Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
  • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
  • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Definitions

• the present disclosure is generally directed to systems and methods for quantification of one or more target proteins in a sample, and particularly to such quantification systems and methods that can provide high sensitivity and selectivity with the target sample contained within a capillary tube of a capillary electrophoresis (CE) system.
• CE capillary electrophoresis
• CE capillary electrophoresis
• the use of a narrow capillary tube, to which high electric field strength can be applied provides an environment enabling highly efficient separations using a minimal sample volume.
• the small capillary inner diameters can create a detection challenge in that they present a short optical path length, thus requiring a large sample load in order to achieve adequate sensitivity for many analytes, especially those present at low concentrations.
• 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 less than that which is required in many applications.
• Laser induced fluorescence (LIF) detection is often used in an attempt to improve concentration sensitivity and extend linear dynamic range.
• commercially available excitation sources generally emit in the visible wavelength range, where proteins are not intrinsically fluorescent. In order to circumvent this LIF detection challenge, fluorophore derivatization has been used but can pose additional problems.
• the labeling sites on proteins have different reactivities and the labeling reaction usually produces a mixture of differentially labeled proteins, resulting in peak broadening or splitting as well as quantitation challenges.
• labeling a protein with fluorescent dyes can lead to a change in the protein net charge and consequently alter the protein’s isoelectric point (pi), resulting in an overall change in a protein’s characteristics relative to its native state.
• the detection sensitivity of the same protein was improved using a 275.4 nm laser line, which was isolated from a water-cooled argon-ion laser.
• Pulsed lasers including ND: YAG (266 nm), KrF (248 nm), He-Ag (224 nm) lasers, were also evaluated as excitation sources for NFD. These lasers are relatively cost-effective, but a concentration sensitivity that can be achieved using such lasers is lower than that achievable using the UV argon-ion lasers, due to unfavorable excitation wavelength and pulse-to-pulse fluctuation.
• Lamps such as xenon and deuterium lamps, provide a continuous spectrum in the deep UV region, thus allowing for flexibility in excitation wavelengths for NFD.
• conventional lamps emit divergent light of low radiant power, presenting a challenge to deliver sufficient energy for fluorescence detection in CE.
• lamp-based fluorescence detection systems have been introduced in which light from a mercury-xenon lamp is filtered and subsequently directed to the capillary via an optical fiber to be focused onto a detection cell using a ball lens. The emission fluorescence is guided along the capillary by total internal reflection to a photomultiplier tube (PMT) positioned at the end of the cell.
• PMT photomultiplier tube
• LODs were in the 10 - 20 nM range, 25 times greater sensitivity 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 the system into a wavelength-resolved fluorescence detector, which can allow for probing protein conformational changes.
• a system for determining concentration of a target protein in a sample, which system includes a light source for generating radiation and an optical system for guiding the radiation onto a capillary tube of a capillary electrophoresis (CE) system through which a sample of interest flows so as to irradiate at least a target protein, when present in the sample.
• the radiation generated by the light source can comprise at least one excitation wavelength suitable for exciting at least one native fluorophore of said at least one target protein in the sample.
• the light source can comprise at least one light emitting diode.
• the light source can comprises a laser driven light source.
• the light source can generate radiation with wavelengths over a spectral range, and the excitation beam can exhibit a spectral bandwidth narrower than the spectral range of the radiation.
• the at least one excitation wavelength can be about 285 nm.
• the capillary tube can comprise a radiation-transparent portion through which the excitation radiation can be introduced into the capillary tube and at least a portion of a fluorescent radiation generated by said target protein in response to the excitation radiation can exit the capillary tube.
• the system can further comprise a detector optically coupled to the transparent portion of the capillary tube for receiving at least a portion of the fluorescent radiation and generating one or more fluorescent detection signals in response to detection of said received fluorescent radiation.
• an analyzer can be in communication with the detector for receiving the detection signal(s) and for processing the detection signal(s) to obtain a concentration of said target protein in said sample.
• the analyzer can be configured to determine the concentration of the target protein based on an intensity of said the fluorescent radiation.
• an optic can be provided for directing the fluorescent radiation onto the detector, and optionally, the optic can comprise a lens for focusing the fluorescent radiation onto the detector, and further optionally, a filter can be positioned in front of the detector to filter out excitation radiation.
• the optical system can comprise an optical fiber extending from a proximal end to a distal end, wherein the proximal end of the optical fiber is optically coupled to the light source for receiving at least a portion of the radiation emitted thereby.
• the system may further comprise one or more lenses disposed between the light source and the proximal end of said optical fiber for transmitting the radiation emitted from the light source to the proximal end of the optical fiber, and optionally in some aspects, the one or more lenses can comprise two convergent lenses placed in tandem so as to image an emitting surface of the LED onto the proximal end of the optical fiber.
• the system can further comprise an optical filter positioned between the two convergent lenses to receive at least a portion of the radiation and select the excitation wavelength for irradiating the sample.
• the optical filter can optionally comprise an optical bandpass or shortpass filter, and further optionally, the optical bandpass or shortpass filter can exhibit a transmission bandwidth in a range of about 270 nm to about 290 nm.
• a system for determining concentration of a target protein in a sample which includes a laser driven light source for generating radiation, an optical system for guiding the radiation onto a capillary tube of a capillary electrophoresis system through which a sample of interest can flow so as to irradiate at least one target protein in the sample, wherein the radiation generated by the radiation source comprises at least one excitation wavelength suitable for exciting at least one native fluorophore of said at least one target protein in the sample.
• the radiation generated by the laser driven light source includes wavelengths extending over a continuous spectral range containing the excitation wavelength.
• the continuous spectral range may extend from about 170 nm to about 2100 nm.
• the system can further include an optical filter that is positioned relative to the laser driven light source to receive at least a portion of the radiation generated by the source and select the excitation wavelength for irradiating the sample.
• the optical filter can be an optical bandpass filter.
• the optical bandpass filter exhibits a transmission bandwidth in a range of about 5 nm to about 50 nm, which can be centered around the excitation wavelength.
