Devices and methods for sensitive detection and quantification of biomolecules

Abstract

The present disclosure provides devices, systems, kits, and methods that can be used to quantify biomolecules such as intact proteins and nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 994,431, filed on March 25, 2020, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure provides devices, systems, kits, and methods that can be used to quantify biomolecules such as intact proteins and nucleic acids. Background Art

[0004] Quantitative protein bioanalysis is typically performed using ligand binding assays (LBAs) or bottom-up surrogate peptide methods for liquid chromatography (LC)-mass spectrometry (MS) analysis. These two approaches can often be used in a complementary manner. However, since the target molecule is bound to an epitope and does not provide structural information (LBAs) or is digested into small fragments (bottom-up LC-MS), it is not possible to determine the metabolic transformation of the molecule.

[0005] To meet this need, analysts can instead utilize MS detection to detect and quantify intact proteins. Recent technological advances in high-resolution mass spectrometry (HRMS) coupled with improvements in sample preparation have enabled the use of reversed-phase liquid chromatography (RPLC) for bioanalysis of intact proteins. Unlike alternative methods, performing RPLC-HRMS on intact proteins can provide a more complete picture of what is accurately quantified in any given assay. Additionally, because no digestion or only a limited amount of digestion is used in the related methods, sample preparation time can be reduced. However, emerging attempts at this analytical modality have shown that intact protein quantification can be quite challenging when both LC and MS dimensions are not properly optimized. Summary of the Invention

[0006] The present disclosure provides devices, systems, kits, and methods for addressing challenges associated with biomolecule quantification (including intact protein quantification) by employing short column devices with optimized stationary and mobile phases, wherein the challenge is the difficulty in achieving good resolution and high protein recovery without losing the stationary phase (i.e., adsorbed particles) or the chromatographic column hardware. The present disclosure also addresses the problem that long runs are detrimental to high-throughput, intact mass analysis. The present disclosure further provides useful LC and MS methods for achieving mass spectrometry sensitivity to further improve detection.

[0007] In various aspects, the present disclosure provides a chromatography system comprising a chromatography separation device comprising (a) adsorbent particles having a width in a range of 0.5 μm to 100 μm, and (b) a housing comprising an inlet, an outlet, and an interior volume between the inlet and the outlet, the interior volume being (i) filled with adsorbent particles having a length in a range of 5 mm to 50 mm and a width in a range of 50 μm to 10 mm, or (ii) configured to be filled with adsorbent particles having a length in a range of 5 mm to 50 mm and a width in a range of 50 μm to 10 mm, wherein a direction of fluid flow from the inlet to the outlet of the housing is along the length of the volume of the adsorbent particles. The volume of the adsorbent particles is also referred to herein as the bed volume.

[0008] In various embodiments that may be used in conjunction with the above-described aspects, the sorbent particles have a length to width ratio in the range of 10:1 to 2:1, and advantageously in the range of 8:1 to 2:1.

[0009] In various embodiments that may be used in conjunction with the above aspects and embodiments, the sorbent particles are, for example, to range (e.g., to to to to to to to to Porous particles having pore sizes within any range (in other words, within the range between any two of the aforementioned values).

[0010] In various embodiments that may be used in combination with any of the above aspects, the sorbent particles are non-porous particles.

[0011] In various embodiments that may be used in combination with any of the above aspects and embodiments, the sorbent particles are spherical particles.

[0012] In various embodiments that may be used in combination with any of the above aspects and embodiments, the sorbent particles have a width in the range of 1.5 μm to 5 μm.

[0013] In various embodiments that may be used in combination with any of the above aspects and embodiments, the volume of the sorbent particles has a length in the range of 5 mm to 20 mm.

[0014] In various embodiments that may be used in combination with any of the above aspects and embodiments, the volume of the sorbent particles has a width in the range of 1 mm to 5 mm.

[0015] In various embodiments that may be used in combination with any of the above aspects and embodiments, the volume of the sorbent particle is a cylindrical volume (in which case the width of the volume of the sorbent particle corresponds to the diameter of the cylindrical volume).

[0016] In various embodiments that can be used in combination with any of the above aspects and embodiments, the adsorbent particles comprise an organic polymer. In some of these embodiments, the organic polymer comprises at least one hydrophobic organic monomer. Specific examples of hydrophobic monomers include, for example, monofunctional and polyfunctional aromatic monomers (such as styrene and divinylbenzene), monofunctional and polyfunctional olefin monomers (such as ethylene, propylene or butene, polycarbonate monomers, ethylene terephthalate), monofunctional and polyfunctional fluorinated monomers (such as vinyl fluoride, 1,1-difluoroethylene, tetrafluoroethylene, chlorotrifluoroethylene, hexafluoropropylene, perfluoropropyl vinyl ether or perfluoromethyl vinyl ether), monofunctional or polyfunctional acrylate monomers, monofunctional or polyfunctional methacrylate monomers or any combination thereof, etc. In specific embodiments, the organic polymer comprises methyl methacrylate, styrene, divinylbenzene or any combination thereof, and in some embodiments, copolymers of styrene and divinylbenzene are preferred.

