Multistage instrument for automated sample preparation and online peptide mapping analysis

Abstract

The present invention relates to a multistage apparatus comprising an injector module, a sample module having a sample tray for holding one or more sample vials, a buffer exchange module having a buffer exchange column, a digestion module having a digestion column downstream of the buffer exchange module, and a separation module having a separation column downstream of the digestion module. The injector module has a needle for extracting a sample from one sample vial and injecting it into another sample vial, and for extracting a sample from a sample vial and injecting it into the buffer exchange module. The sample module is configured to control the temperature of the sample vials. During use, the sample can be reacted by using the needle to introduce reagents and samples into the vials in the sample tray and controlling the temperature before they are injected into the buffer exchange module. The apparatus of the present invention enables the analysis of multiple product attributes in a single, integrated instrument while reducing manual operation and increasing sample throughput compared to known practice methods and other state-of-the-art methods.

Description

Technical Field

[0001] Priority This application claims the priority of European Patent No. 23171877.6, and the entire content thereof is incorporated herein.

Background Art

[0002] Background Proteins such as antibodies are essential and complex biomolecules that play important roles in many biological processes. They consist of one or more peptide chains composed of amino acids that can fold into complex structures and enable them to perform diverse functions within an organism.

[0003] Monoclonal antibodies (mAbs) are powerful proteins that can cure severe diseases and improve patient outcomes. Therefore, mAbs are playing an increasingly important role in the fields of medicine, biotechnology, and the biopharmaceutical industry.

[0004] Understanding the structure of proteins such as mAbs is essential for the development and manufacture of new drugs.

[0005] Generally, multiple attributes (or characteristics) of complex products such as proteins (e.g., mAbs) are necessary to define and characterize the product. The multi-attribute method (MAM) describes an analytical method that has the ability to monitor multiple product characteristics simultaneously. By this approach, MAM can replace a collection of conventional methods that analyze one attribute at a time. In MAM, the required analytical tests are reduced, thereby reducing the workload and potentially the cost, while particularly accelerating the approval process for biopharmaceuticals.

[0006] Therapeutic proteins such as mAbs are heterogeneous products and can change unintentionally during production, downstream processing, formulation, storage, and administration. Structural changes and chemical modifications that affect the safety or efficacy of the product are considered important quality attributes (CQAs).

[0007] To ensure patient safety, it is crucial to verify the reliability and consistency of biopharmaceuticals such as mAbs throughout their entire product lifecycle through quality control. For example, the U.S. Food and Drug Administration (FDA) recommends characterizing and monitoring critical quality attributes (CQAs) to monitor and verify the adequate quality of biopharmaceuticals.

[0008] In quality control (QC) analysis, classical chromatography or electrophoresis (e.g., ion exchange chromatography) is the primary method used for release and stability testing. Such methods cannot obtain site-specific information about PTMs at the peptide level.

[0009] Liquid chromatography-mass spectrometry (LC-MS) based peptide mapping analysis is the preferred method in industry and academia for characterizing protein structures, monitoring modifications, and confirming identity.

[0010] This technique involves breaking down a protein into its constituent peptides, separating them by liquid chromatography, and then analyzing them using mass spectrometry. This technique allows for the direct identification and relative quantification of modifications, such as post-translational modifications (PTMs or CQAs), at the peptide level, enabling the acquisition of site-specific information.

[0011] Peptide mapping analysis can be considered a multi-attribute method (MAM) due to its ability to monitor multiple product characteristics (sequence variants, oxidative variants, charge variants, sugar variants) within a single analysis. However, the preparation of peptide mapping samples is labor-intensive and time-consuming. Manual or separate robotic sample preparation is commonly performed and is a bottleneck in known peptide mapping analytical methods. Furthermore, manual sample preparation is inefficient and prone to (human) error, which can lead to method-induced corrections.

[0012] To provide more drugs and simultaneously reduce the cost of public healthcare, it is desirable to provide improved methods for characterizing proteins such as mAbs and / or improved methods that can be used for CQA and monitoring of other modifications in a QC environment.

[0013] The present invention aims to solve one or more of the above problems. In particular, the present invention provides a multi-stage apparatus that can prepare and analyze multiple attributes of a sample such as an mAb (peptide mapping) in a time-efficient manner with minimal on-site input. The present invention may also provide a method that can be easily adapted to a QC environment. In this way, the present invention can provide rapid and cost-effective analysis of biopharmaceuticals. [Overview of the project]

[0014] Summary of the Invention The present invention provides a multi-stage apparatus for characterizing samples such as protein samples, for example, antibodies, for example, therapeutic monoclonal antibodies.

[0015] The multi-stage device of the present invention is An injector module having a needle, A sample module having a sample tray for holding one or more sample vials, A buffer exchange module having a buffer exchange column, A digestion module having a digestion column downstream of a buffer exchange module, A separation module having a separation column downstream of the digestion module, It holds.

[0016] The injector module is configured so that a needle can extract a sample from one sample vial and inject it into another sample vial, and also extract a sample from a sample vial and inject it into the buffer exchange module. The sample module is configured to control the temperature of the sample vials. During use, the needle is used to introduce reagents and samples into vials in the sample tray, the temperature is controlled, and the sample can be reacted by ensuring that a reaction occurs before the reacted sample is extracted and injected into the buffer exchange module.

[0017] For example, reacting a sample in an automated, offline manner (i.e., not in a column) by reduction means that the sample can be efficiently prepared for digestion.

[0018] In this way, the apparatus of the present invention enables the analysis of multiple product attributes in a robust and efficient manner using a single, integrated instrument. The apparatus of the present invention reduces sample handling compared to known peptide mapping (multi-attribute) methods. The apparatus of the present invention also significantly increases sample throughput compared to known practical methods and other modern technologies.

[0019] The present invention also provides a method for analyzing a sample using any one of the multistage apparatuses described in the preceding sections. In some embodiments, the method is: i. A step of extracting a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module using a needle, ii. The step of extracting a portion of the first sample to be analyzed from the sample vial in the sample module using a needle, iii. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, and a portion of the first sample to be analyzed into a reaction vial in the sample module, iv. A step of controlling the temperature of the reaction vial using a sample module so that the reaction can occur, v. After the reaction occurs, using a needle to extract a part of the reacted sample and injecting the reacted first sample into a buffer exchange module; vi. Flowing the reacted first sample through a buffer exchange column, preferably a reverse phase column, into a digestion module; vii. Digesting the sample on the digestion column and flowing the digested sample into a separation module; viii. Separating the digested sample for analysis. It includes.

Brief Description of the Drawings

[0020] [Figure 1] It shows a schematic diagram of the flow of an embodiment of the present invention. [[ID=I8]] [Figure 2] It shows an embodiment of the multi-stage device of the present invention. Different figures show different configurations of the device at different stages of the method. Figure 2 is divided into two pages and shows a total of five configurations. [Figure 3] It shows an embodiment of the sample module of the present invention. Figure 3A shows the sample module as an overall unit, and Figure 3B shows the sample module separated into different parts. [Figure 4] It shows the total ion chromatogram of mAB1 prepared and analyzed by the multi-stage system ("iSAP-LC-MS") of the present invention. [Figure 5] It shows the data of the linearity test. Figure 5A shows the data at the degree of T22 oxidation, Figure 5B shows the data at the degree of T42 oxidation, and Figure 5C shows the data at the degree of T38 deamidation. [Figure 6] It shows the results of the precision test of the multi-stage system of the present invention, considering T22 oxidation and T38 deamidation. [Figure 7] It shows the results of the robustness test using different digestion columns (7A) and different storage times (7B) considering T22 oxidation. [Figure 8]Figure 8A shows a timescale graph of the method performed using the multistage apparatus of the present invention compared to known methods. Figure 8B shows how this parallel sample preparation provides a time advantage over known methods. Figures 8C and 8D illustrate various environmental and economic aspects of using the multistage apparatus of the present invention and the prior art pipetting robotic method for analytical samples. [Modes for carrying out the invention]

[0021] Detailed explanation The present invention provides a multi-stage apparatus for characterizing samples such as protein samples, for example, antibodies, for example, therapeutic monoclonal antibodies.

[0022] The multi-stage liquid chromatography (LC) apparatus of the present invention is An injector module having a needle, A sample module having a sample tray for holding one or more sample vials, A buffer exchange module having a buffer exchange column, A digestion module having a digestion column downstream of a buffer exchange module, A separation module having a separation column downstream of the digestion module, It holds.

