation generated by the light source can include at least one excitation wavelength suitable for exciting at least one native fluorophore of said at least one target protein in the sample.

[0013] Various light sources can be used according to the present teachings. For example, in some embodiments, the light source can comprise at least one light emitting diode. Alternatively, in some embodiments, the light source can comprise a laser-driven light source. In various embodiments, the light source can generate radiation having wavelengths across a spectral range, and the excitation beam can exhibit a spectral bandwidth narrower than the spectral range of the radiation. In certain embodiments, the at least one excitation wavelength can be about 285 nm.

[0014] In various embodiments, the capillary tube can include a radiation transparent portion through which excitation radiation can be introduced into the capillary tube and at least a portion of the fluorescent radiation generated by the target protein in response to the excitation radiation can exit the capillary tube. Additionally, in some embodiments, the system can further include a detector optically coupled to the transparent portion of the capillary tube to receive at least a portion of the fluorescent radiation and generate one or more fluorescent detection signals in response to detection of the received fluorescent radiation. In some related embodiments, an analyzer can be in communication with the detector to receive a detection signal and process the detection signal to obtain a concentration of the target protein in the sample. For example, the analyzer can be configured to determine a concentration of the target protein based on an intensity of the fluorescent radiation. In some embodiments, an optical element can be provided to direct the fluorescent radiation onto the detector, and optionally the optical element can include a lens to focus the fluorescent radiation onto the detector, and further optionally a filter can be positioned in front of the detector to filter out the excitation radiation.

[0015] In various embodiments, the optical system can include an optical fiber extending from a proximal end to a distal end, the proximal end of the optical fiber optically coupled to the light source to receive at least a portion of the radiation emitted by the light source. In some related embodiments, the system can further include one or more lenses disposed between the light source and the proximal end of the optical fiber to transmit the radiation emitted from the light source to the proximal end of the optical fiber, and optionally, in some embodiments, the one or more lenses can include two converging lenses mounted in tandem to image the emission surface of the LED onto the proximal end of the optical fiber. In some additional related embodiments, the system can further include an optical filter disposed between the two converging lenses to receive at least a portion of the radiation and select an excitation wavelength for illuminating the sample. For example, the optical filter can optionally include an optical bandpass or shortpass filter, and further optionally, the optical bandpass or shortpass filter can exhibit a transmission bandwidth in the range of about 270 nm to about 290 nm.

[0016] In one embodiment, a system for determining the concentration of a target protein in a sample is disclosed that includes a laser-driven light source for generating radiation and optics for directing the radiation onto a capillary tube of a capillary electrophoresis system through which a sample of interest can flow to illuminate at least one target protein in the sample, wherein the radiation generated by the radiation source has at least one excitation wavelength suitable for exciting at least one native fluorophore of the at least one target protein in the sample.

[0017] In some embodiments, the radiation produced by the laser-driven light source comprises wavelengths extending across a continuous spectral range that includes the excitation wavelength, in some such embodiments, the continuous spectral range can extend from about 170 nm to about 2100 nm.

[0018] The system may further include an optical filter positioned relative to the laser-driven light source to receive at least a portion of the radiation generated by the light source and select an excitation wavelength for irradiating the sample. In some embodiments, the optical filter may be an optical bandpass filter. In some such embodiments, the optical bandpass filter exhibits a transmission bandwidth in the range of about 5 nm to about 50 nm, which may be centered on the excitation wavelength.

[0019] The capillary tube can include a radiation-transparent portion (also referred to herein as a transparent or radiation-transparent portion), e.g., a portion made of glass or other suitable material, through which excitation radiation can be introduced into the capillary tube and excite at least one target protein of interest, and through which fluorescent radiation emitted by the excited protein can exit the capillary tube and be detected by a downstream detector. More specifically, the detector can be optically coupled to a back surface of the radiation-transparent portion of the capillary tube to receive at least a portion of the fluorescent radiation and generate one or more detection signals in response to detection of the received fluorescent radiation.

[0020] In some embodiments, an optical component, such as a spherical mirror, is optically coupled to the transparent portion of the capillary tube to capture at least a portion of the fluorescent radiation that exits through the front (also referred to herein as the "front window") of the capillary tube (i.e., the portion that receives the excitation light) and reflect the captured radiation, or at least a portion of it, back into the capillary tube, whereby the reflected radiation, or at least a portion of it, exits through the back surface of the capillary tube and is detected by a detector. In some such embodiments, the spherical mirror can have a radius of curvature and can be positioned relative to the transparent portion of the capillary tube such that the radiation reflected by the spherical mirror is focused at a focal point located in the central portion of the capillary tube. The returned fluorescent radiation can then diverge from that focal point to exit the capillary tube.

[0021] An analyzer in communication with the detector can receive the detection signal generated by the detector and can process the detection signal to calculate the concentration of the target protein in the sample. By way of example, the analyzer can be configured to calculate the concentration of the target protein based on the intensity of the detected fluorescent radiation.

[0022] As mentioned above, a laser-driven radiation source can generate radiation having wavelengths (herein, the wavelength of radiation refers to the vacuum wavelength) spanning a continuous spectral range. This allows for the selection of a desired excitation wavelength from among the wavelengths present in the spectral range of the radiation emitted by the source to excite a target protein of interest. In some embodiments, a device fitted with multiple optical filters exhibiting different transmission bandwidths, e.g., a filter wheel, can be used to switch between optical filters placed in the path of the excitation radiation to select different excitation wavelengths, e.g., to detect different target proteins. In some embodiments, the excitation wavelength can be, for example, in the range of about 200 to about 300 nm.

[0023] In some embodiments, the optical fiber extending from the proximal end to the distal end is positioned relative to the optical filter and the capillary tube such that the optical fiber receives at least a portion of the excitation radiation passing through the filter through its proximal end and transmits at least a portion of the received radiation onto the capillary tube through its distal end to excite at least one target protein present in the sample or suspected of being present in the sample.

[0024] In some embodiments, the optical system configured to direct at least a portion of the excitation radiation to the proximal end of the optical fiber can include one or more lenses for focusing the radiation into the optical fiber. In some embodiments, such lenses can include two converging lenses arranged in tandem. For example, the two lenses can be configured such that a first lens substantially collimates the incident radiation and a second lens focuses the substantially collimated radiation into the proximal end of the optical fiber. In some such embodiments, an optical filter can be disposed between the two lenses to select the excitation wavelength of interest.

[0025] In some embodiments, the f-number of such a lens can be selected such that the convergence angle associated with the radiation focused onto the optical fiber (i.e., the proximal end of the optical fiber) is substantially equal to the numerical aperture of the optical fiber.

[0026] In some embodiments, the system can further include an optical filter disposed between the first lens and the second lens to receive at least a portion of the radiation and select an excitation wavelength for irradiating the sample. In some such embodiments, the optical filter includes an optical bandpass or shortpass filter. By way of example, the optical bandpass or shortpass filter can exhibit a transmission bandwidth in the range of about 200 nm to about 300 nm.

[0027] In some embodiments, the laser-driven light source has a power output of about 0.5 to about 200 mW / mm 2 By way of example, in some such embodiments, the radiance of the emitted radiation in the vacuum wavelength range of about 200 nm to about 300 nm can be from 5 to about 200 mW / mm 2 It can be in the range of .sr.nm.

[0028] In some embodiments, the capillary tube can have an inner diameter in the range of about 10 μm to about 200 μm, for example, in the range of about 20 μm to about 100 μm.