[0017] In various embodiments that may be used in combination with any of the above aspects and embodiments, the housing is a chromatography column.

[0018] In various embodiments that may be used in combination with any of the above aspects and embodiments, an inner surface of the outer shell surrounding the volume of the sorbent particle has a water contact angle of less than 60°.

[0019] In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the enclosure surrounding the volume of adsorbent particles is surrounded by a vacuum jacket or a heating unit. Examples of such enclosures, including vacuum jacketed columns and heating columns, are described in U.S. Patent Application No. 2019 / 0317062 to Fogwill et al., which is hereby incorporated by reference.

[0020] For example, in some of these embodiments, the column may include an insulating member and / or a sheath. In various embodiments, the insulating member may be formed by a vacuum chamber surrounding the column. In some embodiments, the insulating member may include a vacuum chamber in which a gas is arranged. In some embodiments, the gas may be any one of helium, hydrogen, neon, nitrogen, oxygen, carbon dioxide, argon, sulfur hexafluoride, krypton, and xenon. In some embodiments, the vacuum chamber may contain atmospheric gases. In some embodiments, the column and the insulating member may be integrated into a single component forming an insulating chromatographic column. For example, a sheath may surround the column and a vacuum chamber may be formed in the region between the column and the sheath. In some embodiments, the sheath may be made of steel. The vacuum chamber forming the insulating member may provide thermal insulation for the column. The insulating member may substantially prevent radial thermal gradients from forming within the column.

[0021] In some of these embodiments, at least a portion of the post may be arranged in the heater, or the post may be arranged outside the heater. Alternatively or in addition, the heater may be arranged in series with the post so that the mobile phase passes through the heater before arriving at the post. In an exemplary embodiment, various sensors (such as temperature sensors) may be provided to monitor the temperature of the mobile phase entering the post and / or leaving the post. In various embodiments that can be used in combination with any of the above aspects and embodiments, the chromatographic system further includes a detector. In various embodiments, the outlet of the chromatographic separation device is directly connected to the detector. In some of these embodiments, the chromatographic separation device and the detector are arranged in a single device. The example of this construction (particularly, the post is directly coupled to a mass spectrometer ion source device (such as an electrospray ionization emitter)) is described in U.S. Patent Application No. 2019 / 0317062 granted to Fogwill et al.

[0022] In various embodiments that may be used in combination with any of the above aspects and embodiments, the detector is a fluorescence detector or a detector based on UV, visible light, and / or near IR light, or the like.

[0023] In various embodiments that can be used in conjunction with any of the above aspects and embodiments, the detector is a mass spectrometer. In some of these embodiments, the mass spectrometer is a quadrupole-based mass spectrometer and / or an ion mobility mass spectrometer and / or a time-of-flight-based mass spectrometer. In certain embodiments, the mass spectrometer can be a quadrupole-based hybrid high-resolution mass spectrometer (HRMS) or a high-resolution ion mobility mass spectrometer (HDMS, high-definition mass spectrometer).

[0024] In various aspects, the present disclosure provides a method for separating biomolecules, the method comprising: (a) loading an aqueous solution comprising the biomolecules into an inlet of a housing of a chromatographic separation device according to any of the above aspects and embodiments, thereby loading the biomolecules onto adsorbent particles within the interior volume of the housing, and (b) introducing a mobile phase selected from an aqueous acidic mobile phase comprising water and a volatile acid or an aqueous basic mobile phase comprising water and a volatile base into the inlet of the chromatographic separation device in a volume sufficient to elute at least a portion of the loaded biomolecules from the adsorbent in an eluent flow from the outlet of the housing.

[0025] In various embodiments that can be used in conjunction with any of the above aspects and embodiments, the mobile phase further comprises one or more polar organic solvents. Examples of such solvents can be selected from acetonitrile, acetone, tetrahydrofuran, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, dimethyl ether, methanol, and combinations thereof.

[0026] In various embodiments that can be used in combination with any of the above aspects and embodiments, during the elution process, the composition of the mobile phase is changed. In some of these embodiments, during the elution process, the concentration of water in the mobile phase is reduced and the concentration of the polar organic solvent in the mobile phase is increased.

[0027] In various embodiments that may be used in combination with any of the above aspects and embodiments, the temperature of the mobile phase is increased or decreased during the elution process.

[0028] In various embodiments that may be used in combination with any of the above aspects and embodiments, the mobile phase, the chromatographic separation device, or both are heated.

[0029] In various embodiments that can be used in combination with any of the above aspects and embodiments, the mobile phase is an acidic aqueous mobile phase comprising water and a volatile acid. In some of these embodiments, the biomolecule comprises one or more intact proteins. Examples of volatile acids include organic acids (such as formic acid, acetic acid, difluoroacetic acid, trifluoroacetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, glutaric acid) and organic hydroxy acids (such as glycolic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid) and above-mentioned blends, etc.