[0023] The injector module is configured so that a needle can extract a sample from one sample vial and inject it into another sample vial, and also extract a sample from a sample vial and inject it into the buffer exchange module. The sample module is configured to control the temperature of the sample vials. During use, the needle is used to introduce reagents and samples into vials in the sample tray, the temperature is controlled, and the sample can be reacted by ensuring that a reaction occurs before the reacted sample is extracted and injected into the buffer exchange module.

[0024] The currently used approach for multi-attribute measurement of mAbs involves field sample preparation or using a separate pipetting robot to react the sample, such as by reduction, and then transferring it (manually) to an LC-MS system for analysis. Compared to such systems, the apparatus of the present invention reduces sample preparation time and laboratorier input when fewer than approximately 100 samples are measured. For mAbs, the number of samples measured is typically around 10-20. Parallel sample preparation and analysis provided by the apparatus of the present invention are shown in Figure 8A. The time savings relative to the number of samples analyzed compared to several common stepwise protocols are highlighted in Figure 8B. The advantageous environmental and economic aspects of the multi-stage system of the present invention are shown by comparing Figures 8C and 8D.

[0025] The ability to prepare samples for digestion within the sample module of the apparatus before injection provides many advantages to the apparatus of the present invention, as described above. The apparatus of the present invention allows the reaction of one sample, such as reduction and alkylation, to occur independently (in an automated manner) within the sample module, while in parallel, another sample passes through the digestion module for digestion and the separation module for peptide mapping analysis. This allows for improved sample throughput compared to state-of-the-art systems. In particular, the apparatus of the present invention enables improved sample throughput compared to such systems that employ reduction using a separate, for example, pipetting robot (see Figure 8A).

[0026] The apparatus of the present invention also reduces the practical time and interaction required by laboratory workers, and therefore reduces human error.

[0027] Furthermore, integrating sample preparation and analysis into a single instrument, as in the present invention, simplifies reporting and system validation. Specifically, only one sequence table and report are generated, saving considerable time in highly regulated GxP environments. This allows the instrument of the present invention to be used more easily in a QC environment than conventional systems or multiple systems used for sample preparation and analysis.

[0028] In this way, the apparatus of the present invention enables the analysis of multiple product attributes in a robust and efficient manner using a single, integrated instrument. The apparatus of the present invention reduces sample handling compared to known multi-attribute methods. The apparatus of the present invention also significantly increases sample throughput compared to known practical methods and other modern techniques.

[0029] The apparatus of the present invention provides these advantages by having a modified integrated injector module and sample module, such as an autosampler module, as well as a buffer exchange module.

[0030] In some embodiments, the multistage apparatus of the present invention further comprises a biocompatible microfluidic mixer. The biocompatible microfluidic mixer may be located between a needle and a valve for injecting a sample into a buffer exchange module, or between the valve and the buffer exchange module. These locations are, for example, in the direction of flow when the biocompatible microfluidic mixer is located between the valve and the buffer exchange module, and the sample to be analyzed passes through the valve, then through the biocompatible microfluidic mixer, and then into the buffer exchange module.

[0031] The microfluidic mixer may have a volume of 20 μL to 100 μL, preferably 30 μL to 40 μL, for example, about 35 μL.

[0032] The mixer allows for the dilution of the sample to be analyzed. In this way, organic solvents and by-products such as salts from the reaction are reduced before the sample is sent to the buffer exchange column.

[0033] In some embodiments, the multistage apparatus of the present invention further comprises an analytical module for analyzing a sample. The analytical module is fluidly connectable to the separation module and is located downstream of the separation module. The analytical module may include a mass spectrometer such as a high-resolution mass spectrometer (HRMS) or a single quadrupole mass spectrometer, an evaporative light scattering detector (ELSD), a UV detector, or a diode array detector (DAD).

[0034] In some embodiments, the multistage device further comprises at least two column ovens, preferably at least three column ovens. Each column oven may have two independent temperature control zones. Preferably, the multistage device comprises at least two column ovens, and each of the at least two column ovens has two independent temperature control zones. In this way, the temperature of one or more components can be controlled.

[0035] The present invention also provides a method for analyzing a sample using any one of the multistage apparatuses described in the preceding sections. In some embodiments, the method is: i. A step of extracting a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module using a needle, ii. The step of extracting a portion of the first sample to be analyzed from the sample vial in the sample module using a needle, iii. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, and a portion of the first sample to be analyzed into a reaction vial in the sample module, iv. A step of controlling the temperature of the reaction vial using a sample module so that the reaction can occur, v. After the reaction has occurred, a needle is used to extract a portion of the reacted sample, and the reacted first sample is injected into the buffer exchange module. vi. The step of passing the reacted first sample through a buffer exchange column, preferably a reversed-phase column, to a digestion module, vii. The step of digesting the sample on the digestion column and flowing the digested sample into the separation module, viii. The step of separating the digested sample for analysis, Includes.

[0036] definition A “multistage apparatus” refers to an apparatus as defined herein, which is a combination of multiple modules. These modules are interconnected to perform sample preparation and sample analysis steps. The modules include an injector module, a sample module, a buffer exchange module, a digestion module, and a separation module, providing distinct “stages” in an analytical method (e.g., a sample preparation stage and a chromatography stage). These modules are essential to and part of the overall apparatus of the present invention. Some modules are liquid chromatography modules (e.g., a buffer exchange module, a digestion module, and a separation module) that include a column through which the sample passes. Different liquid chromatography modules may be connected via capillaries, columns, and / or valve assemblies. Together, these modules provide an (integrated) liquid chromatography (LC) apparatus that can be connected to an analytical module such as a high-resolution mass spectrometer (HRMS) or a single quadrupole mass spectrometer. Different types of columns can be used in different modules to provide a series of chromatography and processing steps, such as a digestion or separation step that “runs” the apparatus once. Some modules are for preparing samples for liquid chromatography (e.g., an injector module, a sample module, and an optional biocompatible fluid mixer module). Each module may also include components such as a pump and waste discharge. Generally, multistage systems also have several valve assemblies for connecting different modules at different times. The valve assemblies may be 2-position, 10-port valves.

[0037] Liquid chromatography, or LC, is an analytical method that uses an LC column to chromatographically separate a sample, for example, to separate a target analyte from matrix components.

[0038] A “liquid chromatograph or LC apparatus” is an analytical instrument or unit within an analytical instrument for performing liquid chromatography. An LC apparatus may also include a sample injector, valves, a liquid source, fluid connections for, for example, mixing liquids, degassing liquids, or temperature-controlled liquids, one or more sensors such as pressure sensors and temperature sensors, and in particular, at least one LC pump. This list is not exhaustive. According to one embodiment, an LC apparatus is an analytical instrument designed to prepare multiple samples for mass spectrometry and / or to move prepared samples to a mass spectrometer in order to separate the analyte of interest before detection by a mass spectrometer.

[0039] A “valve assembly” refers to a multi-port valve component that controls flow between elements connected to a port. This is typically achieved by a switch mechanism that moves one or more valve conduits to switch communication between different elements. One or more components of an element, such as a module, may be fluidly connected to a port via further conduits such as pipes, tubes, capillaries, or microfluidic channels, and also by fittings such as screws / nuts and ferrules, or alternative liquid-tight seals maintained in place by, for example, a clamping mechanism. In this way, the various components of a module may be connected as defined herein. For example, the pump of a capture module may be connected to a capture column via a third valve assembly.

[0040] "Column" refers to any column, cartridge, capillary, etc., suitable for performing chromatography or for performing reactions such as digestion on substances passing through the column. Preferably, the column is a single column. The column is typically packed or loaded with a stationary phase, through which a mobile phase is pumped to capture and / or separate, elute and / or transfer the analyte of interest under selected conditions, for example, according to their polarity or logP value, size or affinity, as is commonly known. This stationary phase may be particulate, bead-like, or porous monolith. The column may be interchangeable and / or operate in parallel or sequentially with one or more other columns.

[0041] The term “sample” is used in this application to refer to a sample to be analyzed and a sample of reagents used in the apparatus of the present invention. The term “sample to be analyzed” refers to a substance suspected to contain one or more analytes of interest. In this case, the sample is preferably a sample of an analytical antibody, particularly a therapeutic monoclonal antibody. The sample to be analyzed can be pre-treated before use. Treatment methods may include filtration, centrifugation, distillation, concentration, inactivation of interfering components, and addition of reagents. Preferably, the reaction of the sample to be analyzed is carried out online in an automated manner using the apparatus and reagents of the present invention.