[0029] In a related embodiment, a method for protein analysis of a sample in a capillary electrophoresis (CE) system is disclosed, the method comprising: flowing the sample through a capillary tube of the CE system; utilizing a laser-driven light source to generate radiation having wavelengths spanning a spectral range including at least one wavelength suitable for exciting at least one target protein in the sample; and spectrally filtering the radiation to select an excitation wavelength to generate an excitation beam having a spectral bandwidth narrower than the spectral range of the radiation generated by the laser-driven radiation source and including at least one excitation wavelength suitable for exciting at least one target protein of interest. The excitation beam is directed through a transparent portion (e.g., through a radiation-transparent window) into the capillary tube of the capillary electrophoresis system to excite at least one native fluorophore of the target protein passing through the lumen of the transparent portion of the capillary tube to cause the fluorophore to generate fluorescent radiation and to detect at least a portion of the fluorescent radiation emitted by the excited target protein.

[0030] The emitted fluorescent radiation can be detected by directing at least a portion of the fluorescent radiation exiting the capillary tube, for example, through a transparent portion thereof (e.g., through a radiation-transmissive window) onto a detector that generates one or more detection signals in response to detection of the fluorescent radiation.

[0031] In some embodiments, the fluorescent radiation emitted by a plurality of different proteins is detected as a function of the transit time of those proteins from the entrance of the capillary tube to the transparent portion of the tube under the influence of the electric field. In some such embodiments, the distance between the entrance of the capillary tube and its transparent portion can be within a close range, e.g., about 5 cm to about 100 cm, to allow for better resolution in separating the different proteins of interest for interrogation via excitation radiation.

[0032] In some embodiments, the radiation emitted by a laser-driven radiation source can be spectrally filtered by passing the radiation through an optical filter, such as an optical bandpass or shortpass filter, to narrow the spectral range of wavelengths present in the radiation while ensuring that this narrow spectral range includes the excitation wavelength of interest.

[0033] The fluorescent radiation emitted by the excited protein can be analyzed to determine the concentration of the protein in the sample under study. For example, the concentration of the protein of interest can be determined based on the intensity of the detected fluorescent radiation and / or the area under the fluorescent peak.

[0034] In some embodiments, the sample analysis methods described above utilize an optical fiber to direct excitation radiation into the capillary tube (i.e., onto a transparent portion of the capillary tube). In such embodiments, the excitation radiation can be coupled into a proximal end of the optical fiber. The optical fiber can transmit the received radiation to its distal end. The distal end of the optical fiber can be optically coupled to the transparent portion of the capillary tube, such that at least a portion of the radiation exiting the distal end of the optical fiber can be introduced into the capillary tube through the transparent portion to excite at least one protein flowing through the lumen of the transparent portion of the tube.

[0035] In some embodiments, the spectral range of the radiation emitted by the laser-driven light source can range, for example, from about 170 nm to about 2100 nm. Additionally, in some embodiments, the excitation wavelength for exciting one or more target proteins can range from about 200 nm to about 300 nm.

[0036] In a related embodiment, a system for determining the concentration of a target protein in a sample is disclosed, the system comprising: an LED light source for generating radiation; an optical fiber extending from a proximal end to a distal end, the proximal end of the optical fiber being optically coupled to the LED light source for receiving at least a portion of the radiation emitted by the LED light source; and a capillary tube of a capillary electrophoresis system through which a sample of interest flows, the distal end of the optical fiber being optically coupled to a portion of the capillary tube for irradiating at least one target protein in the sample flowing through the capillary tube. The radiation generated by the LED light source comprises at least one excitation wavelength suitable for exciting at least one native fluorophore of at least one target protein in the sample.

[0037] In some embodiments, the radiation produced by the LED light source has a substantially monochromatic wavelength in the UV range of the electromagnetic spectrum. By way of example, the substantially monochromatic wavelength may be about 285 nm.

[0038] In some embodiments, the LED light source provides a light output in the range of about 50 μW to about 10 mW.

[0039] In some embodiments, the capillary tube comprises a radiation transparent portion through which excitation radiation can be introduced into the capillary tube and at least a portion of the fluorescent radiation produced by the target protein in response to the excitation radiation can exit the capillary tube.

[0040] In some embodiments, the system may further include a detector optically coupled to the transparent portion of the capillary tube to receive at least a portion of the fluorescent radiation and generate one or more fluorescent detection signals in response to detection of the received fluorescent radiation. In some such embodiments, an optical element may further be included to direct the fluorescent radiation to the detector. In some such embodiments, the optical element comprises a lens to focus the fluorescent radiation onto the detector. In some embodiments, a filter may be positioned in front of the detector to filter out the excitation radiation.

[0041] The system may further include an optical system disposed between the LED light source and the proximal end of the optical fiber for transmitting radiation emitted from the LED light source to the proximal end of the optical fiber. In some such embodiments, the optical system comprises one or more lenses for transmitting the radiation to the proximal end of the optical fiber. The one or more lenses may include two converging lenses mounted in tandem to image an emission face of the LED to the proximal end of the optical fiber. In some embodiments, the one or more lenses include a first lens and a second lens, the first lens configured and arranged relative to the LED light source to substantially collimate radiation generated by the LED light source, and the second lens configured and arranged relative to the first lens to focus the collimated radiation to the proximal end of the optical fiber.

[0042] In some embodiments, an optical filter can be disposed between the first lens and the second lens to receive at least a portion of the radiation and select an excitation wavelength for illuminating the sample. In some embodiments, the optical filter comprises an optical bandpass or shortpass filter. By way of example, the optical bandpass or shortpass filter can exhibit a transmission bandwidth in the range of about 270 nm to about 290 nm.

[0043] According to various aspects of the present teachings, a method for performing protein analysis in a capillary electrophoresis (CE) system is disclosed. For example, a method for protein analysis can include flowing a sample through a capillary tube of a CE system and utilizing a light source to generate radiation comprising at least one excitation wavelength suitable for exciting at least one natural fluorophore of at least one target protein in the sample. An excitation beam comprising at least one excitation wavelength can be directed to a transparent portion of the capillary tube of the CE system to excite at least one natural fluorophore of the target protein passing through the lumen of the transparent portion to generate fluorescence radiation in the at least one natural fluorophore. At least a portion of the fluorescence radiation emitted by the excited target protein can be detected.

[0044] A variety of light sources can be used in accordance with the present teachings. For example, in some embodiments, the light source can comprise at least one light emitting diode. Alternatively, in some embodiments, the light source can comprise a laser-driven light source. In various embodiments, the light source can generate radiation having wavelengths spanning a spectral range, and the excitation beam can exhibit a spectral bandwidth narrower than the spectral range of the radiation. In some related embodiments, the method can further include spectrally filtering the radiation to generate the excitation beam. For example, an optical bandpass or shortpass filter can be utilized to spectrally filter the radiation. In some related embodiments, the optical bandpass or shortpass filter can exhibit a transmission bandwidth ranging from about 200 nm to about 300 nm.

[0045] In various embodiments, the target protein may comprise an antibody.

[0046] In various embodiments, the at least one excitation wavelength can be about 285 nm. Additionally or alternatively, in some embodiments, the method can include adjusting at least one excitation wavelength contained within the excitation beam.

[0047] In certain embodiments, methods according to the present teachings can utilize a lens to focus the fluorescent radiation onto a detector, hi some related embodiments, the method can further include filtering the excitation wavelengths from the detector.

[0048] A further understanding of the various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are briefly described below. [Brief description of the drawings]

[0049] [Figure 1A] FIG. 1A illustrates a schematic diagram of an example of a system according to one embodiment for NFD quantification of proteins in a CE system.

[0050] [Figure 1B] FIG. 1B shows a schematic cross-sectional view of the capillary tube shown in FIG. 1A, illustrating that a high voltage applied to the capillary tube generates an electric field along the lumen of the tube, which causes the movement of proteins from the inlet to the outlet of the tube.

[0051] [Figure 1C] FIG. 1C shows diagrammatically a filter wheel on which a number of optical filters with different transmission bandwidths can be mounted, in order to make it possible to change the optical filters placed in the path of the radiation emitted by the radiation source and / or the fluorescence radiation.

[0052] [Diagram 2] FIG. 2 presents data showing the signal-to-noise ratio of an example NFD system according to one embodiment as a function of the bias voltage applied to a PMT used in the system as a detector.