[0030] In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the biomolecule comprises one or more intact proteins. In some of these embodiments, the volume of the adsorbent particles is in the range of 10 μL to 100 μL, and linear mass spectrometry detection (R) is obtained in samples having between 0.1 ng and 100 ng of protein. 2>0.90). In some of these embodiments, the volume of the adsorbent particles ranges from 1 μL to 10 μL, and linear mass spectrometric detection (R 2 >0.90).

[0031] In various embodiments that can be used in combination with any of the above aspects and embodiments, the mobile phase is an alkaline aqueous mobile phase comprising water and a volatile base. In some of these embodiments, the biomolecule comprises one or more nucleic acid polymers. Examples of volatile bases include ammonium hydroxide and volatile amines, including organic amines such as methylamine, methanediamine, ethylamine, ethylenediamine, diethylamine, diethanolamine, tromethamine, choline, pyrrolidine, pyrrole, piperazine, or pyridine.

[0032] In various embodiments that may be used in combination with any of the above aspects and embodiments, the method for separating biomolecules further comprises analyzing the eluent stream with a detector.

[0033] In various embodiments, the detector is a fluorescence detector.

[0034] In various embodiments, the detector is a mass spectrometer.

[0035] In various embodiments, the detector is a quadrupole-based mass spectrometer. In some of these embodiments, the quadrupole acts as a mass filter. For example, the quadrupole can be used as a mass filter to filter out ions with m / z values ​​less than 1,000, to filter out ions with m / z values ​​of 1,500, or to filter out ions with m / z values ​​less than 1,800 m / z, etc.

[0036] In various embodiments that may be used in combination with any of the above aspects and embodiments, the method for separating biomolecules further comprises subjecting the eluent stream to gas phase ion mobility separation.

[0037] In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the eluent stream contains or is suspected of containing the target analyte, and the method for separating biomolecules further comprises calibrating the mass spectrometer with a lock spray standard. In some embodiments, the lock spray standard is electrosprayed under conditions suitable for producing lock mass ions having m / z values ​​within + / - 50% of the lock mass ion value exhibited by the target analyte. In certain embodiments, the lock spray standard is electrosprayed under conditions suitable for producing lock mass ions between 1,000 m / z and 10,000 m / z, more particularly between 2,000 m / z and 4,000 m / z.

[0038] Other aspects of the present disclosure relate to a kit comprising a chromatographic separation device comprising (a) an adsorbent particle according to any of the above aspects and embodiments, the adsorbent particle being formed of an organic polymer and having a width in the range of 0.5 μm to 100 μm, and (b) a housing comprising an inlet, an outlet, and an interior volume between the inlet and the outlet, the interior volume being filled with a sorbent having a length in the range of 2 mm to 50 mm (e.g., any range of 2 mm to 5 mm to 10 mm to 20 mm to 30 mm to 40 mm to 50 mm) and a width in the range of 100 μm to 10 mm (e.g., 100 μm to 200 μm). The volume of the housing is configured to be filled with adsorbent particles having a length in the range of 2 mm to 50 mm (e.g., any range of 2 mm to 500 μm to 1 mm to 2 mm to 5 mm to 10 mm), or a volume configured to be filled with adsorbent particles having a length in the range of 2 mm to 50 mm (e.g., any range of 2 mm to 5 mm to 10 mm to 20 mm to 30 mm to 40 mm to 50 mm) and a width in the range of 100 μm to 10 mm (e.g., any range of 100 μm to 200 μm to 500 μm to 1 mm to 2 mm to 5 mm to 10 mm), wherein the direction of fluid flow from the inlet to the outlet of the housing is along the length of the volume of adsorbent particles.

[0039] In various embodiments that may be used in combination with any of the above aspects and embodiments, an inner surface of the outer shell surrounding the volume of the sorbent particle has a water contact angle of less than 60°.

[0040] In various embodiments that can be used in conjunction with any of the above aspects and embodiments, the kit further comprises one or more vials. In some of these embodiments, the inner surface of the one or more vials has a water contact angle of less than 60°. In certain embodiments, the vials comprise an inner surface comprising an O2-plasma treated polymer surface, such as an O2-plasma treated polyolefin (e.g., a polyolefin such as polyethylene and polypropylene, etc.) vial.

[0041] Other aspects and embodiments will become apparent to those skilled in the art upon reading the detailed description and the following claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Chromatographic separation data are presented for the recovery of anti-citrinin murine mAb protein obtained on different stationary phases as observed using a 2.1 x 50 mm column.

[0043] Figure 2 Presented are the effective peak capacity values ​​of the anti-citrinin murine mAb relative to the NIST mAb as observed using columns of varying lengths with a poly(styrene-co-divinylbenzene) (PS-DVB) stationary phase.