• the capillary tube can include a radiation-transmissive portion (herein also referred to as a transparent or radiation transparent portion), e.g., a portion made of glass or other suitable materials, through which the excitation radiation can be introduced into the capillary tube to excite at least one target protein of interest and through which the fluorescent radiation emitted by the excited protein can exit the capillary tube to be detected by a downstream detector. More specifically, the detector can be optically coupled to a back surface of the radiation-transmissive portion of the capillary tube for receiving at least a portion of the fluorescent radiation and generating one or more detection signals in response to the detection of the received fluorescent radiation.
• a radiation-transmissive portion herein also referred to as a transparent or radiation transparent portion
• the detector can be optically coupled to a back surface of the radiation-transmissive portion of the capillary tube for receiving at least a portion of the fluorescent radiation and generating one or more detection signals in response to the detection of the received fluorescent radiation.
• an optical component e.g., a spherical mirror
• a front portion herein also referred to as the “front window”
• the capillary tube i.e., the portion receiving the excitation light
• the captured radiation or at least a portion thereof, back into the capillary tube such that the reflected radiation, or at least a portion thereof, would exit through the back surface of the capillary tube to be detected by the detector.
• 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 thereby is focused into a focal point located in a central portion of the capillary tube.
• the returned fluorescent radiation can then diverge from that focal point to exit the capillary tube.
• An analyzer in communication with the detector can receive the detection signals generated by the detector and can process the detection signals to compute the concentration of the target protein in the sample.
• the analyzer can be configured to compute the concentration of the target protein based on the intensity of the detected fluorescent radiation.
• the laser driven radiation source can generate radiation with wavelengths (herein a wavelength of the radiation refers to a vacuum wavelength) extending over a continuous spectral range. This allows the selection of a desired excitation wavelength from among the wavelengths present in the spectral range of the radiation emitted by the source for exciting a target protein of interest.
• a device e.g., a filter wheel, on which a plurality of optical filters exhibiting different transmission bandwidths are mounted can be employed to switch the optical filter positioned in the path of the excitation radiation in order to select different excitation wavelengths, e.g., for detecting different target proteins.
• the excitation wavelength can be, for example, in a range of about 200 to about 300 nm.
• an optical fiber which extends from a proximal end to a 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 via its proximal end and transmits at least a portion of the received radiation, via its distal end, onto the capillary tube for exciting at least one target protein present in the sample, or suspected of being present in the sample.
• an optical system configured for directing at least a portion of the excitation radiation onto the proximal end of the optical fiber can include one or more lenses for focusing the radiation into the optical fiber.
• such lenses can include two convergent lenses placed in tandem.
• the two lenses can be configured such that the first lens substantially collimates the incident radiation and the second lens focuses the substantially collimated radiation into the proximal end of the optical fiber.
• an optical filter can be positioned between the two lenses for selecting an excitation wavelength of interest.
• the f/number of such lenses can be selected such that the convergence angle associated with the radiation being focused onto the optical fiber (that is, the proximal end of the optical fiber) is substantially equal to the numerical aperture of the optical fiber.
• the system can further include an optical filter positioned between the first lens and the second lens to receive at least a portion of the radiation and select the excitation wavelength for irradiating the sample.
• the optical filter includes an optical bandpass or shortpass filter.
• the optical bandpass or shortpass filter can exhibit a transmission bandwidth in a range of about 200 nm to about 300 nm.
• the laser driven light source can generate radiation with a radiance in a range of about 0.5 to about 200 mW/mm 2 .sr.nm.
• the radiance of the emitted radiation in a vacuum wavelength range of about 200 nm to about 300 nm can be in the range of 5 to about 200 mW/mm 2 .sr.nm
• the capillary tube can have an inner diameter in a range of about 10 pm to about 200 pm, e.g., in a range of about 20 pm to about 100 pm.
• a method for protein analysis of a sample in a capillary electrophoresis (CE) system comprises flowing a sample through a capillary tube of the CE system, utilizing a laser driven light source to generate radiation with wavelengths extending over a spectral range containing at least one wavelength suitable for exciting at least one target protein in the sample, spectrally filtering the radiation to select the excitation wavelength thereby generating an excitation beam with a spectral bandwidth that is narrower than the spectral range of the radiation generated by the laser driven radiation source and contains at least one excitation wavelength suitable for exciting at least one target protein of interest.
• CE capillary electrophoresis
• the excitation beam is directed onto a capillary tube of a capillary electrophoresis system via a transparent portion thereof (e.g., via a radiation-transmissive window) so as to excite at least one native fluorophore of the target protein passing through the lumen of the transparent portion of the capillary tube in order to cause the fluorophore to generate fluorescent radiation, and detecting at least a portion of fluorescent radiation emitted by the excited target protein.
• the emitted fluorescent radiation can be detected by directing at least a portion of the fluorescent radiation exiting the capillary tube, e.g., via a transparent section thereof (e.g., via a radiation-transmissive window), onto a detector, which generates one or more detection signals in response to the detection of the fluorescent radiation.
• the fluorescent radiation emitted by a plurality of different proteins is detected as a function of the migration time of those proteins, under the influence of an electric field, from an inlet of the capillary tube to the transparent portion of the tube.
• the distance between the inlet of the capillary tube and the transparent portion thereof can be, for example, in a range proximity of about 5 cm to about 100 cm, for example, to as to allow better resolution in separating different proteins of interest for interrogation via the excitation radiation.
• the radiation emitted by the laser driven radiation source can be spectrally filtered by passing the radiation through an optical filter, e.g., an optical bandpass or shortpass filter, to narrow the spectral range of wavelengths present in the radiation while ensuring that the narrowed spectral range contains the excitation wavelength of interest.
• an optical filter e.g., an optical bandpass or shortpass filter
• the fluorescent radiation emitted by the excited protein(s) can be analyzed to determine the concentration of the protein(s) in the sample under study.
• 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.
• an optical fiber is utilized to direct the excitation radiation onto the capillary tube (that is, onto the transparent portion of the capillary tube).
• 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 is 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, via its transparent portion, for exciting at least one protein flowing through the lumen of the transparent portion of the tube.