[0053] [Figure 3A] FIG. 3A presents data corresponding to CE-SDS separation of reduced SCIEX IgG control standard with NFD at an excitation wavelength of 280 nm.

[0054] [Figure 3B] FIG. 3B presents data corresponding to CE-SDS separation of reduced SCIEX IgG control standard with NFD at an excitation wavelength of 220 nm.

[0055] [Figure 4A] FIG. 4A presents UV absorbance data obtained using CE-SDS separation of the non-reduced SCIEX IgG control standard using UV absorbance detection at 214 nm and 280 nm.

[0056] [Figure 4B] FIG. 4B presents the NFD data obtained using CE-SDS separation of the non-reduced SCIEX IgG control standard.

[0057] [Figure 5A] FIG. 5A presents UV absorbance detection data using cIEF separation of NIST IgG at 280 nm.

[0058] [Figure 5B] FIG. 5B presents the NFD data using cIEF separation of NIST IgG at 280 nm.

[0059] [Figure 6A] FIG. 6A shows a schematic diagram of an example of a system according to another embodiment for NFD quantification of proteins in a CE system.

[0060] [Figure 6B] FIG. 6B shows a schematic diagram of an example of a system according to yet another embodiment for NFD quantification of proteins in a CE system.

[0061] [Figure 7] FIG. 7 presents an SDS-CGE separation of a reduced IgG control standard with NFD using the direct LED (A) and the filtered LED (B) as the excitation source.

[0062] [Figure 8] 8A and 8B present the effect of PMT control voltage on signal to noise.

[0063] [Figure 9] Figures 9A-D present the effect of sample injection time, detection sensitivity and separation efficiency of SDS-CGE.

[0064] [Figure 10] 10A and 10B present the linearity of SDS-CGE with NFD for the analysis of non-reduced NISTmAb.

[0065] [Figure 11] 11A and 11B present the quantification and purity analysis of etanercept in SDS-CGE using UV absorbance versus NFD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The present disclosure relates to systems and methods that can be used for protein quantification in electrophoretic analysis of various samples without the need for deep UV lasers. As described in more detail below, in many embodiments, the systems and methods of the present teachings allow for performing NFD with at least 10 times higher sensitivity than that exhibited by current UV absorbance detection technology at 214 nm. Furthermore, the systems and methods of the present teachings allow for tuning the excitation wavelength, which can optimize the excitation wavelength for a specific target protein. Thus, in addition to high sensitivity, in many embodiments, the systems and methods of the present teachings allow for flexible selection of excitation wavelengths. For example, in many embodiments, the excitation wavelength can be adjusted by replacing bandpass or shortpass filters with different filters or monochromators without reducing detection sensitivity. As an example, the ability to adjust the excitation wavelength can be advantageously used to optimize quantum yield or to perform wavelength-resolved fluorescence detection.

[0067] As used herein, various terms are used according to their ordinary meaning in the art. As used herein, the term "laser-driven light source" refers to a radiation source that includes a laser that provides laser radiation for heating a gas plasma to a high temperature to produce radiation having a wavelength spanning a continuous spectral region (e.g., deep UV radiation having a wavelength in the range of about 200 nm to about 300 nm).

[0068] As used herein, the term "about" is intended to indicate a variation around a numerical value of up to 10%.

[0069] As used herein, the term "substantially" indicates a departure from perfect state and / or condition, if present, of at most 10%, or at most 5%.

[0070] The terms "light" and "radiation" are used interchangeably herein to refer not only to visible light but more generally to radiation in other regions of the electromagnetic spectrum, including UV radiation.

[0071] The term "radiance" as used herein is defined as the radiant flux emitted per unit solid angle in a given direction by unit area of ​​a radiation source.

[0072] The terms "transparent," "radiation transparent," and similar terms are used herein to denote the property of a material structure (e.g., the wall of a CE capillary tube) that allows the passage of at least 70%, or at least 80%, or at least 90% or 100% of the radiation incident thereon.

[0073] 1A and 1B, a system 100 according to one embodiment of the present teachings for quantifying one or more target proteins in a sample flowing through a capillary tube of a CE system by laser-induced fluorescence measurements includes a radiation source 1 capable of emitting radiation over a spectral vacuum wavelength range spanning from about 170 nm to about 2100 nm. In this embodiment, the radiation source is capable of emitting radiation having a vacuum wavelength spanning from about 200 nm to about 300 nm, with a radiance over this range that is at least 10 times higher than the radiance exhibited by a conventional UV lamp over this range. By way of example, the radiation source 1 can emit radiation having a vacuum wavelength spanning from about 5 to 200 mW / mm over a spectral wavelength range of about 200 nm to about 300 nm. 2 It can emit radiation with a radiance in the range of .sr.nm. By way of example, in some embodiments, a radiation source sold by Energetiq Technology, Inc. of Wilmington, Massachusetts under the trade name LDLS® can be used, which uses a laser to directly heat a xenon plasma to the high temperatures required to produce deep UV radiation.

[0074] As described further herein, it has been discovered that such radioactive sources can be particularly useful in performing NFD analysis of proteins on CE systems.

[0075] In this embodiment, the radiation emitted by radiation source 1 is received by plano-convex lens 2, which is separated from the radiation source by its focal length and which substantially collimates the radiation. As explained further below, bandpass or shortpass filter 3 selects a portion of the spectral range of the emitted radiation to produce an excitation beam having a radiation bandwidth that includes at least one wavelength suitable for exciting at least one native fluorophore of at least one target protein contained in or suspected to be contained in the sample under study. In this embodiment, optical filter 3 is placed between lens 2 and lens 4, although in other embodiments the optical filter may be placed at other positions along the propagation path of the radiation to the sample within the capillary.

[0076] Another plano-convex lens 4 focuses the filtered radiation onto the proximal end (PE) of the optical fiber 5. In this embodiment, the f-numbers of lenses 2 and 4 are selected such that the light focused onto the proximal end of the optical fiber 5 has a convergence angle substantially equal to the numerical aperture of the optical fiber to optimize coupling of the radiation into the optical fiber.

[0077] The optical fiber 5 transmits the received radiation from its proximal end (PE) to its distal end (DE). The distal end (DE) of the optical fiber is optically coupled to the transparent portion 7a of the capillary tube 7, i.e. the portion of the capillary tube with a transparent wall through which the sample can flow. The transparent portion 7a provides a window through which the excitation radiation exiting the distal end of the optical fiber 5 can be introduced into the capillary tube 7 and excite at least one target protein (if present in the sample) as the sample flows through the lumen of the transparent portion of the capillary tube. In other words, the front part of the transparent wall of the capillary tube, i.e. the part facing the distal end of the optical fiber, can function as a window through which the excitation radiation can enter the capillary tube.

[0078] In response to irradiation with excitation radiation, one or more natural fluorophores of a target protein can emit fluorescence radiation. The natural fluorescence of a protein originates from the excitation of three amino acids: phenylalanine, tyrosine, and tryptophan. It is known that the fluorescence emission intensity associated with phenylalanine is approximately 50 times lower than that of tyrosine or tryptophan and can generally be ignored in detecting the natural fluorescence of a protein. In most proteins, the natural fluorescence is mainly due to fluorescence from tryptophan residues, although some proteins may exhibit tyrosine fluorescence.

[0079] Although sensitive to the local environment, the emission peak of tryptophan is typically around 350 nm. In some embodiments, filter 3 can be configured to pass radiation having a wavelength of 280 nm to excite tryptophan amino acids in a target protein. In some such embodiments, the fluorescent radiation emitted by the excited tryptophan amino acids can be collected and analyzed, for example, in the manner described below, to determine the concentration of the target protein in the sample under study.

[0080] More specifically, and still referring to Figure 1A, in this embodiment, the emitted fluorescent radiation can exit the capillary tube through the wall of the transparent portion 7a of the capillary tube. A portion of the emitted fluorescent radiation exits the capillary tube through the rear portion 7aa of the transparent portion 7a, while a portion of the emitted fluorescent radiation exits the capillary tube through the front portion 7bb of the transparent portion 7a.