[0042] Some features of the apparatus of the present invention are defined with respect to their positions relative to other components in the direction of flow (e.g., upstream or downstream). In this case, the direction of flow refers to the flow of liquid, such as a solvent, through the apparatus during operation. For example, if component A is "upstream" of component B, the liquid, such as a solvent, passes component A before component B during the operation of the apparatus. Similarly, if component C is "downstream" of component D, the liquid, such as a solvent, passes component D before component C during the operation of the apparatus.

[0043] Injector module The multistage apparatus of the present invention has an injector module having a needle, the injector module being configured so that the needle extracts a sample from one sample vial and injects it into another sample vial, and extracts a sample from a sample vial and injects it into a buffer exchange module.

[0044] The injector module can be easily controlled by software used to control liquid chromatography equipment (e.g., Chromeleon, Empower, OpenLab).

[0045] In this context, the term "sample" includes reagent samples stored in vials within the sample module and the sample to be analyzed. The reagent samples are used to react with the sample to be analyzed (analyzed).

[0046] In this way, the apparatus of the present invention can extract and react samples from different sample vials. This allows for performing different reactions on separate aliquots of a sample, thereby increasing the amount of information available, for example, by monitoring any changes in the same sample over time.

[0047] The needle can be washed between samples to prevent reagents or samples from being carried over to the next sample. In this way, the apparatus of the present invention helps to significantly reduce the number of disposable consumables required for each sample processed and improve the sustainability of the method.

[0048] In some embodiments, the injector module is a pipette handling robot. For example, the injector module may include a robotic three-axis arm for liquid handling.

[0049] In some embodiments, the injector module is configured to transfer a portion of the sample to a new vial before the reaction occurs.

[0050] In this way, the required amount of sample can be used for analysis without contaminating the sample or without using the entire sample. For example, by having vials in different areas of the sample module, it is also possible to control the temperatures of the "reaction" vial and the "storage" vial (i.e., vials holding the reagent sample and the sample to be analyzed) independently (in combination with the sample module).

[0051] In some embodiments, the injector module is configured to draw a reagent, such as a reducing agent, into the needle, and then draw the sample into the needle. For example, during use, the needle may draw 10 to 50 μL, for example about 31.5 μL, of reagent into the needle, and 1 to 5 μL, for example about 3.5 μL, of sample to be analyzed into the needle. The needle may have a volume of at least 3 μL, for example 3 to 100 μL, preferably at least 20 μL, more preferably at least 40 μL, for example 30 to 60 μL.

[0052] In this way, both the reagent and the sample are present in the needle simultaneously, thereby improving mixing.

[0053] Sample module The multistage apparatus of the present invention has a sample module having a sample tray for holding one or more sample vials. The sample module can hold samples in one or more sample vials. The "sample" may include a sample to be analyzed, such as an mAb sample, and a reagent sample such as a reducing agent or alkylating agent.

[0054] The sample module is adapted to control the temperature of the vial.

[0055] The sample module may be a module for holding a separate vial, such as a glass or plastic vial, or it may hold a multi-well plate, such as a 96-well plate or a 96-deep-well plate. In such cases, the well plate acts as one or more sample vials for holding the sample.

[0056] During use, the reagent and sample can be introduced into vials in the sample tray using a needle, and the sample can be reacted by controlling the temperature before injection into the buffer exchange module. Preferably, during use, the sample to be analyzed, such as an mAb sample, can be reacted by extracting a portion of the sample to be analyzed and a portion of the reagent from their respective sample vials, and introducing the reagent and a portion of the sample to be analyzed into different vials in the sample module using the needle of the injector module. The temperature is controlled by the sample module, and the mixture is reacted before the reacted sample is extracted and injected into the buffer exchange module.

[0057] In this way, the sample module (in combination with the modified injector module) is adapted to allow the sample to be analyzed to react, such as reduction and / or alkylation, within the sample vial before injection. Reagent samples, such as reducing agents and / or alkylating agents, can be injected into the vial containing a portion of the sample to be analyzed by the needle of the injector module. The sample vial in which the reaction occurs can be maintained at a constant high temperature using the sample module to promote reduction and alkylation. The sample vial containing the stock reagents and the sample to be analyzed can be maintained at a lower temperature, for example, to prevent undesirable reactions or decomposition.

[0058] In some embodiments, the sample module comprises two sample trays configured such that the temperature of each sample tray can be controlled independently.

[0059] In this way, vials containing reagents and samples to be analyzed can be kept in one sample tray, for example, which can be kept at a low temperature. The injector module can transfer a portion of the sample to be analyzed and the necessary reagents to vials in other sample trays, for example, which can be kept at a high temperature to accelerate the reaction.

[0060] In some embodiments, the sample module has a heating or cooling device, such as a Peltier element, for controlling the temperature of the vials. Preferably, the sample module has at least two heating or cooling devices, such as two Peltier elements, for controlling the temperature in different areas of the sample module. Preferably, the heating or cooling device is a thermoelectric heating or cooling device, such as a Peltier element. The thermoelectric heating or cooling device works by passing an electric current through the junction of two conductors, resulting in heat being removed at one junction and heat being deposited at the other junction. For cooling applications, the junction from which heat is removed is used to extract heat from the area to be cooled (in this case, the sample vial tray), and the junction from which heat is deposited is located away from the cooled environment and is often kept out of the way using a fan or the like.

[0061] In some embodiments, the sample module includes two sample trays and two heating or cooling devices, such as two Peltier elements. Each sample tray has a corresponding heating or cooling device, such as a Peltier element. In this way, the temperatures of the two sample trays can be controlled independently.

[0062] Heating or cooling elements, such as Peltier elements, can be easily controlled by software used to control (multi-stage) liquid chromatography (LC) modules (e.g., Chromeleon, Empower, OpenLab). They can provide temperature control between 0 and 80°C, for example between 10 and 70°C, and timetables can be set to switch temperatures within a run for specific applications and different incubation temperatures.

[0063] The sample module can control the temperature of one or more sample vials independently of the temperature of the sample tray.

[0064] In some embodiments, the sample tray may hold 54 vials or each tray may have a 96-well plate.

[0065] In some embodiments, the sample module includes sample vials for holding the sample and for holding the reagent.

[0066] The reagent may be a reducing agent, such as tris-(2-carboxyethyl)-phosphine (TCEP) or dithiothreitol (DTT), an alkylating agent such as iodoacetamide, iodoacetic acid (IAA), or N-ethylmaleimide (NEM), an enzymatic reaction agent such as deglycosylation by PNGaseF, or a labeling reagent.

[0067] In some embodiments, the sample module is an autosampler or a multisampler.

[0068] Buffer replacement module The multistage apparatus of the present invention includes a buffer exchange module having a buffer exchange column.

[0069] Including a buffer exchange module upstream of the digestion module is necessary to prepare the reacted sample before it flows into the digestion column. In particular, some of the reduction and / or alkylation byproducts (and any other steps performed in the sample vial) are removed, the buffer is exchanged to make the sample more compatible with the digestion module, and the use of IMER for on-column digestion becomes possible. This extends the instrument's lifespan and makes the data more accurate. Therefore, the use of a buffer exchange module also facilitates high throughput of the instrument and reduces the manual handling of samples before digestion in state-of-the-art systems.

[0070] The buffer exchange column may be a reversed-phase chromatography column. Preferably, the buffer exchange column of the buffer exchange module is a C4, C1, or phenyl stationary phase column or a size exclusion column, and most preferably, the buffer exchange column is a C4, C1, or phenyl stationary phase column.

[0071] The use of reversed-phase columns allows for faster desalting of samples and the use of higher flow rates. Reverse-phase columns can also be used at high temperatures (such as around 80°C) to denature proteins before they move to the digestion module, improving digestion.

[0072] In some cases, the buffer exchange column has a length of 1 to 40 mm, preferably 2 to 10 mm, for example, about 5 mm.

[0073] In some cases, buffer exchange columns have a packing material with an average particle size of 1 to 10 μm, preferably 1.5 to 2.0 μm, for example, about 1.7 μm. The average particle size is typically specified by the column manufacturer. The average particle size can be measured using dynamic light scattering or sieve analysis.