• the spectral range of the radiation emitted by the laser driven light source can extend, for example, from about 170 nm to about 2100 nm. Further, in some embodiments, the excitation wavelength for exciting one or more target proteins can extend from about 200 nm to about 300 nm.
• a system for determining concentration of a target protein in a sample which includes an LED light source for generating radiation; an optical fiber extending from a proximal end to a distal end, wherein the proximal end of the optical fiber is optically coupled to the LED light source for receiving at least a portion of the radiation emitted by the LED light source; a capillary tube of a capillary electrophoresis system through which a sample of interest flows, the distal end of the optical fiber being in optical coupling with a portion of the capillary tube so as to irradiate 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 the at least one target protein in the sample.
• the radiation generated by the LED light source has a substantially monochromic wavelength in the UV range of the electromagnetic spectrum.
• the substantially monochromatic wavelength can be about 285 nm.
• the LED light source provides an optical power in a range of about 50 mW to about 10 mW.
• 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 a fluorescent radiation generated by the target protein in response to the excitation radiation can exit the capillary tube.
• the system can further include a detector optically coupled to the transparent portion of the capillary tube for receiving at least a portion of the fluorescent radiation and generating one or more fluorescent detection signals in response to detection of the received fluorescent radiation.
• an optic can be further included for directing the fluorescent radiation onto the detector.
• the optic comprises a lens for focusing the fluorescent radiation onto the detector.
• a filter can be positioned in front of the detector to filter out excitation radiation.
• the system can further include an optical system disposed between the LED light source and a proximal end of the optical fiber for transmitting the radiation emitted from the LED light source to the proximal end of the optical fiber.
• 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 can comprise two convergent lenses placed in tandem so as to image an emitting surface of the LED onto the proximal end of the optical fiber.
• the one or more lenses comprise a first lens and a second lens, and the first lens is configured and positioned relative to the LED light source to substantially collimate the radiation generated by the LED light source, and the second lens is configured and positioned relative to the first lens to focus the collimated radiation onto the proximal end of the optical fiber.
• an optical filter can be positioned between the first lens and the second lens to receive at least a portion of the radiation and select the excitation wavelength for irradiating the sample.
• the optical filter comprises an optical bandpass or shortpass filter.
• the optical bandpass or shortpass filter can exhibit a transmission bandwidth in a range of about 270 nm to about 290 nm.
• the method for protein analysis may comprise flowing a sample through a capillary tube of the CE system and utilizing a light source to generate radiation containing at least one excitation wavelength suitable for exciting at least one native fluorophore of at least one target protein in the sample.
• An excitation beam containing the at least one excitation wavelength can be directed onto a transparent portion of the capillary tube of the CE system so as to excite said at least one native fluorophore of the target protein passing through a lumen of the transparent portion in order to cause the at least one native fluorophore to generate fluorescent radiation.
• At least a portion of fluorescent radiation emitted by the excited target protein can be detected.
• the light source can comprise at least one light emitting diode.
• the light source can comprises a laser driven light source.
• the light source can generate radiation with wavelengths over a spectral range, and the excitation beam can exhibit a spectral bandwidth narrower than the spectral range of the radiation.
• the method can further comprise spectrally filtering the radiation to generate the excitation beam.
• an optical bandpass or shortpass filter can be utilized to spectrally filter the radiation.
• the optical bandpass or shortpass filter can exhibit a transmission bandwidth in a range of about 200 nm to about 300 nm.
• the target protein can comprise an antibody.
• the at least one excitation wavelength can be about 285 nm. Additionally or alternatively, in some aspects, the method can comprise adjusting the at least one excitation wavelength contained within the excitation beam.
• methods in accordance with the present teachings can utilize a lens to focus the fluorescent radiation onto a detector.
• the method may further comprise filtering the excitation wavelength from the detector
• FIG. 1A schematically depicts an example of a system according to an embodiment for NFD quantification of proteins in a CE system
• FIG. IB schematically depicts a cross-sectional view of the capillary tube shown in FIG. 1A, illustrating that a high voltage applied across the capillary tube generates an electric field along the lumen of the tube, which causes the migration of proteins from an inlet of the tube to an outlet thereof,
• FIG. 1C schematically depicts a filter wheel on which a plurality of optical filters having different transmission bandwidths can be mounted to allow to changing the optical filter that is positioned in the path of the radiation emitted by the radiation source and/or the fluorescent radiation,
• FIG. 2 presents data indicative of signal-to-noise ratio of an example of an NFD system according to an embodiment as a function of the bias voltage applied to PMT employed in the system as a detector
• FIG. 3A presents data corresponding to CE-SDS separation of reduced SCIEX IgG control standard with NFD at excitation wavelength of 280 nm
• FIG. 3B presents data corresponding to CE-SDS separation of reduced SCIEX IgG control standard with NFD at excitation wavelength of 220 nm
• FIG. 4A presents UV absorbance data obtained using CE-SDS separation of non-reduced SCIEX IgG control standard using UV absorbance detection at 214 nm and 280 nm,
• FIG. 4B presents NFD data obtained using CE-SDS separation of non-reduced SCIEX IgG control standard
• FIG. 5A presents UV absorbance detection data using cIEF separation of NIST IgG at 280 nm
• FIG. 5B presents NFD data using cIEF separation of NIST IgG at 280 nm
• FIG. 6A schematically depicts an example of a system according to another embodiment for NFD quantification of proteins in a CE system
• FIG. 6B schematically depicts an example of a system according to yet another embodiment for NFD quantification of proteins in a CE system
• FIG. 7 presents SDS-CGE separations of reduced IgG control standard with NFD using LED directly (A) and filtered LED (B) as the excitation source,
• FIGS. 8A and 8B present effect of PMT control voltage on the signal-to-noise
• FIGS. 9A - 9D present effect of sample injection time the detection sensitivity and separation efficiency of SDS-CGE
• FIGS. 10A and 10B present linearity of SDS-CGE with NFD for analysis of non-reduced NISTmAb
• FIGS. 11A and 11B present quantitation and purity analysis of Etanercept in SDS-CGE with UV absorbance versus with NFD.