[0081] In this embodiment, a spherical mirror 6 positioned near the transparent portion 7a of the capillary tube 7 captures at least a portion of the fluorescence radiation exiting the capillary tube and reflects the captured fluorescence radiation (or at least a portion of it) back into the capillary tube, whereby the reflected fluorescence radiation (or at least a portion of it) exits through a rear portion 7aa of the transparent portion 7a.

[0082] A plano-convex lens 8 is optically coupled to the transparent portion 7a of the capillary tube 7 to receive the fluorescent radiation (or at least a portion thereof) exiting the capillary tube and collimate the fluorescent radiation to produce a substantially collimated fluorescent radiation beam 11. The fluorescent radiation beam 11 passes through a bandpass filter 9 and is received by a detector 10.

[0083] By way of example, a bandpass filter allows the transmission of fluorescent radiation while blocking or reducing the passage of radiation of other wavelengths. By way of example, in some embodiments, a bandpass filter can have a transmission bandwidth centered on the wavelength at which the fluorescent radiation emitted by an excited target protein exhibits its maximum intensity, e.g., can have a transmission bandwidth ranging from about 5 nm to about 50 nm.

[0084] In this embodiment, the detector 10 is a photomultiplier tube, for example, a photomultiplier tube (R5984) commercially available from Hamamatsu. The detector 10 generates one or more detection signals in response to detecting the fluorescent radiation. The analyzer 20 (also referred to herein as an analysis module) receives the detection signals generated by the detector 10 and operates based on those signals to calculate the concentration of the target protein of interest in the sample under investigation. The analyzer 20 can be implemented in software / firmware / hardware in a manner known in the art as informed by the present teachings. In some embodiments, instructions for analysis of the fluorescent data can be stored in a persistent memory of the analyzer and transferred to a transient memory module for execution during execution by the processor of the analyzer.

[0085] For example, in a certain concentration range ("linear dynamic range"), protein concentration is proportional to the area of ​​the protein UV absorbance peak. In some cases, for example, a calibration curve can be established over that range using five or more different concentrations of protein. The concentration of the target protein can then be calculated using the UV absorbance peak area and the calibration curve.

[0086] It should be understood that the present teachings are not limited to determining the concentration of a target protein via excitation of tryptophan residues. For example, in some embodiments, for example when the target protein lacks an appropriate concentration of tryptophan residues, the excitation wavelength of the system can be adjusted to detect tyrosine fluorescence.

[0087] With particular reference to FIG. 1B, a high voltage source 30 applies a high voltage across the capillary tube 7 to generate an electric field through the capillary tube, which can then cause different proteins present in the sample under investigation to move through the tube at different rates (e.g., due to differences in their mobility). The difference in the mobility of the proteins can be at least partially due to differences in their charge. As a result, when a sample containing target proteins introduced into the capillary tube 7 from the inlet 7b of the capillary tube flows under the influence of the electric field to the outlet 7c, the different proteins reach the transparent portion of the capillary tube at different times due to their differences in mobility. Excitation of at least one natural fluorophore of each target protein then allows for quantification of the various proteins in the sample by allowing for quantification of that protein via NFD, e.g., in the manner described herein.

[0088] As shown diagrammatically in FIG. 1C, in some embodiments, a cartridge 30 containing multiple different optical filters 31, 32, 33, and 34 can be placed in the path of the emitted radiation, each optical filter exhibiting a different optical bandpass. The cartridge allows for the selection of different excitation wavelengths for exciting different fluorophores of multiple target proteins. Similarly, a cartridge containing multiple optical filters can be placed in front of the detector 10 to allow for the detection of fluorescent radiation of different wavelengths.

[0089] In some embodiments, an internal standard can be added to a sample to serve as a calibrator for accurate quantification of one or more target proteins present in the sample. Some examples of suitable internal standards include, but are not limited to, peptides and proteins with known molecular weights.

[0090] The present teachings can be used to detect and quantify various types of proteins. By way of example, in some embodiments, the protein can be an antibody, such as a monoclonal or polyclonal antibody, although the present teachings can also be used to quantify the concentration of other proteins in a sample.

[0091] In the following, some other embodiments of a system for NFD quantification of proteins are described with reference to Figures 6A and 6B. More specifically, these embodiments utilize light emitting diodes (LEDs) as light sources.

[0092] Referring to FIG. 6A, a system 600 according to another embodiment of the present teachings for quantifying one or more target proteins in a sample flowing through a capillary tube of a CE system includes a light-emitting diode (LED) radiation source 601 capable of emitting substantially monochromatic radiation in a vacuum wavelength range of about 190 nm to about 310 nm (e.g., about 285 nm). In this embodiment, the LED radiation source can emit a significantly narrower wavelength range than the laser light source described with respect to FIG. 1A. By way of example, in some embodiments, an LED commercially available under the trade name M280L6 by Thorlabs, Inc. of Newton, NJ can be used. In some embodiments, the LED light source 601 can be powered by a DC power supply commonly included in commercially available CE systems. Thus, the power efficiency of the system can be improved over laser-based systems that typically require a dedicated power supply.

[0093] The LED radiation source 601 can be directly coupled to the proximal end of the optical fiber 605, for example, via a butt coupling technique. The distal end of the optical fiber 605 is then optically coupled to a transparent portion 607a of the capillary tube 607, e.g., a portion of the capillary tube 607 having a transparent wall through which a sample can flow, similar to the embodiment shown in FIG. 1A. The transparent portion 607a provides a window through which excitation radiation exiting the distal end of the optical fiber 605 can be introduced into the capillary tube 607 and excite at least one target protein (if present in the sample) as the sample flows through the lumen of the transparent portion of the capillary tube 607.

[0094] In some embodiments, a spherical mirror 606 is positioned near the transparent portion 607a of the capillary tube 607 to capture at least a portion of the fluorescent radiation exiting the capillary tube and reflect the captured fluorescent radiation (or at least a portion thereof) back into the capillary tube 607, whereby the reflected fluorescent radiation (or at least a portion thereof) exits through the rear of the transparent portion 607a.

[0095] A plano-convex lens 608 is optically coupled to the transparent portion 607a of the capillary tube 607 to receive the fluorescent radiation exiting the capillary tube 607 (or at least a portion thereof) and collimate the fluorescent radiation to generate a substantially collimated fluorescent radiation beam 611. The fluorescent radiation beam 611 passes through a bandpass filter 609 and is received by a detector 610.

[0096] In some embodiments shown in FIG. 6B, the bandpass filter 609 can have a transmission bandwidth centered on the wavelength at which the fluorescent radiation emitted by the excited target protein exhibits its maximum intensity, for example, can have a transmission bandwidth ranging from about 5 nm to about 50 nm.

[0097] Detector 610 can be implemented as a photomultiplier tube, for example, a photomultiplier tube (R5984) commercially available from Hamamatsu. Detector 610 can generate one or more detection signals in response to detecting the fluorescent radiation. Analyzer 620 (also referred to herein as an analysis module) receives the detection signals generated by detector 610 and operates based on those signals to calculate the concentration of a target protein of interest in the sample under investigation.

[0098] In some related embodiments, a filter (e.g., a bandpass filter) 603 can be placed in front of the LED radiation source 601 to more precisely limit the wavelengths of radiation emitted from the LED radiation source 601. In some such embodiments, for example, a 280 nm bandpass filter can be used. In some such embodiments, the bandpass filter 603 can have a transmission band (e.g., >65%) in the range of about 270 nm to about 290 nm. In such embodiments, as shown in FIG. 6B, the radiation emitted by the LED radiation source 601 is received by a plano-convex lens 602, which is separated from the LED radiation source by its focal length and which substantially collimates the radiation.