[0074] In some cases, the buffer exchange column has an inner diameter of 1 to 10 mm, preferably 1 to 4 mm, for example, about 2.1 mm.

[0075] In some cases, buffer exchange columns have an average pore size of approximately 100 Å to 1000 Å, preferably 200 Å to 350 Å, for example, about 300 Å. The average pore size is typically specified by the column manufacturer. The pore size can be measured by gas adsorption, for example, using the Brunauer-Emmett-Teller theory.

[0076] Digestive module The multistage apparatus comprises a digestion module having a digestion column containing immobilized proteolytic enzymes for digesting the reduced sample and providing the digested sample. The digestion module is located downstream of the first splitter and is fluid-connectable to the second outlet of the second splitter.

[0077] In some embodiments, the digestion column is selected from a trypsin-immobilized enzyme reactor, a LysC-immobilized enzyme reactor, or an aspN-immobilized enzyme reactor. Preferably, the digestion column is selected from a trypsin-immobilized enzyme reactor or a LysC-immobilized enzyme reactor.

[0078] In some embodiments, the digestion module has a first mixer, such as a static mixer or a zero-delay volume T-piece downstream of the digestion module in the direction of flow. In some such embodiments, the first mixer can be further fluid-connected to a pump in the separation module or capture module, if present, to allow solvent exchange before the sample is sent to the separation or tapping module.

[0079] In some embodiments, the digestion module has a second mixer, such as a static mixer or a zero-delay volume T-piece. In some cases, the second mixer is located before or upstream of the digestion column in the direction of flow, so as to provide a homogeneous mixture before the sample passes through the mixer and is mixed with, for example, an additional solvent before entering the digestion column. The second mixer may be fluidly connectable to a digestion pump if a digestion buffer is pumped through the T-piece to dilute the sample before it enters the digestion column.

[0080] In some embodiments, the digestion module comprises two digestion columns. In some such cases, the two digestion columns are connected in parallel so that the sample flow is split between the two columns when in use.

[0081] In some such cases, the two digestion columns are independently selected from a trypsin-immobilized enzyme reactor and a LysC-immobilized enzyme reactor, preferably the two digestion columns are a trypsin-immobilized enzyme reactor and a LysC-immobilized enzyme reactor.

[0082] A parallel digestion setup offers unique peptide combinations, allowing for, for example, the acquisition of unique trypsin and LysC peptides. This increases the likelihood of post-translational modification (PTM) characterization and enhances sequence coverage. Specifically, using two digestion columns provides a broader range of digested products, enabling greater sequence coverage. This parallel digestion can be particularly advantageous for increasing the number of complex bispecific mAbs.

[0083] In some embodiments, the digestion module includes a digestion pump. The digestion pump may be a binary pump or a quaternary pump.

[0084] Capture module The multistage apparatus of the present invention may further comprise a capture module having a capture column for capturing a digested sample. The capture module is located downstream of the digestion module and upstream of the separation module.

[0085] The capture column is selected to have a stationary phase that holds the analyte of interest, such as digested antibodies, but does not retain any salts, buffers, surfactants, or other matrix components, which can be washed away.

[0086] A capture column enables the decoupling of digestion and separation columns. In protein mapping, a capture column can also help protect the separation column by capturing undesirable components, such as undigested proteins.

[0087] In some embodiments, the capture column has a length of 3 to 30 mm, for example, 5 to 10 mm. In this way, the capture column allows for the capture of the sample and dilution of acetonitrile without significantly increasing the back pressure on the digestion column.

[0088] In some cases, the capture column has a length of 3 to 10 mm, for example, 5 to 10 mm, preferably about 5 mm. In this way, sufficient capture can be achieved and back pressure can be reduced.

[0089] In some cases, the capture column has a length of 25-30 mm. This results in increased capture, which is particularly useful when capture performance is critical.

[0090] In some embodiments, the capture column has a packing material with a particle size of 1.0 to 3.0 μm, for example, 1.0 to 2.0 μm, preferably 1.5 to 1.7 μm.

[0091] In some embodiments, the capture column has an inner diameter of 1.5 to 5 mm. In some cases, the inner diameter is 1.5 to 2.5 mm, such as about 2.1 mm.

[0092] In some embodiments, the capture column has the same packing material as the peptide mapping column. Preferably, the capture column has a C18 stationary phase.

[0093] In some embodiments, the capture module includes a capture pump. The capture pump may be a binary pump or a quaternary pump.

[0094] Separation module The multistage apparatus includes a separation module with a separation column for separating analytes such as peptide mapping samples.

[0095] The separation module is configured to receive the digested sample from the digestion column. In some embodiments, the separation column is not directly fluid-connectable to the digestion column. In such cases, the transfer of the digested sample is carried out via another module, such as the capture module described above.

[0096] In some embodiments, the isolation module includes an isolation pump. The isolation pump may be a binary pump or a quaternary pump. Preferably, the isolation module has a binary pump.

[0097] In some embodiments, the separation column is selected from peptide mapping columns (e.g., UHPLC columns, HPLC columns, reversed-phase chromatography columns, or hydrophilic interaction chromatography columns), and preferably the separation column is a UHPLC column. Preferably the separation column is a UHPLC column.

[0098] In some cases, the separation column has a C18 stationary phase. The C18 stationary phase is typically used for the chromatographic separation of peptides. In this way, good peptide retention and separation can be achieved.

[0099] In some cases, the separation column may have a length of 100-200 mm, such as approximately 150 mm.

[0100] In some cases, the separation column has a packing material with a particle size of 1.0 to 3.0 μm, for example, 1.0 to 2.0 μm, preferably 1.5 to 2.0 μm.

[0101] In some cases, the separation column has an inner diameter of 1.5 to 5 mm, preferably 1.5 to 2.5 mm, for example, about 2.1 mm.

[0102] In some cases, the separation column contains a C18 stationary phase.

[0103] Valve Assembly The multistage device of the present invention may further comprise at least one valve assembly, preferably at least two valve assemblies. The valve assemblies may be configured to connect and disconnect two or more modules during operation.

[0104] In some embodiments, the apparatus has a first valve assembly configured such that the buffer exchange module is fluidly connected to the digestion module in a first position, and the buffer exchange module is not fluidly connected to the digestion module in a second position.

[0105] In some embodiments, the apparatus has a second valve assembly configured such that the capture column and the separation column are fluidly connected in a first position, and are not fluidly connected in a second position.

[0106] In some embodiments, the apparatus comprises a first valve assembly and a second valve assembly. The first and second valve assemblies are configured such that the digestion column and the capture column are fluid-connectable, the capture column and the separation column are fluid-connectable, the separation module and the digestion module are not fluid-connectable, and the separation module and the buffer exchange module are not fluid-connectable.

[0107] In some such embodiments, the buffer exchange column, digestion column, and capture column are fluid-connected when the first valve assembly is in the second position and the second valve assembly is in the first position. The capture column and digestion column are not fluid-connected in any other combination of valve assembly positions. The capture column and separation column are fluid-connected when the second valve assembly is in the second position. The capture column and separation column are not fluid-connected in any other combination of valve assembly positions. The separation module and digestion module are not fluid-connected in any combination of valve assembly positions. The separation module and buffer exchange module are not fluid-connected in any combination of valve assembly positions.

[0108] A “valve assembly” refers to a multi-port valve component that controls flow between elements connected to a port. This is typically achieved by a switch mechanism that moves one or more valve conduits to switch communication between different elements. One or more components of an element, such as a module, may be fluidly connected to a port via further conduits such as pipes, tubes, capillaries, and microfluidic channels, and also by fittings such as screws / nuts and ferrules, or alternative liquid-tight seals maintained in place by, for example, a clamping mechanism. In this way, various components of a module may be connected as defined herein. For example, the pump of a capture module may be connected to a capture column via a first valve assembly.

[0109] The first and second valve assemblies may be any multiport valves having 2-7 direction switching, preferably 2 direction switching.

[0110] The first and second valve assemblies may be multiport valves having 10, 12, or 14 port valves. Preferably, the first valve assembly is a 10-port valve with bidirectional switching. Preferably, the second valve assembly is a 10-port valve with bidirectional switching.

[0111] In some embodiments, the flow through the capture column may be reversed when the second valve assembly is switched from a first position to a second position.

[0112] The first and second mixers may be fluid-connected to the capture pump when the first valve is in the second position and the second valve is in the first position.