[0044] Figures 3A-3D The extracted ion chromatogram (XIC) ( Figure 3A ), sub-nanogram detection MS data from NIST reference material 8671 ( Figure 3B , raw MS data; Figure 3C , deconvoluted MS data) and a calibration curve plotting the mass loading of NIST mAb from 0.025 ng to 200 ng ( Figure 3D )'s wide dynamic detection range of a 2.1×15 mm PS DVB column.

[0045] Figures 4A-4C Indicates that if a column with a length of less than 1 mm ID and 5 cm is used, a thermal gradient ( Figure 4A ) and elution gradient ( Figure 4B ) for 1 ng mass loading and separation of trastuzumab using BSA as carrier protein ( Figure 4C ) obtained using a thermal gradient combined with an elution gradient.

[0046] Figures 5A-5B Shown in the case of active preheating and column heating ( Figure 5A ) and in the case of active preheating without column heating ( Figure 5B ) Comparison of LCMS chromatographic selectivity for the separation of anti-citrinin murine mAb using a 2.1×15 mm PSDVB column with a mass load of 100 ng.

[0047] Figures 6A-6B Presents the use IMS QToF mass spectrometer and use automatic configuration ( Figure 6A ) and manually configured quadrupoles ( Figure 6B ) Set up for comparison of mass spectra as detected by infusion of anti-citrinin mAb.

[0048] Figures 7A-7C Figure 5 shows the comparison of ion mobility spectra of a co-eluting mixture of trastuzumab (25 ng mass load) and bovine serum albumin. Figure 7A ) and amplified spectrum ( Figure 7B ) and after IMS extraction using amplified spectra ( Figure 7C ) shows the mass spectrum of trastuzumab.

[0049] Figure 8 Presented is the mass spectrum of enolase T37 obtained from the total ion chromatogram of the locked spray channel during the separation of anti-citrinin murine mAb. DETAILED DESCRIPTION

[0050] Many different types of columns and adsorbent particles can be used for chromatographic separation and analysis of proteins. In various embodiments, a column or device that exhibits minimal secondary interactions may be beneficial. Although not limited to theory, it is believed that during the analysis of low mass loads, secondary interactions can have a negative impact on peak shape and peak recovery. If there are high-affinity secondary interaction sites, such as those consisting of both ion binding sites and hydrophobic binding sites, an undesirable large amount of protein may be adsorbed too strongly, and peak shape and recovery may be affected. The inventors have found ways to minimize these effects. The first way is to use adsorbent particles formed from organic polymers, and the second way is to use no more adsorbent particles in the column than are required to provide sufficient retention and selectivity to distinguish the target analyte. In various embodiments, it is desirable to use adsorbent particles with a sufficiently large pore size to allow the protein of interest to diffuse through. In various embodiments, adsorbent particles formed from organic polymers are selected that are suitable for use under high temperature and acidic conditions, because protein LC-MS is typically run at high temperatures using hydrophobic acidic mobile phase additives for ion pairing. Organic acids, including formic acid (FA), can be used in bioanalysis of proteins to provide high MS sensitivity, but weaker acids with lower resolving power than trifluoroacetic acid (TFA) can also be used effectively.

[0051] A variety of different adsorbent particles have been used in protein bioanalysis. However, the present inventors have discovered that the performance of these adsorbent particles can vary significantly depending on the application and the desired detection limit. For example, as described above, for very low detection limits, it has been shown that minimizing secondary interactions can be important. Analysis of silica, organosilica, and polymeric adsorbent particles has shown that adsorbent particles containing silanols can be detrimental to trace level detection of protein analytes (see Example 1 below). While organosilica has shown itself to be a better choice than 100% silica particles, in various embodiments, 100% polymer-based adsorbent particles are superior. In some embodiments, the adsorbent particles are based on polymers containing styrene, divinylbenzene, methacrylate, methyl methacrylate, or mixtures thereof. These adsorbent particles can be non-porous in nature, or can be porous or superficially porous, containing to Advantageously to These adsorbent particles may also exhibit a particle size of 1 μm to 100 μm, more desirably 1.5 μm to 5 μm.

[0052] While not limited to theory, it appears that the number of secondary interactions in a column arrangement is proportional to the amount of stationary phase or the size of the column bed, which in turn affects protein peak shape and recovery under conditions of low mass loading. To demonstrate this, the inventors have compared separations using column arrangements of 2.1 mm ID x 5 mm, 10 mm, 15 mm, 20 mm, and 50 mm lengths (see Example 2 below). From 10 mm to 50 mm length, the separation resolving power appears comparable, and there is a significant reduction in resolution using only the 5 mm arrangement. Therefore, in certain preferred embodiments, packed columns having an internal diameter size of 100 μm to 4.6 mm ID and a length in the range of 5 mm to 50 mm, more preferably 10 mm to 20 mm, are used.