[0099] A bandpass filter 603 or shortpass filter eliminates residual emission at wavelengths above about 300 nm. Another plano-convex lens 604 focuses the filtered radiation into the proximal end of optical fiber 605. In this embodiment, bandpass filter 603 is placed between lens 602 and lens 604, although in other embodiments bandpass filter 603 may be placed at other locations along the propagation path of the radiation to the sample flowing within capillary tube 607.

[0100] In the embodiment shown in FIG. 6B, the spherical mirror 606, plano-convex lens 608, bandpass filter 609, detector 610, and analyzer 620 can be similarly arranged and can function similarly to the embodiments described above to excite at least one native fluorophore of at least one protein in the sample and to collect and analyze fluorescent radiation emitted by the fluorophore in response to that excitation.

[0101] The following examples are provided to further illustrate various aspects of the present teachings and are not necessarily presented to represent the best way to practice the present teachings and / or the best results that may be obtained.

[0102] Example 1: Use of a laser-driven light source A laser-driven light source (LDLS®) developed by Energetiq Technology, Inc., Wilmington, Massachusetts, was used to excite the natural fluorophores of several proteins in the CE system. The LDLS® generates a high-intensity plasma in xenon gas, which produces radiation across a continuous spectrum ranging from 170 to 2100 nm. In the 200-300 nm region, the radiance of the generated radiation is about 10 times that of conventional lamps, with a reported typical lifetime of about 10,000 hours. The continuous spectrum allows greater flexibility than lasers in the selection of excitation wavelengths.

[0103] The excitation wavelength was selected using a bandpass filter, which was mounted on a filter wheel, allowing interchangeable selection of different filters. A photomultiplier tube (PMT) was used as the detector. Using a 200 ng / mL tryptophan solution as a standard, with an excitation of 280 nm and an emission of 333 nm to 375 nm, the effect of the PMT bias voltage on the signal-to-noise ratio was investigated. The contribution of tryptophan and tyrosine residues to the intrinsic fluorescence of a 10 kDa internal standard and reduced IgG1 was investigated by adjusting the excitation and emission wavelengths.

[0104] The developed NFD system was successfully applied to the analysis of monoclonal antibodies (mAbs) in CE-SDS and cIEF. In CE-SDS, NFD showed a 15-fold improvement in concentration sensitivity compared with UV absorbance detection at 214 nm. Furthermore, the detection data showed less baseline fluctuation or interference from other absorbing species. In cIEF, NFD showed 178-fold higher sensitivity than UV absorbance detection at 280 nm, possibly due to the reduced background of UV-sensitive amphoteric carrier cells.

[0105] Reagents and Materials SDS-MW analysis kit, 10 kDa internal standard, IgG control standard, cIEF peptide marker kit, sample loading solution, cIEF gels were obtained from SCIEX (Brea, CA). Tryptophan (Sigma-Aldrich, St. Louis, MO) and IgG control standard (SCIEX, Brea, CA) were used to evaluate the optical components and optimize parameters of the NFD prototype. USP monoclonal IgG system compatibility reference standard (USP IgG) was obtained from the United States Pharmacopeia (Rockville, MD). NISTmAb, humanized IgG1 k Monoclonal antibodies (NIST IgG) were purchased from the National Institute of Standards and Technology (Gaithersburg, MD). Pharmalyte 3-10 was obtained from Cytiva (Marlborough, MA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0106] (CE-SDS conditions) The reduced IgG standard mixture was prepared by mixing 96 μL of SCIEX IgG control standard solution, 2 μL of 10 kDa internal standard, and 2 μL of 2-mercaptoethanol. The subsequent solution was then heated at 70° C. for 10 minutes to promote dissociation of non-covalently bound species and SDS-protein binding. The non-reduced SCIEX IgG control standard was also heated at 70° C. for 10 minutes before use.

[0107] All CE-SDS separations were performed using bare fused silica capillaries (Polymicro Technologies, Phoenix, AZ) with an inner diameter of 50 μm, an outer diameter of 375 μm, and a total / effective length of 30 / 20 cm. Before each separation, the capillary was flushed successively with 0.1 M sodium hydroxide, 0.1 M hydrochloric acid, and DI water at 50 psi for 5, 3, and 3 min, respectively. SDS-MW gel buffer (SCIEX, Brea, CA) was filled into the capillary at 50 psi for 10 min. Electrokinetic injection was performed at −5 kV for 10 s. Separations were performed by applying a voltage of −15 kV with a pressure of 20 psi to reduce the risk of forming air bubbles.

[0108] (cIEF condition) A cIEF master mix was prepared by combining the following solutions: (1) 3.75 M urea in 1 mL of cIEF gel; (2) 60 μL of Pharmalyte 3-10; (3) 500 mM arginine in 100 μL of DI water; (4) 10 μL of 200 mM iminodiacetic acid in DI water. A NIST IgG standard mixture was prepared for UV absorbance detection by combining 200 μL of cIEF master mix, 8 μL of 4 mg / mL NIST IgG, and 2 μL of each pI marker (10.0, 7.0, 9.5, 5.5). NIST IgG samples were prepared for NFD by diluting 150-fold with cIEF master mix.

[0109] All cIEF separations were performed using neutral coated capillaries (SCIEX, Brea, CA) with an inner diameter of 50 μm, an outer diameter of 375 μm, and a total / effective length of 30 / 20 cm. Prior to use, the capillaries were conditioned by rinsing with 350 mM acetic acid, DI water, and cIEF gel (SCIEX, Brea, CA) at 50 psi for 5, 2, and 5 min, respectively. Before each separation, the capillaries were rinsed with sample packing solution (SCIEX, Brea, CA) and water at 20 psi for 3 and 2 min, respectively. NIST IgG samples were loaded into the capillaries at 25 psi for 99 s. Focusing was performed by applying a voltage of 25 kV for 15 min while the capillary inlet was immersed in 200 mM phosphoric acid and the outlet was immersed in 300 mM sodium hydroxide. The 300 mM sodium hydroxide was replaced by 350 mM acetic acid and chemical mobilization was carried out by applying a voltage of 30 kV for 20 min.

[0110] (Detection System) An NFD system as described above in connection with FIG. 1A was used to perform the measurements. A light beam from a radiation source 1 (e.g., a laser-driven plasma source) was sequentially collimated using a plano-convex lens 2, filtered with a band-pass or short-pass filter 3, and focused with a second plano-convex lens 4 into an optical fiber 5 and delivered to the capillary window 7 for excitation. Fluorescence emission was collimated with a plano-convex lens 8, filtered with a band-pass filter 9, and detected with a detector 10 (e.g., a photomultiplier tube (PMT)). A spherical mirror 6 was placed proximal to the detection window to increase the collection efficiency of the light emitted by the analyte in the capillary.

[0111] (Results and Discussion) (1. PMT voltage) Noise was defined as the standard deviation of the response of the blank sample over a period of 20 seconds. The effect of PMT voltage on the signal-to-noise ratio was investigated at four levels: 350 V, 475 V, 650 V, and 890 V. A 200 ng / mL tryptophan solution was injected for 5 seconds at 0.5 psi, and the pressure-driven separation was operated at 5 psi with water as background. As shown in Figure 2, the signal-to-noise ratio improved from 110 to 380 as the PMT voltage increased from 350 V to 475 V. The signal-to-noise ratio was 1,229 at a PMT voltage of 650 V, and a further increase in the PMT voltage to 890 V resulted in a higher signal-to-noise ratio of 1,681. However, at PMT voltages above 650 V, a 2.5 mg / mL solution of USP IgG produced a voltage signal that saturated the analog-to-digital module used (data not shown). This limited the application of the NFD scheme in this example for protein analysis at high concentration levels. As a compromise between concentration sensitivity and linear dynamic range, a PMT voltage of 650 V was selected for further experiments.