• the present disclosure is directed to systems and methods that can be employed for protein quantification in electrophoresis analysis of a variety of samples, without a need for deep UV lasers.
• the systems and methods according to the present teachings allow performing NFD with a sensitivity that is at least 10 times greater than that exhibited by current UV absorbance detection techniques at 214 nm.
• the systems and methods of the present teachings allow adjusting the excitation wavelength, which can in turn allow optimizing the excitation wavelength for a particular target protein.
• the systems and methods according to the present teachings allow flexible selection of the excitation wavelength.
• the excitation wavelength can be adjusted by replacing a bandpass or a shortpass filter with a different filter or a monochromator without degrading the detection sensitivity.
• the ability to adjust the excitation wavelength can be advantageously employed to optimize the quantum yield or can be employed for performing wavelength-resolved fluorescence detection.
• 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 so as to generate radiation having wavelengths that extend over a continuous spectral region, e.g., for generating deep UV radiation, e.g., radiation with wavelengths in a range of about 200 nm to about 300 nm.
• substantially indicates a deviation, if any, from a complete state and/or condition of at most 10%, or at most 5%.
• light and “radiation” are used herein interchangeably to refer to not only to visible light but more generally to radiation in other regions of the electromagnetic spectrum, including UV radiation.
• radiation is defined as the flux of radiation emitted per unit solid angle in a given direction by a unit area of a radiation source.
• transparent e.g., a wall of a CE capillary tube
• radiation-transmissive e.g., a radiation-transmissive
• a system 100 for quantifying one or more target proteins in a sample flowing through a capillary tube of a CE system via laser-induced fluorescence measurements includes a radiation source 1 that can emit radiation over a spectral vacuum wavelength range extending from about 170 nm to about 2100 nm.
• the radiation source is capable of emitting radiation having vacuum wavelengths over a range of about 200 nm to about 300 nm with a radiance over this range that is at least 10 times higher than that exhibited by conventional UV lamps over this range.
• the radiation source 1 is capable of emitting radiation with a radiance in a range of about 5 to 200 mW/mm 2 .sr.nm. over the spectral wavelength range of about 200 nm to about 300 nm.
• a radiation source marketed by Energetiq Technology, Inc. of Wilmington, MA under the tradename LDLS® which uses a laser to directly heat a xenon plasma to the high temperatures that are needed for production of deep UV radiation, can be employed.
• LDLS® which uses a laser to directly heat a xenon plasma to the high temperatures that are needed for production of deep UV radiation
• the radiation emitted by the radiation source 1 is received by a plano convex lens 2, which is separated from the radiation source by its focal length and substantially collimates the radiation.
• a bandpass or a shortpass filter 3 selects a portion of the spectral range of the emitted radiation so as to generate an excitation beam having a radiation bandwidth containing at least one wavelength suitable for exciting at least one native fluorophore of at least one target protein contained in a sample, or suspected of being contained in a sample, under study, as discussed further below.
• the optical filter 3 is placed between the lenses 2 and 4, in other embodiments, the optical filter can be placed at other locations along the propagation path of the radiation to the sample in the capillary.
• Another plano-convex lens 4 focuses the filtered radiation onto a proximal end (PE) of an optical fiber 5.
• the f/number of the lenses 2 and 4 are selected such that the light focused onto the proximal end of the optical fiber 5 has a convergence angle that is substantially equal to the numerical aperture of the optical fiber so as to optimize the coupling of the radiation into the optical fiber.
• 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 a transparent portion 7a of a capillary tube 7, i.e., a portion of the capillary tube that has a transparent wall, in which a 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 so as to excite at least one target protein when present in the sample, as the sample flows through the lumen of the transparent portion of the capillary tube.
• a front portion of the transparent wall of the capillary tube i.e., the portion facing the distal end of the optical fiber, can function as a window through which the excitation radiation can enter the capillary tube.
• one or more native fluorophores of the target protein can emit fluorescent radiation.
• Native fluorescence of proteins originates from the excitation of three amino acids: phenylalanine, tyrosine, and tryptophan. It is known that the fluorescent emission intensity associated with phenylalanine is about 50 times lower than that of tyrosine or tryptophan and is generally negligible in native fluorescence detection of proteins. In most proteins, the native fluorescence is principally due to fluorescence by tryptophan residues, although some proteins can exhibit tyrosine fluorescence.
• the filter 3 can be configured to allow the passage of radiation with a wavelength of 280 nm therethrough for exciting the tryptophan amino acids in the target protein.
• the fluorescent radiation emitted by the excited tryptophan amino acids can be collected and analyzed, e.g., in a manner discussed below, to determine the concentration of the target protein in the sample under study.
• the emitted fluorescent radiation can exit the capillary tube through the wall of its transparent portion 7a. Some of the emitted fluorescent radiation leaves the capillary tube via a back section 7aa of the transparent portion 7a while some of the fluorescent radiation leaves the capillary tube via the front section 7bb of the transparent portion 7a.
• a spherical mirror 6 positioned in proximity of the transparent portion 7a of the capillary tube 7 captures at least a portion of the fluorescent radiation exiting the capillary tube and reflects the captured fluorescent radiation (or at least a portion thereof) back into the capillary tube such that the reflected fluorescent radiation (or at least a portion thereof) exits through the back section 7aa of the transparent portion 7a.
• a plano-convex lens 8 is optically coupled to the transparent portion 7a of the capillary tube 7 to receive the fluorescent radiation exiting the capillary tube (or at least a portion thereof) and collimate the fluorescent radiation to generate 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.
• the bandpass filter allows transmission of the fluorescent radiation while blocking or reducing the passage of radiation at other wavelengths.
• the bandpass filter can have a transmission bandwidth that is centered at a wavelength at which the fluorescent radiation emitted by the excited target protein exhibits its maximum intensity and can have a transmission bandwidth that extends, for example, from about 5 nm to about 50 nm.
• the detector 10 is a photomultiplier tube, e.g., a photomultiplier tube marketed by Hamamatsu (R5984).