[0053] In theory, adsorbent particles with large pores combined with sufficiently small particle size should provide improved separation performance and detection capabilities. In this regard, the present inventors have found that by studying the porosity of polymer polystyrene-divinylbenzene (PS-DVB) particles, greater than A pore size of 3 μm can provide resolution benefits. In addition, smaller particle sizes may also be beneficial. PS-DVB particles have been shown to provide The particles have higher peak capacities and have been shown to demonstrate low limits of detection (as low as 0.025 ng mass loading for a pure solution of NIST mAb without the use of a carrier protein), indicating that these adsorbent particles provide a good balance of particle size and pore size for protein bioanalysis (see Example 3 below). In some embodiments, a column device with a 10 μL to 100 μL void volume chamber is filled with 1 μm to 5 μm diameter adsorbent particles and used to achieve linear mass spectrometric detection (R) between 0.1 ng and 100 ng of protein. 2 In other embodiments, a column device having a 1 μL to 10 μL void volume chamber is filled with adsorbent particles of 1 μm to 5 μm diameter and used to achieve linear mass spectrometric detection (R) between 0.01 ng and 10 ng of protein. 2 >0.90).

[0054] Use the chromatographic column with specially short sorbent particle bed, liquid chromatography technique has also been optimized.For example, column temperature can be used to improve sensitivity and improve peak shape, accelerate running time and elution analyte (such as protein or other biomolecules).The inventor has found that the short column of the chromatographic material with reduction is more sensitive to temperature change than long column or the column containing more stationary phase materials, and can arrive high temperature faster and cool down from high temperature.By combining thermogradient with mobile phase composition gradient, it is possible to obtain enhanced selectivity, resolution and the sensitivity (referring to embodiment 4) of protein analyte.In some embodiments, set temperature can be combined with multiple isocratic mobile phase composition distribution, or thermogradient can be combined with multiple isocratic mobile phase composition distribution coupling, or multiple isocratic temperature distribution can be combined with constant mobile phase composition coupling, or multiple isocratic temperature distribution can be combined with mobile phase composition gradient coupling, or multiple isocratic temperature distribution can be combined with multiple isocratic mobile phase composition distribution coupling, this may be useful in the separation (for example, relating to those separations of carrier protein) of relating to multiple protein substances.

[0055] In other embodiments, isocratic gradients or thermal gradients are used, showing that by using active preheating alone, or simply heating the mobile phase when the mobile phase enters the post, selectivities equivalent to or even advantageous for separations using a column heater can be produced (see Example 5). By avoiding the use of a column heater, active preheating can be used to directly couple the chromatographic column to the detector inlet. In this way, sensitivity can be improved by reducing the piping leading to the detector and therefore minimizing dispersion. In addition, a short bed length column directly integrated into the MS interface with optimized temperature control can minimize dispersion and even further increase MS sensitivity. Relatedly, with or without optimized temperature control, coupling a fluorescence detector directly or integrated into a short bed length column device can also improve feasible FLR sensitivity below nanogram quantification.

[0056] It has also been found that problematic sample loss can occur when monoclonal antibodies and antibody drug conjugates are serially diluted into glass sample containers. To mitigate these sample losses, O plasma-treated polypropylene vials can be used. Furthermore, lower detection limits and improved linearity can be obtained over a wider dynamic range (see Example 3). Polymer or vapor deposition-coated sample containers, mobile phase flow paths, column hardware, and detector components can also improve analyte recovery.

[0057] In order to accurately detect low concentrations of protein analytes, a high-sensitivity MS can be used in combination with the above-mentioned column technology and liquid chromatography conditions. In particular, using a quadrupole-based hybrid mass spectrometer, analysts can increase sensitivity by operating the quadrupole as a mass filter that is selective only for high m / z ions. Therefore, quadrupole tuning can be very useful in optimizing protein detection by HRMS, especially when it is used to achieve low detection limits. For peptide and protein analysis, a quadrupole time-of-flight mass spectrometer (QTOF MS) can be used. The quadrupole manages the transmission of ions based on the RF and DC voltages applied to its rods, where the movement of ions can be characterized by the Mathieu equation. By adjusting these voltages, ions can pass through the quadrupole or collide with the rods, thereby being discharged without being detected. This selective filtering of ions can be used to tune ions of lower m / z or alternatively tune ions of higher m / z.

[0058] To date, HRMS protein quantification has been performed using the automatic setting of the quadrupole, which provides full scans and transmissions of both low and high m / z ions. However, the inventors have observed that tuning the quadrupole of the hybrid instrument has significant advantages, so that it only transmits ions corresponding to the natural charge state distribution of the intact protein analyte of interest. In fact, this greatly improves mass spectrometry sensitivity and detection. For example, when infusing and detecting anti-citrinin murine mAb, tuning the quadrupole to transmit only 2,000m / z to 5,000m / z ions significantly improved the signal-to-noise ratio (see Example 6). Furthermore, this allows for higher sensitivity to be achieved when running LC-MS separations, as the inventors have shown that the extracted ion chromatogram (XIC) peak area increases by two times. In some embodiments, the method of the present disclosure is therefore combined with a quadrupole mass filtration that neutralizes and filters out ions with m / z values ​​less than 1,000, beneficially less than 1,500 and more beneficially less than 1,800m / z.