[0113] others Other aspects and embodiments of the present invention provide the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting of", and the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting essentially of".

[0114] It should be understood that this application discloses all combinations of any of the above aspects and embodiments, unless otherwise required by context. Similarly, this application discloses all combinations of preferred and / or any features, either individually or in combination with any of the other aspects, unless otherwise required by context.

[0115] Modifications to the above embodiments, further embodiments, and modifications thereof will be apparent to those skilled in the art upon reading this disclosure, and therefore they are within the scope of the present invention.

[0116] All documents and array database entries referenced herein are incorporated herein by reference in their entirety for all purposes.

[0117] Where used herein, “and / or” should be interpreted as a specific disclosure of each of the two features or components specified therein, the other of which may or may not be present. For example, “A and / or B” should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were described separately herein.

[0118] method The present invention also provides a multidimensional LC method for analyzing samples such as therapeutic antibody samples.

[0119] This method is carried out using the apparatus of the present invention. The terms provided above for the apparatus are used below to define the method. Optional preferred features of the apparatus can be implemented in the method of the present invention. For example, preferred buffer exchange column features of length, inner diameter, or particle size disclosed above are also preferred for use in the method of the present invention.

[0120] The method is, 1. Using a needle, extract a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module. 2. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, into a reaction vial in the sample module and bringing the reagent into contact with the sample to be analyzed, 3. A step of controlling the temperature of the reaction vial using a sample module to allow the reaction to occur, 4. After the reaction has occurred, a needle is used to extract a portion of the reacted sample, and the reacted first sample is injected into the buffer exchange module. 5. The first reacted sample is passed through a buffer exchange column, preferably a reversed-phase column, to a digestion module. 6. The step of digesting the sample on the digestion column and flowing the digested sample to the separation module, 7. The step of separating the digested sample for analysis, Includes.

[0121] Within the sample module, "reagent vial" refers to a vial that contains a stock solution of a reagent.

[0122] Within the sample module, "sample vial" refers to the vial containing the stock solution of the sample to be analyzed.

[0123] In the sample module, the "reaction vial" refers to the vial in which the desired reaction (e.g., reduction and / or alkylation) takes place. The reaction vial is not a vial containing a stock solution of the reagent. In some cases, the reaction vial may be a vial containing a stock solution of the sample to be analyzed. That is, the reaction vial and the sample vial are the same vial. Preferably, the reaction vial is not a vial containing a stock solution of the sample to be analyzed. That is, preferably, the reaction vial and the sample vial are different vials.

[0124] Preferably, the method is i. A step of extracting a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module using a needle, ii. The step of extracting a portion of the first sample to be analyzed from the sample vial in the sample module using a needle, iii. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, and a portion of the first sample to be analyzed into a reaction vial in the sample module, iv. A step of controlling the temperature of the reaction vial using a sample module so that the reaction can occur, v. After the reaction has occurred, a needle is used to extract a portion of the reacted sample, and the reacted first sample is injected into the buffer exchange module. vi. The step of passing the reacted first sample through a buffer exchange column, preferably a reversed-phase column, to a digestion module, vii. The step of digesting the sample on the digestion column and flowing the digested sample into the separation module, viii. The step of separating the digested sample for analysis, Includes.

[0125] In some preferred embodiments, steps (i) and (ii) are performed before step (iii). In this way, the reagents and the sample to be analyzed are contained in the needle simultaneously before being deposited in the reaction vial for the reaction. Preferably, step (i) is performed before step (ii). In this way, reagents such as reducing agents are extracted first to avoid contamination of the reagent vial.

[0126] The sample is passed through a buffer exchange module, a digestion module, an optional capture module, and a separation module using a solvent and one or more pumps.

[0127] In some embodiments, steps (i) to (iv) are performed on a second sample to be analyzed when the first sample to be analyzed begins step (v). In this way, the method can be streamlined and the overall analysis time for multiple samples can be reduced.

[0128] In some embodiments, the separated samples are analyzed using mass spectrometry.

[0129] In some embodiments, the reagent is selected from reducing agents such as tris-(2-carboxyethyl)-phosphine (TCEP) or dithiothreitol (DTT), alkylating agents such as iodoacetamide, iodoacetic acid (IAA) or N-ethylmaleimide (NEM), enzymatic reaction agents such as deglycosylation with PNGaseF or digestion with papain, pepsin, IdeS, IdeZ protease, trypsin or LysC, and labeling reagents. Preferably, the reagent is a reducing agent. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). Preferably, the reducing agent is in a solution in the reagent vial, for example, in a solution of 50% acetonitrile and water. The concentration of the reducing agent may be 10 to 40 mM, for example, about 25 mM.

[0130] In some embodiments, additional reagents are introduced into the reaction vial, preferably alkylating agents. The additional reagents are added by using a needle to extract a portion of the additional reagent from the additional reagent vial within the sample module. After the (first) reagent has reacted with the sample to be analyzed, additional reagents may be added to the reaction vial. The additional reagents are added before step (v) and allowed to react before step (v) is carried out.

[0131] In some embodiments, alkylating agents such as iodoacetamide, iodoacetic acid (IAA), or N-ethylmaleimide (NEM) are used. Preferably, the alkylating agent is N-ethylmaleimide (NEM). Preferably, the alkylating agent is in a solution such as 50% acetonitrile and aqueous solution in a further reagent vial. The alkylating agent may be at a concentration of 30 to 90 mM, for example, about 60 mM.

[0132] In some embodiments, the sample vial is located in a first region of the sample holder. In some embodiments, the reagent vial is located in a first region of the sample holder. The reagent vial includes further reagent vials. In some embodiments, the reaction vial for carrying out the reaction is located in a second region of the sample holder.

[0133] In some embodiments, a first region of the sample holder is cooled. In some embodiments, a second region of the sample holder is heated to a temperature for reacting the sample and reagent.

[0134] The sample holder may have the aforementioned cooling or heating elements, such as a Peltier element, for controlling the temperature of the vial. Preferably, the sample module has at least two heating or cooling elements, such as two Peltier elements, for separately and independently controlling the temperature in first and second regions of the sample module.

[0135] In this way, the temperature of the reaction vial can be controlled independently of the temperatures of the sample and reagent vials. This means that the reaction can be accelerated by heating the reaction vial as needed, while maintaining the sample and reagent stock solutions at lower temperatures to prevent undesirable reactions. The temperature of the reaction vial can be controlled to be between 0°C and 100°C, for example, between 0°C and 70°C or between 35°C and 65°C. The temperatures of the sample and reagent vials may be between 0°C and 30°C, for example, between 2°C and 8°C. That is, the sample and reagent vials can be cooled or maintained at a temperature close to room temperature.

[0136] In some embodiments, the needle is washed between samples. In this way, cross-contamination of samples or reagents is avoided, and the method can be performed on multiple different samples or with different reagents to provide reliable results.

[0137] In some embodiments, the sample flows through a biocompatible mixer after injection and before entering the buffer exchange module. The mixer allows for dilution of the sample to be analyzed. In this way, organic solvents and by-products from the reaction are reduced before the sample is sent to the buffer exchange column.

[0138] In some embodiments, the sample flows out of the buffer exchange column through a T-piece before entering the digestion column. In some such embodiments, the T-piece introduces digestion buffer to the sample as it passes through the T-piece in order to dilute the sample before it enters the digestion column. In this way, the sample solution is optimized before passing through the digestion column, providing improved digestion and extending the life of the digestion column.

[0139] The sample to be analyzed may be any type of protein or polypeptide sample. For example, the sample to be analyzed may be an enzyme; hormone; small protein; multi-subunit protein; viral protein for gene therapy, such as capsid protein from adeno-associated virus group (AAV); antibody, such as therapeutic antibody for monoclonal antibodies; or a sample of a protein mixture containing multiple different proteins. In a preferred embodiment, the sample to be analyzed is a sample of a biological pharmaceutical, such as a therapeutic antibody.

[0140] This method uses the first valve assembly and the second valve assembly, It may provide a fluid connection between the buffer exchange module and the digestion module, and This can prevent fluid connection between the separation column and the digestion column.

[0141] In some embodiments, the method further includes the step of analyzing the fraction of the sample flowing out of the separation column by mass spectrometry. [Examples]

[0142] This application provides a novel multi-stage (LC) apparatus that enables the acquisition of multi-attribute measurements from a single sample.