[0112] (2. Excitation and Emission Wavelengths) The natural fluorescence of proteins originates from the excitation of three aromatic amino acids: phenylalanine, tyrosine, and tryptophan. The emission intensity of phenylalanine is approximately 50 times lower than that of tyrosine or tryptophan and is generally negligible in detecting the natural fluorescence of proteins. In most proteins, the natural fluorescence is primarily due to tryptophan residues, but some proteins exhibit tyrosine fluorescence.

[0113] Although the emission peak of tryptophan is sensitive to the local environment, the emission peak of tryptophan is typically around 350 nm. By adjusting the excitation and emission filters in the setup (described in relation to Figure 1A above), the NFD measurement process was first evaluated with excitation at 280 nm and collection of emission in the range of 333-375 nm. A reduced SCIEX IgG control standard mixed with a 10 kDa internal standard was analyzed by CE-SDS using the NFD setup. The three peaks representing the IgG light chain (LC), nonglycosylated heavy chain (NGHC), and heavy chain (HC) showed enhanced signal-to-noise ratio compared to UV absorbance detection at 214 nm. However, the response to the 10 kDa internal standard was low, likely due to the lack of tryptophan residues.

[0114] The tyrosine emission peak was shown to be insensitive to the local environment, with an emission maximum seen at approximately 304 nm. The contribution of tyrosine residues to the native fluorescence was investigated by collecting emission spectra in the 305-315 nm band following excitation at 220 nm. Separation was performed in an uncoated capillary with an inner diameter of 50 μm, an outer diameter of 375 μm and a total / effective length of 30 / 20 cm. The applied voltage was -15 kV with 20 psi pressure at both ends. Sample injection was achieved at a voltage of -5 kV for 10 s.

[0115] The magnitude of the 10 kDa internal standard peak was enhanced (Figure 3B), whereas the three peaks representing LC, NGHC, and HC were less than 10-fold stronger (Figure 3B vs. Figure 3A). Overall tyrosine fluorescence was weak, likely due to overlap in emission with tryptophan absorbance and energy transfer from tyrosine to tryptophan residues.

[0116] To maximize the concentration sensitivity of the CE-SDS and cIEF analysis of mAbs, the NFD was used with excitation at 280 nm and emission in the 333-375 nm band. However, the excitation and emission wavelengths of the NFD system can be adjusted to detect tyrosine fluorescence if the target protein lacks an appropriate concentration of tryptophan residues.

[0117] (Analysis of mAbs using CE-SDS with NFD) Due to its high resolution, ease of use and high reproducibility, CE-SDS is replacing SDS-PAGE as a routine tool in the biopharmaceutical industry for the analysis of therapeutic mAbs. In CE-SDS, UV absorbance detection is often operated in the 210-220 nm region, where maximum concentration sensitivity is achieved. However, detection is not selective and suffers from interference by other absorbing species or baseline anomalies.

[0118] A non-reduced SCIEX IgG control standard was separated using CE-SDS with UV absorbance detection at 214 nm (see trace 1 in Figure 4A). IgG samples were heated at 70°C for 10 min prior to use. IgG samples were used directly (Figure 4A) and also at a 10-fold dilution in SDS-MW sample buffer (SCIEX, Brea, CA) (Figure 4B). All other CE-SDS conditions were the same as those used in connection with Figure 3.

[0119] The main peak yielded a signal-to-noise ratio of 3,389. In an attempt to eliminate baseline anomalies, the same IgG sample was separated using CE-SDS with absorbance detection at 280 nm (see trace 2 in FIG. 4A). However, the response was much lower, which yielded a signal-to-noise ratio of 67 for the main peak, representing a sensitivity of 1 in 51 with UV absorbance detection at 214 nm (trace 2 vs. trace 1).

[0120] A non-reduced SCIEX IgG control standard was diluted 10-fold in SDS sample buffer (SCIEX, Brea, CA) and analyzed using the NFD prototype system (see Figure 4B). The signal-to-noise ratio of the main peak was 5,568, representing a 16-fold improvement in sensitivity compared to UV absorbance detection at 214 nm. Additionally, the baseline was less aberrant and peak integration was less interfered with than absorbance detection at 214 nm (Figure 4B vs. trace 1 in Figure 4A).

[0121] (Analysis of mAbs using cIEF with NFD) cIEF is an important CE-based technique that separates protein molecules based on their pI and is routinely used in the biopharmaceutical industry for mAb charge isoform analysis. In cIEF, absorbance detection is generally performed at 280 nm, since carrier amphoteric electrolytes tend to be UV-sensitive, especially at 214 nm. cIEF with LIF detection is not widely used, since fluorophore derivatization often leads to changes in the charge of various protein isoforms, which can result in separations not comparable to those of their native protein counterparts.

[0122] Separation of NIST IgG using cIEF with absorbance detection at 280 nm was performed (FIG. 5A). Separation was performed in a neutral coated capillary (SCIEX, Brea, CA, with inner diameter 50 μm, outer diameter 375 μm, and total / effective length 30 / 20 cm). NIST IgG samples were prepared as described above. For NFD, samples used for UV absorbance detection were diluted 150-fold. Sample injection was achieved at 25 psi for 99 seconds, focusing at 25 kV for 15 minutes, and chemical mobilization at 30 kV for 20 minutes.

[0123] The signal-to-noise ratio of the main peak was 956. Using the NFD workflow, the same NIST IgG solution produced a signal that saturated the analog-to-digital module on our system (data not shown). The IgG solution was diluted 150-fold with cIEF master mix, and the signal-to-noise ratio of the IgG sample was 1,137 (Figure 5B). Considering the 150-fold sample dilution, analysis using NFD provided a 178-fold improvement in sensitivity for the main peak over absorbance detection at 280 nm.

[0124] (Example 2: Use of LED light source) Fiber-coupled 285 nm LED modules were obtained from Thorlabs, Inc. (Newton, NJ). The LED light source contained a single LED mounted on a metal core circuit board in direct contact with a heat sink for thermal stability. The LED was driven with a compact controller (Thorlabs, Inc., Newton, NJ) capable of modulating the LED's forward DC current in the range of 0-1,200 mA. In this experiment, the LED was operated with a forward DC current of 500 mA; at this current, the typical lifetime of an LED is >10,000 hours.

[0125] The performance of the developed NFD scheme was evaluated using an IgG control standard (SCIEX, Brea, CA) consisting of glycosylated and non-glycosylated IgG from mouse serum. The total mass concentration is 1 mg / mL, and the amount of non-glycosylated form is about 9.5%. 95 μL of sample was mixed with 5 μL of β-mercaptoethanol, and then the IgG sample was reduced by heating at 70° C. for 10 min. Prior to analysis, the reduced IgG control standard was diluted 10-fold with SDS sample buffer (SCIEX, Brea, CA). As shown in FIG. 7, the three peaks represent the IgG light chain (LC), non-glycosylated heavy chain (NGHC), and heavy chain (HC). NGHC and HC were baseline separated with a resolution of 1.62. Here, the noise was defined as the standard deviation of the response of the blank sample over 30 s. The signal-to-noise ratios (S / N) were 3240, 860, and 5458 for LC, NGHC, and HC, respectively.

[0126] To identify residual emission of the LED above 300 nm, fluorescence was collected in the range of 325-375 nm, because residual emission in this band can significantly increase the background signal and consequently reduce the S / N. To further improve the detection sensitivity, residual emission was eliminated by collimating the light with a plano-convex lens, filtering the light through a 280 nm bandpass filter, and focusing the filtered light into an optical fiber with a second plano-convex lens, as described above with reference to Figure 6B. The 280 nm bandpass filter (IDEX, Rochester, NY) exhibits >65% transmission in the 270-290 nm band and an average cutoff of >5 OD in the 310-380 nm band. With the combination of two lenses and one optical filter, the light power received by the optical fiber was reduced by approximately 50%, and therefore the fluorescence emitted by the reduced IgG sample was reduced by approximately 45% (see line A vs line B). Despite the overall reduction in received power, the background signal was significantly reduced, so the S / N increased to 7409, 1917, and 12566 for LC, NGHC, and HC, respectively. This represents about a 2.3-fold enhancement in detection sensitivity over the configuration without the 280 nm bandpass filter. Therefore, in Example 2, an LED light source combined with two lenses and a 280 nm bandpass filter was used for excitation.