[0059] The present inventors have also studied complementary gas phase separation and data processing software. Using these techniques, gas phase ion mobility separation can be combined, which can distinguish protein conformations and key analytes based on their collision cross sections and millisecond time scale separations. When ion mobility is added between LC separation and MS detection (such as can be done with an (IMS)-QTOF mass spectrometer with ion mobility spectroscopy capability (such as When IMS-QTOF (Waters Corporation, Milford, MA) is used, the drift time analysis of ions can be used to improve the selectivity of the separation. Mass spectrometers are able to resolve trastuzumab and albumin (bovine serum albumin) based in part on differences in their gas phase ion mobility (see Example 7). The ion mobility resolution is lower than that of other commercially available IMS instruments (such as the SYNAPT G2-Si system (Waters Corporation)). Therefore, it is reasonable to assume that the benefits of IMS will become even more apparent when using some other types of instruments. In some embodiments, a cyclic ion mobility separator can be used to further increase the IMS resolution. In various embodiments, the method of the present disclosure includes using ion mobility separation to filter out ions with undesirable drift times or to facilitate more thorough data analysis. In this regard, various algorithms can be used to deconvolute the charge state distribution into a single uncharged ion peak. Quantification can then be applied to the deconvoluted data. Alternatively, the signal can be quantified in the form of a single charge state, multiple combined charge states, or extracted ion chromatograms. Generally speaking, the common practice is to use raw data and measure the intensity or extracted ion chromatogram corresponding to the highest charge state of the protein analyte; however, this sacrifices ion signal and selectivity relative to noise. Maximum entropy has been the main example of a processing tool for deconvolution, and other algorithms, such as those based on parsimony analysis, have also been used. Optimizing deconvolution signal processing can produce even more accurate protein quantification.

[0060] Mass calibration techniques can also be employed in some embodiments. Accurate mass measurements can be obtained by running a reference or lock spray standard along with the analyte. However, the standards used are typically those that yield much lower m / z values ​​than would be expected based on electrospray of intact proteins. To provide a more accurate calibration, reference compounds with MS profiles within the range of the target analyte can be used. Therefore, in the present disclosure, lock spray compounds and electrospray conditions have been specifically selected to yield a more appropriate lock spray signal. In one embodiment, the inventors selected the enolase T37 peptide as the lock spray standard because its molecular weight is 2827.3 g / mol, which would yield an m / z of 2828.3 in the +1 charge state. Other compounds containing values ​​within the m / z window of interest are also considered, including but not limited to melittin, ubiquitin, and insulin. For enolase T37, it is optimal to electrospray the peptide at a dilute concentration starting with a mixture of 10 mM ammonium acetate and 0.2% ammonium hydroxide. In Example 8, an optimized quadruple setup was also used to increase sensitivity for ions in the +1 charge state. Using vapor from an acid or base (such as triethylamine) to change the desolvation gas flow can also further optimize the 1+ charge state ions and even provide a charge state reduction advantage for the MS mass of biomolecules. In some embodiments, the electrospray lock spray compound is sprayed under appropriate conditions to produce lock mass ions between 2,000 m / z and 4,000 m / z. In some embodiments, the electrospray can be exchanged with the effluent of the column device, for example using a baffle, so that a more accurate real-time mass calibration can be achieved.

[0061] Utilize the optimization of column device, liquid chromatography gradient condition, mass spectrometer method and / or treatment algorithm, present disclosure provides the detailed method that can be used for using LC-MS quantitative analysis protein.Can be paired with the device of wide-pore polymer stationary phase with specially small bed volume, so that improve protein recovery, provide high throughput capability and provide better peak shape in wider mass loading dynamic range.Join with quadruple and lock spray tuning, can realize higher MS sensitivity, lower detection limit and more reliable peak detection.In other embodiments, these devices can be used for separating other biomolecules (such as nucleic acid polymers), for separating and analyzing small molecules (in this case, can adopt the adsorbent particles of smaller pore size) or can be used for other applications, such as for other applications such as protein titration analysis, protein desalination, multidimensional analysis.

[0062] Example 1: Protein recovery using different adsorbent particles

[0063] Anti-citrinin murine mAb was obtained as a Waters Intact mAb Mass Check Standard. The contents of the vial were reconstituted in 0.1% (w / v) formic acid in water and serially diluted using the same sample diluent. This sample was analyzed using a Waters Corporation ACQUITY UPLC H-Class Bio LC system and the separation method outlined below. Figure 1 Chromatographic data obtained with various RPLC columns are presented.