[0143] The novel instrument enables time-efficient multi-attribute measurement of mAb samples.

[0144] As used in this application and particularly in the examples, the term “iSAP-LC-MS” refers to the apparatus of the present invention that combines online integrated sample preparation ("iSAP") and integrated LC-MS. In the examples, an overview of the specific apparatus used in the experiments is shown in Figure 1, and in more detail in Figure 2.

[0145] material reagent The following reagents were used in the examples: TIFF2026515907000002.tif113170

[0146] Monoclonal antibodies mAb1 refers to a Phase 3 IgG monoclonal antibody formulation provided by F. Hoffmann-La Roche.

[0147] Apparatus and method iSAP-LC-MS equipment The iSAP-LC-MS instrument is based on the Agilent Technologies 1290 Infinity II Bio LC module, which incorporates an improved multisampler, two binary pumps, two quaternary pumps, two UV detectors, and two column ovens with integrated two-position 10-port valves (see Table S1). The instrument is controlled by Thermo Fisher Scientific's GMP-compliant software Chromeleon 7.2.10 ES.

[0148] To enable reduction and alkylation within the multisampler, a custom incubator was integrated into the upper sample tray by manufacturing a software-integrated incubator with two Peltier elements positioned beneath the sample tray. This multisampler was invented, developed, and manufactured in-house and is shown in Figures 3A and 3B. A modified sample tray is shown in Figure 3. Figure 3A shows a sample module having a tray (1) with two sample holder trays (3 and 4), the sample module having a heat exchanger module (2) beneath the sample tray with an insulating cover (5). The heat exchanger module is shown in more detail in Figure 3B with a Peltier element (6) for temperature control and a heat exchange funnel (7) to also assist in temperature control. An inlet fan (8) and an outlet fan (9), along with a fan deflector (10) covering the fans, are provided for heat exchange from the device to the environment.

[0149] By connecting to the software, the temperature can be set between 0°C and 70°C, and this is automatically maintained by the integrated temperature sensor and control elements. Furthermore, the timetable within the software allows the temperature to be changed while a specific application is running.

[0150] The multisampler was further modified by installing a biocompatible microfluidic mixer (see "Mixer" in Figure 2; Jetweaver 100uL, Agilent Technologies) between the needle seat and the injector valve to reduce the concentration of the organic solvent after injection.

[0151] As shown in Figures 1 and 2, custom valve configurations and flow paths were also used. The valve setup is shown in Figure 2 and consists of two valve assemblies, namely valve 1 and valve 2.

[0152] The iSAP-LC-MS instrument further enhances elution efficiency by using a novel backflush mode for the pre-C18 capture column.

[0153] iSAP-LC-MS method The iSAP-LC-MS method (Figure 1) consists of a modified multisampler liquid processing program and an LC module for online sample preparation and analysis. The operating conditions for each module are shown in the table below.

[0154] Sample preparation begins with a multisampler injector program (ip) by aliquoting the sample into a new vial and diluting it with denaturation and reduction buffer (ip-Buffer1: 25 mM tris-(2-carboxyethyl)-phosphine (TCEP) in 50% acetonitrile (ACN) in Milli-Q water). After incubation at 40°C for 20 minutes, the reduced sample is alkylated by transferring it to a reaction sample vial with alkylation buffer (ip-Buffer2: 60 mM N-ethylmaleimide (NEM) in acetonitrile (ACN)) and reacting it at 40°C for 20 minutes.

[0155] The reduced and alkylated sample is injected into the (LC) instrument for a 1-minute online buffer exchange using a C4 pre-column (ACQUITY UPLC Protein BEH C4 VanGuard Pre-column 2.1×5 mm, Waters Corporation). Then, valve 1 (see Figure 2) is switched, and the reduced and alkylated sample is eluted from the C4 pre-column for 2 minutes. Before entering the trypsin or LysC immobilized enzyme reactor (IMER, trypsin or LysC 2.1×33 mm, Perfinity Biosciences), the eluted mAb chains are diluted with digestion buffer (50 mM TRIS, 10 mM CaCl2) via a T-piece. The sample is digested for 1 minute, and the received peptides are captured on a C18 pre-column (ACQUITY UPLC BEH C18 VanGuard Pre-column 2.1×5 mm, Waters Corporation). Next, valve 1 (see Figure 2) is switched back, and the captured peptides are desalted for 1 minute.

[0156] Subsequently, by switching valve 2, peptide mapping analysis is initiated (see Figure 2), and a series connection of the pre-column, analytical C18 column (ACQUITY UPLC BEH C18 Column (2.1 × 150 mm, Waters Corporation)) and the MS instruments used (I-IV) is obtained. At the same time, sample preparation for the next sample is started in parallel and completed simultaneously with the peptide mapping analysis of the previous sample.

[0157] The iSAP-LC-MS system modules—all liquid chromatography modules—are manufactured by Agilent Technologies and controlled by Thermo Scientific Chromeleon 7.2.10 ES software. All solvents are continuously degassed using an in-line degasser. TIFF2026515907000003.tif152170

[0158] Online Reduction Autosampler: The following table shows the sample preparation steps for the iSAP-Multisampler for automated sample reduction. Automated sample reduction using the 1290 BioMultisampler. Before each step, including solvent changes, an external rinse of the needle is performed to reduce carryover (not shown in the table). The reduction buffer contained 20 mM TCEP in 50% ACN and 50 mM ammonium acetate buffer. TIFF2026515907000004.tif85170

[0159] Online buffer exchange: Online buffer exchange was performed at 80°C using an Acquity UPLC Protein BEH C4 BEH column (2.1 × 5 mm, 1.7 μm, 300 Å Waters). Elution of the reduced mAb sample was monitored at 214 nm and 280 nm using a 1290 Infinity II UV detector. The following table shows the flow rates and flow compositions of the corresponding binary pumps. TIFF2026515907000005.tif142170

[0160] Online Trypsin Digestion: The following table shows the flow rate and flow composition of the quaternary pump for online trypsin digestion of mAb samples. For this purpose, a 2.1 × 33 mm trypsin-immobilized enzyme reactor (Perfinity) was used at 40°C. TIFF2026515907000006.tif85170

[0161] Peptide Capture: Peptide capture prior to reverse-phase separation was performed at 80°C using an Acquity UPLC Protein BEH C4 BEH column (2.1 × 5 mm, 1.7 μm, 300 Å Waters). For pre-column peptide capture, the temperature was reduced to 20°C after the capture and washing steps to improve column lifetime. The corresponding flow rates and flow compositions are shown in the table below. TIFF2026515907000007.tif132170

[0162] Reverse-phase separation: Reverse-phase separation for online peptide mapping was performed at 40°C using an Acquity Premier Peptide BEH C18 column (2.1 × 150 mm, 1.7 μm, 300 Å, Waters) with the gradients shown in the table below. TIFF2026515907000008.tif97170

[0163] Mass Spectrometer Parameters for MS Acquisition: The following table shows the settings for a Waters Xevo G2-S QTof high-resolution mass spectrometer for MS acquisition. High-resolution MS was controlled using MassLynx software (Waters). MS acquisition was initiated by a digital contact closing signal from an Agilent Technologies Universal Interface Box (UIBII). TIFF2026515907000009.tif79170

[0164] Mass Spectrometer Settings for MS / MS Acquisition: The following table shows the settings for a Waters Xevo G2-S QTof high-resolution mass spectrometer for MS acquisition. High-resolution MS was controlled using MassLynx software (Waters). MS acquisition was initiated by a digital contact closing signal from an Agilent Technologies Universal Interface Box (UIBII). TIFF2026515907000010.tif98170

[0165] Single Quadrupole Mass Spectrometer Parameters for MS Acquisition: The following table shows the settings for the Thermo Scientific ISQ EM single quadrupole mass spectrometer for MS acquisition. MS was controlled using Chromeleon software. TIFF2026515907000011.tif42170

[0166] experiment A 5 mg / mL sample of mAB1 (recombinant monoclonal IgG-antibody produced in CHO cells with a human glycosylation profile) was prepared in a sample vial by diluting it with a buffer and then placed in a sample holder.

[0167] The sample was processed according to the method described above. The total ion chromatogram of mAB1 prepared and analyzed by iSAP-LC-MS is shown in Figure 4.

[0168] The linearity of the system was tested. Linearity evaluation was performed using mAB1 trypsin peptide oxidation (T22 or T42) or deamination quantification. Ten samples of mAB1 with different oxidation levels and ten samples with different deamination levels were prepared and measured.