• the detector 10 generates one or more detection signals in response to the detection of the fluorescent radiation.
• An analyzer 20 (herein also referred to as an analysis module) receives the detection signals generated by the detector 10 and operates on those signals to compute a concentration of the target protein of interest in a 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.
• the instructions for analysis of the fluorescent data can be stored in a permanent memory of the analyzer and can be transferred into a transient memory module during runtime by analyzer’s processor to be executed.
• the protein concentration is proportional to the area of the protein UV absorption peak.
• a calibration curve can be established, e.g., with 5 or more different concentrations of proteins over the range. The concentration of a target protein can then be calculated using the UV absorption peak area and the calibration curve.
• the present teachings are not limited to determining the concentration of target proteins via excitation of tryptophan residues.
• the excitation wavelength of the system can be adjusted to detect tyrosine fluorescence, e.g., in cases in which the target proteins lack an adequate concentration of tryptophan residues.
• a high-voltage source 30 applies a high voltage across the capillary tube 7 to generate an electric field through the capillary tube that can in turn cause different proteins present in a sample under investigation to migrate through the tube at different rates, e.g., due to differences in their mobilities.
• the differences of the mobilities of the proteins can be at least partially due to differences in their electrical charges. Consequently, when a sample containing different target proteins introduced into the capillary tube 7 via its inlet 7b flows, under the influence of the electric field, to the outlet 7c, the different proteins arrive at the transparent portion of the capillary tube at different times due to the differences in their mobilities.
• the excitation of at least one native fluorophore of each target protein can in turn allow the quantification of that protein via NFD, e.g., in a manner discussed herein, thereby allowing the quantification of various proteins in the sample.
• a cartridge 30 containing a plurality of different optical filters 31, 32, 33, and 34 can be placed in the path of the emitted radiation, where each optical filter exhibits a different optical bandpass.
• the cartridge allows the selection of different excitation wavelengths for exciting different fluorophores of a plurality of target proteins.
• the cartridge containing a plurality of optical filters can be placed in front of the detector 10 to allow detecting fluorescent radiation at different wavelengths.
• an internal standard can be added to a sample to function as a calibrant for accurate quantification of one or more target proteins present in the sample.
• suitable internal standards include, without limitation, peptides and proteins with known molecular weight.
• the present teachings can be employed for the detection and quantification of different types of proteins.
• the protein can be an antibody, such as a monoclonal or polyclonal antibody, though the present teachings can be used to quantify concentrations of other proteins in the sample, as well.
• LEDs light emitting diodes
• a system 600 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 that can emit a substantially monochromic radiation in a vacuum wavelength range of about 190 nm to about 310 nm (e.g., about 285 nm).
• the LED radiation source is capable of emitting a significantly narrower wavelength range than the laser light source discussed with regard to FIG. 1A.
• an LED marketed by Thorlabs, Inc., Newton, NJ under the trade designation M280L6 can be employed.
• the LED light source 601 can be powered by a DC power supply, which is generally included in a commercial CE system. Accordingly, the power efficiency for the system can be improved over the laser-based systems that typically require dedicated power supplies.
• the LED radiation source 601 can be directly coupled to a proximal end of an optical fiber 605 via, e.g., a butt-coupling technique. Thereafter, similar to the embodiment shown in FIG. 1A, a distal end of the optical fiber 605 is optically coupled to a transparent portion 607a of a capillary tube 607, e.g., a portion of the capillary tube 607 that has a transparent wall, in which a sample can flow.
• a transparent portion 607a of a capillary tube 607 e.g., a portion of the capillary tube 607 that has a transparent wall, in which a sample can flow.
• the transparent portion 607a provides a window through which the excitation radiation exiting the distal end of the optical fiber 605 can be introduced into the capillary tube 607 so as to excite at least one target protein when present in the sample, as the sample flows through the lumen of the transparent portion of the capillary tube 607.
• a spherical mirror 606 is positioned in proximity of the transparent portion 607a of the capillary tube 607 to capture at least a portion of the fluorescent radiation exiting the capillary tube and to reflect the captured fluorescent radiation (or at least a portion thereof) back into the capillary tube 607 such that the reflected fluorescent radiation (or at least a portion thereof) exits through the back section of the transparent portion 607a.
• 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.
• the bandpass filter 609 can have a transmission bandwidth that is centered at a wavelength at which the fluorescent radiation emitted by the excited target protein exhibits its maximum intensity and can have a transmission bandwidth that extends, for example, from about 5 nm to about 50 nm.
• the detector 610 can be implemented as a photomultiplier tube, e.g., a photomultiplier tube marketed by Hamamatsu (R5984).
• the detector 610 can generates one or more detection signals in response to the detection of the fluorescent radiation.
• An analyzer 620 (herein also referred to as an analysis module) receives the detection signals generated by the detector 610 and operates on those signals to compute a concentration of the target protein of interest in a sample under investigation.
• a filter e.g., a bandpass filter
• a filter can be disposed in front of the LED radiation source 601 to more precisely limit the wavelengths of the emitted radiation from the LED radiation source 601.
• a 280 nm bandpass filter can be used.
• the bandpass filter 603 can have a transmission band (e.g., >65%) in a range of about 270 nm to about 290 nm.
• 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 substantially collimates the radiation.
• the bandpass filter 603 or a shortpass filter rejects the residual emission in wavelengths beyond about 300 nm.
• Another plano-convex lens 604 focuses the filtered radiation onto a proximal end of the optical fiber 605.
• the bandpass filter 603 is placed between the lenses 602 and 604, in other embodiments, the bandpass filter 603 can be placed at other locations along the propagation path of the radiation to the sample flowing in the capillary tube 607.
• a spherical mirror 606, a plano-convex lens 608, a bandpass filter 609, a detector 610, and an analyzer 620 can be similarly arranged and can function similarly as the embodiments described above to excite at least one native fluorophore of at least one protein within the sample, collect and analyze fluorescent radiation emitted by the fluorophore in response to its excitation.