[0064] LC conditions

[0065] Column: Surface porous C4, 2.7μm, 2.1×50mm

[0066] The surface is porous, 2.7μm Polyphenyl BioResolve TM RP mAb polyphenyl column), 2.1×50 mm

[0067] Organic silica 3.5μm (Waters BEH (Ethylene Bridged Hybrid) Technology TM ), 2.1×50mm

[0068] Polymer PS-DVB 4μm, 2.1×50mm

[0069] Polymer PS-DVB 5μm, 2.1×50mm

[0070]

[0071]

[0072] Gradient Table :

[0073]

[0074] Example 2: Peak Capacity with Varying Column Length

[0075] Anti-citrinin murine mAb was obtained as a Waters Corporation Intact mAb Mass Check Standard. NIST mAb was obtained as a Waters Corporation Humanized mAb Mass Check Standard. The contents of the vial were reconstituted in 0.1% (w / v) formic acid in water. These samples were analyzed using a Waters Corporation ACQUITY UPLC H-Class Bio LC System and the separation method outlined below. Figure 2 The resolution of the separation as obtained with columns of various lengths of polymeric PS-DVB stationary phase is presented.

[0076] LC conditions:

[0077]

[0078] Gradient Table :

[0079]

[0080] Example 3: MS with dynamic detection range

[0081] NISTmAb was obtained as a Waters Corporation Humanized mAb Mass Check Standard and reconstituted in 0.1% (w / v) formic acid in water. Serial dilutions were performed using the same sample diluent. Quantification was performed using a Waters Corporation ACQUITY UPLC H-Class Bio LC system and Analysis of these samples was performed using an IMS-QTOF mass spectrometer and the separation method outlined below. Figures 3A-3D The dynamic range of the separation as obtained with the short prototype polymer PS-DVB stationary phase is presented.

[0082] LC conditions:

[0083]

[0084] Gradient Table :

[0085]

[0086]

[0087] MS conditions:

[0088]

[0089] Example 4: Thermal Gradient

[0090] Trastuzumab was spiked into rat plasma and immunoprecipitated using Protein A. Trastuzumab was released from the antibody and BSA was added at a concentration of 0.1 mg / mL. Figure 4 shows data collected using a thermal gradient combined with an elution gradient as obtained using a sub-1 mm ID, 5 cm long column setup.

[0091] LC conditions:

[0092]

[0093]

[0094] Example 5: MS with direct column connection

[0095] Anti-citrinin murine mAb was obtained as a Waters Corporation Intact mAb Mass Check Standard. The contents of the vial were reconstituted in 0.1% (w / v) formic acid in water. Analysis was performed using a Waters Corporation ACQUITY UPLC H-Class Bio LC system and the separation method outlined below. Figure 5 presents the resolution of the separation achieved using active preheating in conjunction with column heating and using active preheating alone, as with a short prototype polymer PS-DVB stationary phase.

[0096] LC conditions:

[0097]

[0098] Gradient Table :

[0099]

[0100] MS conditions:

[0101]

[0102] Example 6: MS with quadruple tuning

[0103] Anti-citrinin murine mAb was obtained as a Waters Corporation Intact mAb Mass Check Standard and purified over Protein A to a 2.5 mg / mL solution in 10 mM ammonium acetate. The sample was diluted to 0.1 mg / mL with water and infused into the MS using the MS settings outlined below. Figure 6 presents a comparison of mass spectra obtained using the automated and manual quadrupole options under the MS curve.

[0104] MS conditions:

[0105]

[0106] Example 7: MS with ion mobility

[0107] Trastuzumab spiked into mouse plasma at 5 μg / mL was immunopurified using a goat anti-human Fc capture antibody. Trastuzumab was released from the antibody using 0.1% TFA and BSA was added at a concentration of 0.1 mg / mL. Figure 7 shows a comparison of data collected with and without ion migration during data acquisition.

[0108] LC conditions:

[0109]

[0110] Gradient Table :

[0111]

[0112] MS conditions:

[0113]

[0114] Quadrupole Configuration Options: Automatic

[0115] Example 8: MS with targeted locking spray

[0116] Enolase T37 peptide was obtained from New England BioLabs (Ipswich, MA) and reconstituted in 50:50 water:acetonitrile modified with 0.1% formic acid. The sample was diluted to 0.1 mg / mL with 10 mM ammonium acetate modified with 0.2% ammonium hydroxide and analyzed using the method outlined below and a Waters Corporation ACQUITY UPLC H-Class Bio LC system. The IMS-QTOF mass spectrometer was infused as a lock spray reference standard for the isolation of anti-citrinin murine mAb.Anti-citrinin murine mAb was obtained as a Waters Corporation Intact mAb Mass Check Standard and reconstituted in 0.1% (w / v) formic acid in water. Figure 8 Presented is the mass spectrum of enolase T37 obtained from the total ion chromatogram of the locked spray channel during the separation of anti-citrinin murine mAb.