[0169] For forced oxidation, the mAbs were incubated in 20 mM histidine buffer (pH 6.0, 0.2% H2O2) at 25°C for 24 hours. For forced deamidation, the samples were incubated in 200 mM Tris-HCl buffer (pH 9.0) for 7 days. After incubation, each stress was stopped to a final concentration of 0.5 mg / ml by buffer exchange with fresh 20 mM histidine and 20 mM methionine buffer (pH 6.0) using an NAP 5 column. These stressed samples were brought to 100% stress level.

[0170] A reference standard without stress was defined as a 0% stress level. A mixture of 0% and 100% stress levels results in intermediate stress levels (0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5%, 10%, 20%, 40%) wherever stress occurs. Each level is corrected by oxidation measured for level 0.00 (2.43%). Each level is then measured and compared to the theoretically corrected oxidation level.

[0171] The T22 oxidation degree data is shown in the table and Figure 5A below. TIFF2026515907000012.tif89170

[0172] The fitted linearity of the degree of oxidation measured for T22 is R 2 The correlation coefficient is 0.9987. The measured data are plotted against the normalized theoretical degree of oxidation. Normalization was performed using the degree of oxidation from unstressed samples (2.43% for T22).

[0173] The T42 oxidation degree data is shown in Figure 5B. The fitted linearity of the oxidation degree measured for T42 is R 2 The correlation coefficient is 0.9992. Normalization was not necessary because T42 is not oxidized in unstressed samples. The lowest theoretical oxidation level was 0.63%, confirming the oxidation of T42.

[0174] The data for the degree of deamination is shown in Figure 5C. The fitted linearity of the measured degree of deamination is R 2 The correlation coefficient is 0.995. The measured data is plotted against the theoretical degree of deamidation.

[0175] In summary, the results show very good linearity within the test range.

[0176] The accuracy of the system was tested by injecting the same mAB1 sample with a 0% stress level (reference standard) six times. For comparison, two modification degrees (T22 and T38) were obtained for each sample. The results are shown in the table and Figure 6 below. TIFF2026515907000013.tif46170

[0177] The oxidation degree of the unstressed mAb at T22 had an RSD of 2.51% (see pentagon in Figure 6). The first replica had a higher measured oxidation degree compared to the latter five replicas. The deamidation stress at T38 had an RSD% of 3.62% (see triangle in Figure 6). Overall, the relative standard deviations calculated for oxidation and deamination were low, less than 10%, indicating good accuracy of the instruments and methods used.

[0178] The robustness of digestion in the system was tested by storing mAB1 samples for 0, 12, and 24 hours before sample preparation using three different lots of trypsin columns. This method was performed (in triplicate) using mAB1 in iSAP-autosampler samples through the three different trypsin columns. The oxidation of T22 was compared for each trypsin column, and the results are shown in Figure 7A. This method was also performed (in triplicate) using mAB1 samples prepared and used immediately (i.e., with zero storage time), as well as with storage times of 12 and 24 hours, and approximately 30 hours. The oxidation of T22 was compared for columns with different storage times. The results are shown in the table and Figure 7B below. No significant changes in oxidation were observed. TIFF2026515907000014.tif52170TIFF2026515907000015.tif52170

[0179] Consideration The iSAP-LC-MS platform enables the preparation of a second sample while digesting and mapping a first sample, thereby improving the instrument's throughput. This is schematically illustrated in Figure 8A. In particular, the iSAP-LC-MS platform improves sample throughput compared to the use of known instruments such as pipetting robot protocols. This is illustrated in Figure 8B.

[0180] Compared to known pipetting robot protocols (Figures 8C and 8D), the multi-stage apparatus of the present invention offers improvements in both cost and environmental impact.

[0181] The iSAP-LC-MS platform demonstrated good linearity, accuracy, and repeatability. Similar results were obtained compared to our manual and robotic workflows, but the iSAP LC-MS system showed better robustness in terms of autosampler storage time and IMER efficiency.

[0182] iSAP-LC-MS instruments can help accelerate analysis, reduce workload, and make more drugs available at a lower cost.

[0183] term The following numbered sections provide several specific embodiments of the present invention.

[0184] 1. A multi-stage device, An injector module having a needle, A sample module having a sample tray for holding one or more sample vials, A buffer exchange module having a buffer exchange column, A digestion module having a digestion column downstream of a buffer exchange module, A separation module having a separation column downstream of the digestion module, Equipped with, The injector module is configured so that a needle extracts a sample from one sample vial and injects it into another sample vial, and extracts a sample from a sample vial and injects it into the buffer exchange module. The sample module is configured to control the temperature of the sample vial. This multi-stage device allows for the reaction of the sample by using a needle to introduce reagents and samples into vials in a sample tray during use, and by controlling the temperature before they are injected into the buffer exchange module.

[0185] 2. The multi-stage apparatus of item 1, wherein the injector module is a pipette operating robot.

[0186] 3. The injector module is a multi-stage device according to item 1 or 2, configured to transfer a portion of the sample into a new vial.

[0187] 4. A multi-stage device according to any one of items 1 to 3, wherein the injector module is configured to draw in a reagent such as a reducing agent into the needle, and then draw in a sample into the needle.

[0188] 5. A multistage apparatus according to any one of items 1 to 4, wherein the needle has a volume of at least 3 uL.

[0189] 6. A multistage apparatus according to any one of items 1 to 5, wherein the sample module has a heating or cooling element such as a Peltier element for controlling the temperature of the vial.

[0190] 7. The multistage apparatus of item 6, wherein the sample module has at least two heating or cooling elements, such as two Peltier elements, for controlling the temperature in different regions of the sample module.

[0191] 8. A multistage apparatus according to any one of items 1 to 7, wherein the sample module comprises two sample trays configured to allow independent control of the temperature of each sample tray.

[0192] 9. A multi-stage apparatus according to any one of items 1 to 8, wherein the sample tray can hold 54 vials or has 96 well plates per tray.

[0193] 10. A multistage apparatus according to any one of items 1 to 9, wherein the sample module comprises sample vials for holding samples and reagents such as reducing agents.

[0194] 11. A multistage apparatus according to any one of items 1 to 10, wherein the sample module is an autosampler or a multisampler.

[0195] 12. A multistage apparatus according to any one of items 1 to 11, wherein the buffer exchange column is a reversed-phase column such as a C4 column, C1 column, or phenyl stationary-phase column.

[0196] 13. A multistage apparatus according to any one of items 1 to 12, further comprising a capture module having a capture column for retaining a digested sample within the digestion module, wherein the capture module is located between the digestion module and the separation module in the flow direction.

[0197] 14. A multi-stage apparatus according to item 13, wherein the capture column has a length of 5 to 30 mm.

[0198] 15. A multistage apparatus according to item 13 or 14, wherein the capture column has a packing material with a particle size of 1.0 to 3.0 μm, for example 1.0 to 2.0 μm, preferably 1.5 to 2.0 μm.

[0199] 16. A multistage apparatus according to any one of items 13 to 15, wherein the capture column has an inner diameter of 1.5 to 5 mm.

[0200] 17. A multistage apparatus according to any one of items 13 to 16, wherein the capture column has the same packing material as the peptide mapping column.

[0201] 18. A multistage apparatus according to any one of items 1 to 17, wherein the separation column is selected from a peptide mapping column, such as a UHPLC column or an HPLC column, and preferably the separation column is a UHPLC column.

[0202] 19. A multistage apparatus according to any one of items 1 to 18, wherein the separation column has a C18 stationary phase.

[0203] 20. A multi-stage apparatus according to any one of items 1 to 19, with a separation column having a length of 100-200 mm, such as approximately 150 mm.

[0204] 21. A multistage apparatus according to any one of items 1 to 20, wherein the separation column has a packing material with a particle size of 1.0 to 3.0 μm, for example 1.0 to 2.0 μm, preferably 1.5 to 2.0 μm.

[0205] 22. A multistage apparatus according to any one of items 1 to 21, wherein the separation column has an inner diameter of 1.5 to 5 mm, preferably 1.5 to 2.5 mm, for example, about 2.1 mm.

[0206] 23. A multistage apparatus according to any one of items 1 to 22, wherein the separation column contains a C18 stationary phase.