• Example 1 Using a laser driven light source
• the LDLS® generates high-intensity plasma in a xenon gas, which in turn generates radiation over 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 higher than that of traditional lamps, combined with a reported typical lifetime of about 10,000 hours.
• the continuous spectrum allows for greater flexibility than lasers in selection of the excitation wavelength.
• the excitation wavelength was selected using a bandpass filter, which was mounted on a filter wheel and allowed interchangeable selection of different filters.
• a photomultiplier tube (PMT) was used as the detector.
• PMT photomultiplier tube
• the effect of the PMT bias voltage on the signal-to-noise ratio with 280 nm excitation and 333 - 375 nm emission was investigated.
• the contribution of tryptophan and tyrosine residues to the intrinsic fluorescence of a 10 kDa internal standard and reduced IgGl was investigated by adjusting the excitation and emission wavelengths.
• NFD monoclonal antibodies
• SDS-MW analysis kit 10 kDa internal standard, IgG control standard, cIEF peptide marker kit, sample loading solution, and cIEF gel were obtained from SCIEX (Brea, CA). Tryptophan (Sigma-Aldrich, St Louis, MO) and IgG control standard (SCIEX, Brea, CA) were used to evaluate optical components and optimize parameters for the NFD prototype.
• USP Monoclonal IgG system suitability Reference Standard USP IgG was obtained from US Pharmacopeia (Rockville, MD).
• NISTmAb, Humanized IgG lk Monoclonal Antibody (NIST IgG) was purchased from National Institute of Standards & Technology (Gaithersburg, MD). Pharmalyte 3 - 10 was obtained from Cytiva (Marlborough, MA). All other chemicals were sourced from Sigma- Aldrich (St Louis, MO).
• the reduced IgG standard mixture was prepared by mixing 96 pL SCIEX IgG control standard solution, 2 pL of 10 kDa internal standard, and 2 pL of 2-mercaptoethanol. The subsequent solution was then heated at 70 °C for 10 minutes in order to accelerate dissociation of non-covalently bound species and SDS-protein binding. Non-reduced SCIEX IgG control standard was also heated at 70 °C for 10 minutes before use.
• CE-SDS separations were performed using bare fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with a 50 pm i.d., a 375 pm o.d., and total/effective lengths of 30/20 cm. Prior to each separation, the capillary was sequentially rinsed with 0.1 M sodium hydroxide, 0.1 M hydrochloric acid, and DI water at 50 psi for 5, 3, and 3 minutes, respectively. SDS-MW gel buffer (SCIEX, Brea, CA) was loaded into the capillary at 50 psi for 10 minutes. Electrokinetic injection was performed at -5 kV for 10 seconds. Separations were conducted by applying a voltage of -15 kV, with 20 psi pressure at both ends to reduce the risks of forming air bubbles. cIEF Conditions
• a cIEF master mix was prepared by combining the following solutions: (1) 1 mL of 3.75 M urea in cIEF gel; (2) 60 pL of Pharmalyte 3-10; (3) 100 pL of 500 mM arginine in DI water; (4) 10 pL of 200 mM iminodiacetic acid in DI water.
• a NIST IgG standard mixture was prepared for UV absorbance detection by combining 200 pL cIEF mater mix, 8 pL of 4 mg/mL NIST IgG, and 2 pL each pi markers (10.0, 7.0, 9.5, and 5.5).
• a NIST IgG sample was prepared for NFD by diluting 150-fold with cIEF master mix.
• cIEF separations were performed using neutral coated capillaries (SCIEX, Brea, CA) with a 50 pm i.d., 375 pm o.d., and total/effective lengths of 30/20 cm. Before use, the capillary was conditioned by being rinsed with 350 mM acetic acid, DI water, and cIEF Gel (SCIEX,
• the light beam from the radiation source 1 was sequentially collimated using a plano-convex lens 2, filtered with a bandpass or shortpass filter 3, 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 bandpass filter 9, and detected with a detector 10 (e.g., a photomultiplier tube (PMT)).
• a spherical mirror 6 was positioned proximal to the detection window to increase the collection efficiency of light emitted by analytes inside the capillary.
• the noise was defined as the standard deviation of responses of a blank sample over a period of 20 seconds. Effect of the 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/mF tryptophan solution was injected at 0.5 psi for 5 seconds and pressure-driven separation was operated at 5 psi with water as the background. As shown in FIG. 2, the signal-to-noise ratio was enhanced from 110 to 380 when the PMT voltage increased from 350 V to 475 V.
• the signal-to-noise ratio was 1,229 with the PMT voltage at 650 V, and further increase of the PMT voltage to 890 V yielded a higher signal- to-noise ratio of 1,681.
• a PMT voltage above 650 V a 2.5 mg/mF solution of USP IgG generated a voltage signal which 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.
• a PMT voltage of 650 V was selected for further experiments. 2.
• Native fluorescence of protein originates from the excitation of three aromatic amino acids: phenylalanine, tyrosine, and tryptophan.
• the emission intensity of phenylalanine is about 50 times lower than that of tyrosine or tryptophan and is generally negligible in native fluorescence detection of proteins.
• native fluorescence is principally due to tryptophan residues while some proteins exhibit tyrosine fluorescence.
• the emission peak of tryptophan is usually near 350 nm.
• the excitation and emission filters in the setup were first evaluated with excitation at 280 nm and emission collected in the range of 333 to 375 nm.
• Reduced SCIEX IgG control standard mixed with 10 kDa internal standard was analyzed by CE-SDS using the NFD setup.
• the three peaks, representing IgG light chain (LC), non-glycosylated heavy chain (NGHC), and heavy chain (HC) exhibited enhanced signal-to-noise ratios relative to UV absorbance detection at 214 nm.
• the response for 10 kDa internal standard was low, presumably due to the lack of tryptophan residues.
• the tyrosine emission peak was shown to be insensitive to the local environment and the emission maximum was found at around 304 nm.
• the contributions of tyrosine residues to native fluorescence were investigated by collecting the emission spectrum in the 305 - 315 nm band following excitation at 220 nm. Separation was performed in an uncoated capillary having a 50 pm i.d., 375 pm o.d., and total/effective lengths of 30/20 cm.