[0117] LC conditions:

[0118]

[0119] Gradient Table :

[0120]

[0121] MS conditions:

[0122]

[0123]

Claims

1. A method for isolating and analyzing one or more intact proteins, the method comprising: A sample comprising the one or more intact proteins is introduced into an inlet of a chromatographic separation device comprising (a) adsorbent particles formed from an organic polymer formed from one or more monofunctional or polyfunctional aromatic monomers and having a width in the range of 0.5 μm to 100 μm, and (b) the chromatographic separation device comprises an inlet, an outlet, and an interior volume between the inlet and the outlet, the interior volume being filled with a volume of adsorbent particles, wherein the interior volume has a length in the range of 2 mm to 50 mm and a width in the range of 50 μm to 10 mm, wherein a direction of fluid flow from the inlet to the outlet is along the length of the volume of adsorbent particles, and wherein a ratio of the length to the width of the volume of adsorbent particles is in the range of 10:1 to 2:1, thereby loading the one or more intact proteins onto the adsorbent particles; and introducing an acidic mobile phase comprising formic acid, acetic acid, and / or a fluorinated carboxylic acid into the inlet of the chromatographic separation device in a volume sufficient to elute at least a portion of the loaded one or more intact proteins from the adsorbent in an eluent stream from the outlet of the chromatographic separation device; and The eluent stream was analyzed with a mass spectrometer calibrated with a lock spray standard electrosprayed under conditions suitable to produce lock mass ions between 2,000 and 4,000 m / z.

2. The method of claim 1, wherein the one or more monofunctional or polyfunctional aromatic monomers comprise styrene and divinylbenzene.

3. The method according to any one of claims 1 to 2, wherein the adsorbent particles are porous particles or superficially porous particles having a pore size in the range of 300Å to 5000Å.

4. The method of claim 1, wherein an interior surface of the interior volume has a water contact angle of less than 60°. The method according to claim 1 , wherein the mobile phase further comprises one or more polar organic solvents. The method of claim 1 , wherein the composition of the mobile phase is changed during the elution process.

7. The method of claim 1, wherein the mobile phase further comprises one or more polar organic solvents and water.

8. The method according to claim 7, wherein during the elution process, the concentration of the water in the mobile phase decreases, and the concentration of the polar organic solvent in the mobile phase increases.

9. The method of claim 7, wherein the temperature of the mobile phase is increased during the elution process.

10. The method of claim 7, wherein during the elution process, (a) a constant temperature profile is combined with a multi-isocratic mobile phase composition profile, (b) a temperature gradient is combined with a multi-isocratic mobile phase composition profile, (c) a multi-isocratic temperature profile is combined with a constant mobile phase composition, (d) a multi-isocratic temperature profile is combined with a mobile phase composition gradient, or (e) a multi-isocratic temperature profile is combined with a multi-isocratic mobile phase composition profile.

11. The method of claim 1, wherein the mass spectrometer is a quadrupole-based mass spectrometer, and wherein the quadrupole serves as a mass filter.

12. The method of claim 11, wherein the quadrupole is used to filter out ions with m / z values ​​less than 1,000.

13. The method of claim 1 , wherein the volume of the adsorbent particles is in the range of 10 μL to 100 μL, and linear mass spectrometric detection is obtained in an injected sample having between 0.1 ng and 100 ng of the one or more intact proteins, R 2 >0.90, or wherein the volume of the adsorbent particles is in the range of 1 μL to 10 μL and linear mass spectrometric detection is obtained in an injected sample having between 0.01 ng and 10 ng of the one or more intact proteins, R 2 >0.

90.

14. A kit comprising: A chromatographic separation device comprising (a) adsorbent particles formed from an organic polymer and having a width in the range of 0.5 μm to 100 μm, the organic polymer being formed from one or more of styrene, methacrylate, methyl methacrylate, or a mixture thereof, and (b) a housing comprising a housing inlet, a housing outlet, and an interior volume between the housing inlet and the housing outlet, the interior volume being filled with, or configured to be filled with, the volume of the adsorbent particles having a length in the range of 2 mm to 50 mm and a width in the range of 50 μm to 10 mm, wherein the adsorbent particles are formed from one or more of styrene, methacrylate, methyl methacrylate, or a mixture thereof. a direction of fluid flow from the housing inlet to the housing outlet along the length of the volume of sorbent particles, and wherein a ratio of the length to the width of the volume of sorbent particles is in the range of 10:1 to 2:1, and (b) a housing comprising an inlet, an outlet, and an interior volume between the inlet and the outlet, the interior volume being filled with, or configured to be filled with, a volume of sorbent particles having a length in the range of 2 mm to 50 mm and a width in the range of 100 μm to 10 mm, wherein the direction of fluid flow from the inlet to the outlet of the housing is along the length of the volume of sorbent particles; and A container for an acidic solution comprising formic acid, acetic acid, and / or a fluorinated carboxylic acid.

15. The kit of claim 14, wherein an interior surface of the interior volume of the housing has a water contact angle of less than 60°.

16. The kit of claim 14, further comprising one or more vials, wherein an inner surface of the one or more vials has a water contact angle of less than 60°.

17. The kit of claim 14, further comprising one or more vials, wherein the one or more vials are O2-plasma treated polyolefin vials.

18. The kit of claim 17, wherein the polyolefin is polyethylene or polypropylene.

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