[0207] 24. A multistage apparatus according to any one of items 1 to 23, wherein the digestion column is selected from a trypsin-immobilized enzyme reactor, a LysC-immobilized enzyme reactor, or an aspN-immobilized enzyme reactor.

[0208] 25. A multistage apparatus according to any one of items 1 to 24, the digestion module having a first mixer such as a static mixer or a zero-delay volume T piece after the digestion column in the direction of flow.

[0209] 26. A multistage apparatus of item 25, wherein the first and second mixers are fluid-connectable to a capture pump.

[0210] 27. A multistage apparatus according to any one of items 1 to 26, wherein the digestion module has a second mixer such as a static mixer or a zero-delay volume T piece prior to the digestion column in the direction of flow.

[0211] 28. A multistage apparatus according to any one of claims 1 to 27, wherein the digestion module comprises two digestion columns, preferably the two digestion columns connected in parallel.

[0212] 29. A multistage apparatus according to any one of items 1 to 28, wherein two digestion columns are connected in parallel so that the sample flow is divided between the two columns during use.

[0213] 30. A multistage apparatus according to item 28 or 29, wherein the two digestion columns are a trypsin-immobilized enzyme reactor and a LysC-immobilized enzyme reactor.

[0214] 31. A multistage apparatus according to any one of items 1 to 30, further comprising an analytical module for analyzing a sample after it has passed through a separation column.

[0215] 32. The multistage apparatus of item 31, comprising a mass spectrometer such as a high-resolution mass spectrometer (HRMS) or a single quadrupole mass spectrometer, an evaporative light scattering detector (ELSD), a UV detector, or a diode array detector (DAD).

[0216] 33. A multistage apparatus according to any one of items 1 to 32, further comprising a biocompatible microfluidic mixer between a needle and a valve for injecting a sample into a buffer exchange module, or between an injector valve and a buffer exchange module.

[0217] 34. The present invention also provides a method for analyzing a sample using a multistage apparatus according to any one of items 1 to 33.

[0218] 35. i. A step of extracting a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module using a needle, ii. The step of extracting a portion of the first sample to be analyzed from the sample vial in the sample module using a needle, iii. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, and a portion of the first sample to be analyzed into a reaction vial in the sample module, iv. A step of controlling the temperature of the reaction vial using a sample module so that the reaction can occur, v. After the reaction has occurred, a needle is used to extract a portion of the reacted sample, and the reacted first sample is injected into the buffer exchange module. vi. The step of passing the reacted first sample through a buffer exchange column, preferably a reversed-phase column, to a digestion module, vii. The step of digesting the sample on the digestion column and flowing the digested sample into the separation module, viii. Steps to separate the digested sample for analysis and The method of item 34, including the method of item 34.

[0219] 36. The method of item 35, wherein steps (i) and (ii) are performed before step (iii), preferably step (i) is performed before step (ii).

[0220] 37. The method of item 35 or 36, wherein steps (i) to (iv) are performed on a second sample to be analyzed when step (v) is initiated on a first sample to be analyzed.

[0221] 38. The method of any one of items 35 to 37, wherein the separated sample is analyzed using mass spectrometry.

[0222] 39. The method of any one of claims 35 to 38, wherein the reagent is selected from reducing agents, alkylating agents, or labeling agents, and preferably the reagent is a reducing agent.

[0223] 40. The method of any one of claims 35 to 39, wherein a further reagent is introduced into the reaction vial, preferably the further reagent being an alkylating agent.

[0224] 41. The method according to any one of items 35 to 40, wherein the sample vial is located in the first area of ​​the sample holder.

[0225] 42. The method according to any one of items 35 to 41, wherein the reagent vial is located in the first area of ​​the sample holder.

[0226] 43. The method according to any one of items 35 to 42, wherein the reaction vial for carrying out the reaction is located in a second area of ​​the sample holder.

[0227] 44. The method according to any one of items 41 to 43, wherein the first region of the sample holder is cooled.

[0228] 45. The method of any one of items 41 to 44, wherein the second area of ​​the sample holder is heated to a temperature for reacting the sample with the reagent.

[0229] 46. ​​The method according to any one of items 35 to 45, wherein the needle is washed between samples.

[0230] 47. The sample is flowed through a biocompatible mixer after injection and before entering the buffer exchange module, according to any one of the methods in items 35 to 46.

[0231] 48. The method of any one of items 35 to 47, wherein the sample is drained from the buffer exchange column through a T-piece before entering the digestion column.

[0232] 49. The method of section 48, wherein the T-piece introduces digestion buffer into the sample as the sample passes through the T-piece in order to dilute the sample before it enters the digestion column.

[0233] 50. The method according to any one of items 35 to 49, wherein the sample to be analyzed is a sample of a biological product, such as a therapeutic antibody.

Claims

1. It is a multi-stage device, An injector module having a needle, A sample module having a sample tray for holding one or more sample vials, A buffer exchange module having a buffer exchange column, A digestion module having a digestion column downstream of the buffer exchange module, A separation module having a separation column downstream of the digestion module, Equipped with, The injector module is configured such that the needle extracts a sample from one sample vial and injects it into another sample vial, and also extracts a sample from a sample vial and injects it into the buffer exchange module. The sample module is configured to control the temperature of the sample vial. This multi-stage device allows the reagent and sample to be reacted by using the needle to introduce them into the vial in the sample tray and controlling the temperature before they are injected into the buffer exchange module.

2. The multi-stage apparatus according to claim 1, wherein the injector module is a pipette operating robot, and preferably the injector module may include a three-axis robotic arm for liquid processing.

3. The multi-stage apparatus according to claim 1 or 2, wherein the injector module is configured to move a portion of the sample into a new vial.

4. The multi-stage apparatus according to any one of claims 1 to 3, wherein the injector module is configured to draw in the reagent, such as a reducing agent, into the needle, and then draw in the sample into the needle.

5. The multistage apparatus according to any one of claims 1 to 4, wherein the sample module has a heating or cooling element such as a Peltier element for controlling the temperature of the vial.

6. The multistage apparatus according to claim 5, wherein the sample module has at least two heating or cooling elements, such as two Peltier elements, for controlling the temperature in different regions of the sample module.

7. The multi-stage apparatus according to any one of claims 1 to 6, wherein the sample module comprises two sample trays configured such that the temperature of each sample tray can be controlled independently.

8. The multistage apparatus according to any one of claims 1 to 7, wherein the sample tray can hold 54 vials, or each tray can have 96 well plates.

9. The multistage apparatus according to any one of claims 1 to 8, wherein the buffer exchange column is a reversed-phase column such as a C4 column, a C1 column, or a phenyl stationary-phase column.

10. The multistage apparatus according to any one of claims 1 to 9, further comprising a biocompatible microfluidic mixer between the needle and a valve for injecting the sample into the buffer exchange module, or between the injector valve and the buffer exchange module.

11. The multistage apparatus according to any one of claims 1 to 10, further comprising an analytical module for analyzing the sample after the sample has passed through the separation column, preferably the analytical module comprising a mass spectrometer such as a high-resolution mass spectrometer (HRMS) or a single quadrupole mass spectrometer, an evaporative light scattering detector (ELSD): UV detector, or a diode array detector (DAD).

12. A method for analyzing a sample using a multi-stage apparatus according to any one of claims 1 to 11.

13. i. A step of using the needle to extract a portion of the reagent, such as a reducing agent or alkylating agent, from the reagent vial in the sample module, ii. The step of extracting a portion of the first sample to be analyzed from the sample vial in the sample module using the needle, iii. A step of injecting a portion of the reagent, such as a reducing agent or alkylating agent, and a portion of the first sample to be analyzed into a reaction vial in the sample module, iv. A step of controlling the temperature of the reaction vial using the sample module so that the reaction can occur, v. After the reaction has occurred, the needle is used to extract a portion of the reacted sample, and the reacted first sample is injected into the buffer exchange module. vi. The step of passing the reacted first sample through the buffer exchange column, preferably a reversed-phase column, to the digestion module, vii. The step of digesting the sample on the digestion column and flowing the digested sample into the separation module, viiii. The step of separating the digested sample for analysis, The method according to claim 12, including the method described in claim 12.

14. The method according to claim 13, wherein steps (i) and (ii) are performed before step (iii), preferably step (i) is performed before step (ii).

15. The method according to claim 13 or 14, wherein steps (i) to (iv) are performed on a second sample to be analyzed when the first sample to be analyzed begins step (v).

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