• the applied voltage was -15 kV with 20 psi pressure at both ends.
• the injection of the sample was achieved at a voltage of -5 kV for 10 seconds.
• NFD NFD was employed with excitation at 280 nm and emission in the 333 - 375 nm band.
• the excitation and emission wavelength of the NFD system can be adjusted to detect tyrosine fluorescence in cases where target proteins lack an adequate concentration of tryptophan residues.
• CE-SDS is replacing SDS-PAGE as a routine tool in the biopharmaceutical industry for the analysis of therapeutic mAbs.
• UV absorbance detection is often operated in the region of 210 - 220 nm, where maximum concentration sensitivity is achieved. However, detection is not selective and suffers from interference by other absorbing species or baseline anomalies.
• Non-reduced SCIEX IgG control standard was separated using CE-SDS with UV absorbance detection at 214 nm (See, trace 1 in FIG. 4A).
• the IgG sample was heated at 70 °C for 10 minutes before use.
• the IgG sample was directly used (FIG. 4A) and was also used with a 10-fold dilution with SDS-MW sample buffer (SCIEX, Brea, CA) (FIG. 4B). All other CE- SDS conditions were the same as those used in connection with FIG. 3.
• the main peak yielded a signal-to-noise ratio of 3,389.
• the same IgG sample was separated using CE-SDS with absorbance detection at 280 nm (See, trace 2 in FIG. 4A).
• the response was much lower and it yielded a signal-to-noise of 67 for the main peak, representing 51 times lower sensitivity than with UV absorbance detection at 214 nm (trace 2 vs. trace 1).
• the non-reduced SCIEX IgG control standard was diluted 10-fold with SDS sample buffer (SCIEX, Brea, CA) and analyzed (See, FIG. 4B).
• the signal-to-noise ratio was 5,568 for the main peak, representing 16-fold sensitivity improvement relative to UV absorbance detection at 214 nm.
• the baseline exhibited less anomalies and peak integration suffered less interference than with absorbance detection at 214 nm (FIG. 4B vs. trace 1 in FIG. 4A).
• Analysis of mAbs using cIEF with NFD cIEF separates protein molecules based on their pis and is an important CE-based technique routinely used in the biopharmaceutical industry for mAh charge isoform analysis.
• cIEF absorbance detection
• absorbance detection is generally performed at 280 nm because carrier ampholytes tend to be UV-sensitive, especially at 214 nm.
• cIEF with LIF detection is not widely employed because fluorophore derivatization often can lead to a change in the charge of various protein isoforms, resulting in separation not comparable to those of their native proteins counterparts.
• NIST IgG A 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 having a 50 pm i.d., 375 pm o.d., and total/effective lengths of 30/20 cm).
• SCIEX neutral coated capillary
• the NIST IgG sample was prepared as described above.
• the sample used in UV absorbance detection was 150-fold diluted. Injection of the sample was achieved at 25 psi for 99 seconds, and focusing was achieved at 25 kV for 15 minutes, and chemical mobilization at 30 kV for 20 minutes.
• the signal-to-noise ratio was 956 for the main peak.
• the same NIST IgG solution generated 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 IgG sample yielded a signal-to-noise ratio of 1,137 (FIG. 5B).
• analysis using NFD provided 178 times sensitivity improvement for the main peak over absorbance detection at 280 nm.
• Example 2 Using an LED light source
• a fiber-coupled 285nm FED module was obtained from Thorlabs, Inc. (Newton, NJ).
• the FED light source included a single FED mounted on a metal-core circuit board, which is in direct contact with a heat sink for thermal stability.
• the FED was driven with a compact controller (Thorlabs, Inc., Newton, NJ), which can modulate the FED’s forward DC current in the range of 0 - 1 ,200 mA.
• the FED was operated with a forward DC current of 500 mA, at which its typical lifetime is >10000 hours.
• Performance of the developed NFD scheme was evaluated using an IgG control standard (SCIEX, Brea, CA), which is composed of glycosylated and non-glycosylated IgG from mouse serum.
• the total mass concentration is 1 mg/mL and quantity of the non-glycosylated form is -9.5%.
• the IgG sample was reduced by mixing 95 pL sample with 5 pL b-mercaptoethanol and then heating at 70 °C for 10 minutes. Before analysis, the reduced IgG control standard was 10- fold diluted with SDS sample buffer (SCIEX, Brea, CA). As shown in FIG. 7, the three peaks represent 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. Herein, the noise was defined as the standard deviation of responses of a blank sample over a period of 30 seconds.
• the signal-to-noise ratio (S/N) was 3240, 860, 5458 for LC, NGHC, and HC, respectively.
• the light power received by the optical fiber decreased -50% and accordingly fluorescence emitted by the reduced IgG sample decreased -45% (see, line A vs. line B).
• S/N increased to 7409, 1917, and 12566 for LC, NGHC, and HC, respectively. This represents about 2.3-fold enhancement of detection sensitivity over the configuration without a 280 nm bandpass filter. Therefore, the LED light source combined with two lenses and a 280 nm bandpass filter was used for excitation in Example 2.
• IgG control standard, SDS-MW gel separation buffer, SDS sample buffer, 0.1 N HC1 acidic wash, 0.1 N NaOH basic wash, and CE grade water were from SCIEX (Brea, CA).
• the NIST monoclonal antibody (NISTmAb) was purchased from National Institute of Standards & Technology (Gaithersburg, MD). It is a solution of 10 mg/mL humanized IgGl k monoclonal antibody in 12.5 mM L-histidine, 12.5 mM L-histidine HC1 (pH 6.0). Iodoacetamide and 2- mercaptoethanol were sourced from Sigma- Aldrich (St Louis, MO). Etanercept was purchased from Myonex (Norristown, PA).
• 250 mM iodoacetamide was prepared by dissolving 46.2 mg in 1 mL of CE grade water (SCIEX, Brea, CA). Before use, the stock solution was diluted to 20 mM with SDS sample buffer (SCIEX, Brea, CA).