[0127] Reagents and Materials IgG control standard, SDS-MW gel separation buffer, SDS sample buffer, 0.1 N HCl acid wash, 0.1 N NaOH basic wash, and CE grade water were from SCIEX (Brea, CA). NIST monoclonal antibody (NISTmAb) was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). It is a humanized IgG1 at 10 mg / mL in 12.5 mM L-histidine, 12.5 mM L-histidine HCl (pH 6.0). kMonoclonal antibody solutions. Iodoacetamide and 2-mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, MO). Etanercept was purchased from Myonex (Norristown, PA).

[0128] 250 mM iodoacetamide was prepared by dissolving 46.2 mg in 1 mL of CE grade water (SCIEX, Brea, CA). Prior to use, the stock solution was diluted to 20 mM with SDS sample buffer (SCIEX, Brea, CA).

[0129] (Equipment) A PA800 Plus CE system (SCIEX, Brea, CA) configured with LIF detection was used in this experiment. The excitation source was designed based on a 285 nm LED (Thorlabs, Inc., Newton, NJ) and customized optics to allow compatibility with the LIF detector. The light source was powered and self-contained in the PA800 Plus CE system. All data was collected and processed using 32Karat software (version 10.1).

[0130] (Sample Preparation) A reduced IgG control standard was prepared by mixing 95 μL of SCIEX IgG control standard solution with 5 μL of 2-mercaptoethanol. The mixture was heated at 70° C. for 10 minutes and then cooled to room temperature. Before use, the sample was diluted 10-fold with SDS sample buffer (SCIEX, Brea, CA).

[0131] Non-reduced SDS-NISTmAb complexes were prepared by diluting 10 mg / mL NISTmAb to the desired concentration with SDS sample buffer (SCIEX, Brea, CA) containing 20 mM iodoacetamide, and the solution was then heated at 70°C for 10 min and then cooled to room temperature before use.

[0132] Etanercept, 25 mg / vial, was reconstituted with 1 mL of diluent and the stock solution was diluted to the desired concentration with SDS sample buffer containing 20 mM iodoacetamide (SCIEX, Brea, CA). Sample solutions were heated at 70°C for 10 min and then cooled to room temperature before use.

[0133] (SDS-CGE conditions) All SDS-CGE separations were performed using bare fused silica capillaries (P / N 338451, SCIEX, Brea) with an inner diameter (ID) of 50 μm and an outer diameter (OD) of 375 μm. The capillaries were installed in cartridges (SCIEX, Brea, CA) with a total length of 30.2 cm and a length from inlet to detection window of 20.0 cm. Prior to first use, the capillaries were conditioned by a 0.1 N NaOH basic wash, a 0.1 N HCl acidic wash, and rinsing with CE grade water at 20 psi for 10, 5, and 2 min, respectively. The capillaries were then filled with SDS-MW gel separation buffer at 70 psi for 10 min and then equilibrated by applying a voltage of −15 kV at 20 psi to both the inlet and outlet ends for 15 min.

[0134] Before each separation, the capillary was rinsed successively with a 0.1 N NaOH basic wash, a 0.1 N HCl acidic wash, and CE grade water at 70 psi for 3, 1, and 1 min, respectively. SDS-MW gel separation buffer was filled into the capillary at 70 psi for 10 min. Samples were electrokinetically injected into the capillary at -5 kV for 40 s. Separations were performed at a voltage of -15 kV. To reduce the risk of forming air bubbles, a pressure of 20 psi was applied to both the inlet and outlet ends during the separation.

[0135] (Results and Discussion) (1.PMT gain) The manufacturer, Hamamatsu, specifies a maximum rating for the PMT at 1250V. To provide reliability against voltage fluctuations, the PMT was operated at a control voltage of less than 1000V, 20% lower than the specified maximum rating. In this experiment, the effect of the PMT control voltage on the S / N was investigated at seven levels: 386, 458, 530, 628, 727, 862, and 997V. A 1mg / mL IgG control standard was reduced as above and diluted 10-fold before analysis by SDS-CGE. As shown in Figure 8A, when the control voltage was increased from 386V to 727V, the fluorescent signal increased approximately 100-fold. For all three peaks representing LC, NGHC, and HC, the S / N was significantly enhanced (see Figure 8B). Further increase of the control voltage to 997V resulted in a further improvement of the fluorescent signal by 10-fold, but the S / N had reached a plateau. Furthermore, at PMT voltages above 727V, a 1mg / mL solution of IgG produced a voltage signal outside the input voltage range of the analog-to-digital converter used, which limited the application of SDS-CGE with NFD for protein analysis at high concentration levels. In this study, we developed a solution to quantify trace-level impurities in mAb samples while pursuing both detection sensitivity and linear dynamic range. Therefore, a PMT control voltage of 727V was selected for further experiments.

[0136] (2. Sample Injection) In SDS-CGE, the separating gel buffer can be viscous, but the sample solution can be gel-free. This mismatch often leads to problems with pressure injection, and sample injection is generally performed electrokinetically. Because SDS-denatured proteins have a uniform charge-to-size ratio regardless of their molecular weight, various protein molecules migrate with the same mobility in the gel-free sample solution, mitigating injection bias.

[0137] In general, increasing the sample injection volume improves the S / N ratio, but a long sample plug injection can contribute to peak broadening and reduce separation efficiency. In this experiment, the sample injection volume was adjusted by changing the injection time from 5 to 120 s while the injection voltage was fixed at -5 kV. Figure 9A shows representative electropherograms of a 100 μg / mL reduced IgG sample with injection times of 5, 20, 60, and 100 s. As the injection time increased, the corrected peak areas increased with correlation coefficients (r 2 ) increased linearly with increasing injection time (see Figure 9B). As shown in Figure 9C, longer injection times resulted in higher S / N for all three peaks representing LC, NGHC, and HC, but also resulted in peak broadening, reducing the separation efficiency. When the injection time was longer than 40 s, the resolution between NGHC and HC was <1.5 (see Figure 9D). In this study, to compromise between the detection sensitivity and separation efficiency of the SDS-CGE assay, sample injection was performed for 40 s at -5 kV.

[0138] (3. Linearity and Sensitivity) Detection of low-level impurities is one of the main applications of SDS-CGE assays. In this experiment, the linear dynamic range of SDS-CGE with NFD was investigated using NISTmAb. Non-reduced SDS-NISTmAb complexes were prepared by diluting 10 mg / mL NISTmAb in SDS sample buffer containing 20 mM iodoacetamide (SCIEX, Brea, CA) and heating at 70 °C for 10 min. Non-reduced NISTmAb samples were prepared at 11 concentration levels ranging from 0.03 to 500 μg / mL, and Figure 10A shows the overlap of their SDS-CGE electropherograms. The inset is a magnified view of the electropherogram at 0.05 μg / mL. Each sample was injected in triplicate, and a linear regression was established by plotting the average of the corrected peak areas versus concentration (see Figure 10B). The regression equation was y = 631.63x, where y was the corrected peak area and x was the NISTmAb concentration. The correlation coefficient (r 2) was 0.9999, indicating an excellent linear relationship between the corrected peak area and NISTmAb concentration.

[0139] In this experiment, the limit of detection (LOD) and limit of quantification (LOQ) were defined as the protein concentration that resulted in an S / N of 3 and 10, respectively. The noise was estimated by measuring the magnitude of the background response of blank samples and calculating the standard deviation of these responses over a 30-second period. Under optimized conditions, the LOD and LOQ of non-reduced NISTmAb were 8.3 and 27 ng / mL, respectively. This is comparable to the concentration sensitivity obtained with SDS-CGE-LIF and SDS-PAGE with silver staining.