• 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 enable compatibility with the LIF detector.
• the light source was powered and self-contained in the PA800 Plus CE system. All data were collected and processed with 32 Karat software (Version 10.1).
• the reduced IgG control standard was prepared by mixing 95 pL SCIEX IgG control standard solution and 5 pL 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).
• Non-reduced SDS-NISTmAb complexes were prepared by diluting 10 mg/mL NISTmAb to the desired concentration with SDS sample buffer (SCIEX, Brea, CA), which contained 20 mM iodoacetamide. The subsequent solution was heated at 70 °C for 10 minutes and then cooled to room temperature before use.
• SDS sample buffer SDS sample buffer
• the 25 mg/vial Etanercept was reconstituted with 1 mL diluent, and the stock solution was diluted to the desired concentration with SDS sample buffer (SCIEX, Brea, CA), which contained 20 mM iodoacetamide. Before use, the sample solution was heated at 70 °C for 10 minutes and then cooled to room temperature.
• SDS-CGE Conditions SDS-CGE Conditions
• the capillary was sequentially rinsed with 0.1 N NaOH basic wash, 0.1 N HC1 acidic wash, and CE grade water at 70 psi for 3, 1, and 1 minutes, respectively.
• SDS-MW gel separation buffer was loaded into the capillary at 70 psi for 10 minutes.
• the sample was electrokinetically injected into the capillary at -5 kV for 40 seconds. Separations were conducted with a voltage of -15 kV. To reduce the risks of forming air bubbles, 20 psi pressure was applied at both inlet and outlet ends during separation.
• the manufacturer Hamamatsu specify the PMT maximum rating to be 1250 V. To provide reliability against voltage fluctuations, the PMT was operated with a control voltage below 1000 V, 20% lower than the specified maximum rating. In this experiment, the effect of the PMT control voltage on S/N was investigated at seven levels: 386, 458, 530, 628, 727, 862, and 997 V. 1 mg/mL IgG control standard was reduced as described above and 10-fold diluted before being analyzed in SDS-CGE. As shown in FIG. 8A, as the control voltage was increased from 386 to 727 V, the fluorescence signal increased -100 times. For all three peaks, representing LC, NGHC and HC, S/N was significantly enhanced (see, FIG. 8B).
• the separation gel buffer can be viscous while the sample solution can be gel-free. This mismatch often presents issues with pressure injection, and sample injection is generally performed electrokinetically. Since the SDS-denatured proteins have uniform charge - to-size ratios, regardless of their molecular weight, various protein molecules migrate in the gel- free sample solution with the same mobility, mitigating injection bias.
• FIG. 9A shows representative electropherograms of 100 pg/mL reduced IgG samples with the injection time at 5, 20, 60, and 100 seconds. As the injection time increased, corrected peak area increased linearly with a correlation coefficient (r 2 ) in the range of 0.9993 - 0.9999 (see, FIG. 9B). As shown in FIG.
• Detection of low-level impurities is one of the primary uses of SDS-CGE assays.
• the non-reduced SDS-NISTmAb complexes were prepared by diluting 10 mg/mL NISTmAb with SDS sample buffer (SCIEX, Brea, CA) containing 20 mM iodoacet amide and heating the mixture at 70 °C for 10 minutes.
• SDS sample buffer SDS sample buffer
• the non-reduced NISTmAb sample was prepared at 11 concentration levels in the range of 0.03 to 500 pg/mL, and FIG. 10A shows overlapping of their SDS-CGE electropherograms.
• the inset is an expanded view of the electropherogram at 0.05 pg/mL.
• Each sample was injected in triplicate, and linear regression was established by plotting the mean of corrected peak areas versus the concentration (see, FIG. 10B).
• the correlation coefficient (r 2 ) was 0.9999, indicating an excellent linear relationship between the corrected peak area and the NISTmAb concentration.
• the limit of detection (LOD) and limit of quantitation (LOQ) were defined as the protein concentration which generates a S/N of 3 and 10, respectively.
• the noise was estimated by measuring the magnitude of background response of a blank sample and calculating the standard deviation of these responses over a period of 30 seconds.
• LOD and LOQ for non-reduced NISTmAb were 8.3 and 27 ng/mL, respectively. This is comparable to the concentration sensitivity obtained in SDS-CGE-LIF and SDS-PAGE with silver staining.
• Etanercept is a therapeutic Fc-fusion protein, approved by FDA in 1998 to treat psoriatic arthritis and rheumatoid arthritis. It is highly glycosylated and with an apparent molecular weight of -150 kDa. To evaluate purity and the high-molecular- weight (HMW) content in Etanercept and its biosimilar, Cho et al. (Cho et ah, "Evaluation of the structural, physicochemical, and biological characteristics of SB4, a biosimilar of etanercept.
• SEC-MALLS size exclusion chromatography-multi-angle laser light scattering
• FIG. 11A shows the SDS-CGE-UV electropherogram of 1 mg/mL non-reduced Etanercept.
• the baseline waviness around the main peak complicated peak integration and posed challenges in purity analysis of Etanercept, especially in the HMW region.
• FIG. 11B shows the SDS-CGE-NFD electropherogram of 100 pg/mL non-reduced Etanercept.
• the HMW and LMW peaks were both well distinguished from the noise and baseline fluctuation, allowing for quantitation of HMW and LMW content using a single assay.
• the percentage of corrected peak area was as shown in Table 2.
• the determined HMW content was consistently in the range of 2.57 - 2.63%, comparable to the reported HMW content in a reference product analyzed with SEC-MALLS.
• the LMW content was determined to be 1.19 - 1.27%, consistent over the range of 50 - 150% of the target concentration (200 pg/mL).
• a calibration curve was established over the range of 50 - 150% of the target concentration, 200 pg/mL, for the main peak.
• the correlation coefficient (r 2 ) was 0.9990, indicating good linearity of the SDS-CGE-NFD method for quantitation of Etanercept.
• the mean of corrected peak areas was substituted into the regression equation to calculate the experimental concentration, and recoveries were in the range of 97.02 - 101.6%.

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