[0140] (4. Precision and Accuracy) Repeatability was assessed by 18 determinations at three concentration levels, with six determinations at each concentration. The RSDs of the corrected peak areas were 2.35%, 0.75%, and 0.56% at 0.05, 5, and 500 μg / mL, respectively.

[0141] Since samples of impurities in NISTmAb could not be obtained, accuracy was assessed by comparing the experimental concentrations of non-reduced NISTmAb samples with their theoretical concentrations. Non-reduced IgG samples were prepared at five concentration levels: 0.05, 0.5, 5, 50, and 200 μg / mL. Each sample was injected five times, and the average corrected peak area was substituted into the regression equation to calculate the experimental concentration. As shown in Table 1, recoveries ranged from 91.5% to 104.9%. [Table 1]

[0142] 5. Application of SDS-CGE-NFD to Quantification and Purity Analysis of Etanercept Etanercept is a therapeutic Fc fusion protein approved by the FDA in 1998 to treat psoriatic arthritis and rheumatoid arthritis. It is heavily glycosylated with an apparent molecular weight of approximately 150 kDa. To evaluate the purity and high molecular weight (HMW) content in etanercept and its biosimilars, Cho et al. (Cho et al., ''Evaluation of the structural, physicochemical, and biological characteristics of SB4, a biosimilar of etanercept. mAbs'', 2016, 8(6), 1136-1155) used size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and the determined HMW content in the reference product was 2.3-3.1%. However, due to insufficient SEC separation of the major and low molecular weight (LMW) species, quantification of the LMW content in SEC-MALLS was not accurate, which was achieved using SDS-CGE-UV.

[0143] Absorbance detection at 214 nm is the most widely used detection strategy in SDS-CGE, but it is not selective and is often subject to interference from baseline anomalies. Figure 11A shows the SDS-CGE-UV electropherogram of 1 mg / mL non-reduced etanercept. The baseline undulations around the main peak complicated peak integration and posed challenges to the purity analysis of etanercept, especially in the HMW region.

[0144] NFD eliminated baseline anomalies and improved concentration sensitivity. Figure 11B shows the SDS-CGE-NFD electropherogram of 100 μg / mL non-reduced etanercept. Both HMW and LMW peaks were well distinguished from noise and baseline fluctuations, allowing quantification of HMW and LMW content using a single assay. The corrected peak area percentages were as in Table 2. The determined HMW content was consistently in the range of 2.57-2.63% and comparable to the reported HMW content of the reference product analyzed by SEC-MALLS. The LMW content was determined to be 1.19-1.27%, consistently over a range of 50-150% of the target concentration (200 μg / mL). [Table 2]

[0145] A calibration curve was established over the range of 50–150% of the target concentration of 200 μg / mL for the main peak. The regression equation was y = 353.43x, where y represents the corrected peak area and x represents the etanercept concentration. The correlation coefficient (r 2 The linearity coefficient (RCL) was 0.9990, indicating good linearity of the SDS-CGE-NFD method for the quantification of etanercept. The average corrected peak areas were substituted into the regression equation to calculate the experimental concentrations, and the recoveries ranged from 97.02 to 101.6%.

[0146] Those skilled in the art will appreciate that various modifications can be made to the above-described embodiments without departing from the scope of the present teachings.

Claims

1. 1. A method for protein analysis of a sample in a capillary electrophoresis (CE) system, the method comprising: flowing the sample through a capillary tube of the CE system; utilizing a light source to generate radiation, said radiation comprising at least one wavelength suitable for exciting at least one native fluorophore of at least one target protein in said sample; directing an excitation beam comprising the at least one excitation wavelength into a transparent portion of the capillary tube of the CE system to excite the at least one natural fluorophore of the target protein passing through a lumen of the transparent portion, thereby causing the at least one natural fluorophore to produce fluorescent radiation; detecting at least a portion of the fluorescent radiation emitted by the excited target protein; A method comprising:

2. The method of claim 1 , wherein the light source comprises at least one light emitting diode (LED).

3. The method of claim 1 , wherein the light source comprises a laser-driven light source.

4. 4. The method of claim 1, wherein the light source produces radiation having wavelengths spanning a spectral range, and the excitation beam exhibits a spectral bandwidth narrower than the spectral range of the radiation.

5. The method of claim 4 , further comprising spectrally filtering the radiation to generate the excitation beam.

6. The method of claim 5 , wherein spectrally filtering the radiation comprises utilizing an optical bandpass or shortpass filter.

7. The method of claim 6, wherein the optical bandpass or shortpass filter exhibits a transmission bandwidth in the range of about 200 nm to about 300 nm.

8. The method of claim 1 , wherein the target protein comprises an antibody.

9. The method of claim 1 , wherein the at least one wavelength is about 285 nm.

10. The method of claim 1 , further comprising utilizing a lens to focus the fluorescent radiation onto a detector.

11. The method of claim 10, further comprising filtering the excitation wavelength from the detector.

12. The method of claim 1 , further comprising adjusting the at least one excitation wavelength contained within the excitation beam.

13. 1. A system for determining a concentration of a target protein in a sample, the system comprising: a light source for generating radiation; an optical system for directing said radiation onto a capillary tube of a capillary electrophoresis system; Equipped with a sample of interest is flowed through said capillary tube to illuminate at least a target protein if present in said sample; A system wherein the radiation produced by the light source comprises at least one excitation wavelength suitable for exciting at least one native fluorophore of the at least one target protein in the sample.

14. The system of claim 13 , wherein the light source comprises at least one light emitting diode (LED).

15. The system of claim 13 , wherein the light source comprises a laser-driven light source.

16. 16. The system of claim 13, wherein the light source is configured to generate radiation having wavelengths spanning a spectral range, and the excitation beam exhibits a spectral bandwidth narrower than the spectral range of the radiation.

17. 14. The system of claim 13, wherein the at least one excitation wavelength is about 285 nm.

18. 14. The system of claim 13, wherein the capillary tube comprises a radiation transparent portion through which the excitation radiation can be introduced into the capillary tube and at least a portion of the fluorescent radiation produced by the target protein in response to the excitation radiation can exit the capillary tube through the radiation transparent portion.

19. 20. The system of claim 18, further comprising a detector optically coupled to the transparent portion of the capillary tube, the detector receiving at least a portion of the fluorescent radiation and generating one or more fluorescent detection signals in response to detecting the received fluorescent radiation.

20. 20. The system of claim 19, further comprising an optical element for directing the fluorescence radiation onto the detector, optionally comprising a lens for focusing the fluorescence radiation onto the detector, and optionally further comprising a filter positioned in front of the detector to filter out excitation radiation.

21. 21. The system of any one of claims 19 and 20, further comprising an analyzer in communication with the detector, the analyzer receiving the one or more detection signals and obtaining a concentration of the target protein in the sample by processing the detection signals.

22. 22. The system of claim 21, wherein the analyzer is configured to determine the concentration of the target protein based on the intensity of the detected fluorescent radiation.

23. 14. The system of claim 13, wherein the optical system comprises an optical fiber extending from a proximal end to a distal end, the proximal end of the optical fiber optically coupled to the light source to receive at least a portion of the radiation emitted by the light source.

24. 24. The system of claim 23, further comprising one or more lenses disposed between the light source and the proximal end of the optical fiber, the one or more lenses transmitting the radiation emitted from the light source to the proximal end of the optical fiber, and optionally the one or more lenses comprising two converging lenses mounted in tandem to image an emission surface of the LED onto the proximal end of the optical fiber.

25. 25. The system of claim 24, further comprising an optical filter disposed between the two converging lenses, the optical filter receiving at least a portion of the radiation and selecting the excitation wavelength for illuminating the sample, optionally the optical filter comprising an optical bandpass or shortpass filter, further optionally the optical bandpass or shortpass filter exhibiting a transmission bandwidth in the range of about 270 nm to about 290 nm.

Images