Systems and methods for sample processing

Abstract

The present invention is directed to systems and methods for sample processing. Systems and methods of the invention provide improved sample processing for advanced therapy workflows that accelerates sample clean-up and conditioning for real-time analysis. Systems of the invention include one or more microfluidic chip devices, and are capable of both bioreactor-agnostic integration and direct / indirect interfacing with analysis systems. As such, systems and methods of the invention provide a simple, cost-effective, and fast direct-from-sample-to- analysis process.

Description

[0001] SYSTEMS AND METHODS FOR SAMPLE PROCESSING

[0002] Field of the Invention

[0003] The invention generally relates to systems and methods for sample processing.

[0004] Background

[0005] Advanced therapies refer to new medical products that use gene therapy, cell therapy, and tissue engineering to repair or replace damaged human cells, tissues, or organs to supplement or restore function. The field of advanced therapies goes beyond existing treatments to address the underlying causes of disease or to provide treatments for previously untreatable diseases and conditions. In particular, cell-based therapeutics are an emerging modality with the potential to treat many currently intractable diseases through unique modes of action.

[0006] However, cell-based therapies continue to face numerous challenges that limit their widespread translation and commercialization. These challenges include identification of the appropriate cell source, generation of a sufficiently viable, potent and safe product that meets patient- and disease-specific needs, and the development of scalable manufacturing processes. Such potentially transformative treatments have yet to reach broad patient populations due in part because, in the nascent cell therapy space, the entire cell is the final product, comprising thousands of different biochemicals. That difference makes characterization and control during product manufacturing far more challenging to ensure the safety, efficacy, and potency of cellular therapies.

[0007] Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). A critical issue in real-time monitoring and feedback is the extensive sample preparation necessary before analysis. Current procedures are time-consuming and can result in poor recovery. As a result advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. Thus, to ensure the safety, efficacy, and potency of advanced therapies during product manufacturing, and to provide cost-efficient and time-saving characterization, novel solutions for sample preparation for analysis are needed. Summary

[0008] The present invention recognizes the drawbacks of conventional sample processing systems which limit the characterization and control of advanced therapies during product development and manufacture. Accordingly, systems and methods of the invention provide improved sample processing for advanced therapy workflows that accelerates sample clean-up and conditioning for real-time analysis.

[0009] Aspects of the invention provide systems comprising multiple flow channels in fluidic communication. The fluidic communication may be via one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. Molecules within a sample are selectively filtered between channels, thereby separating specific molecules into each flow channel without ever interrupting the flow of fluid within each channel. Additionally, the systems provide for concurrent sample conditioning for downstream analysis. For example, the fluid within each channel may comprise reagents or solvents specific to the molecules filtered into each specific flow channel. This allows for a sample to be continuously flowed through the system such that real-time size-specific separation of molecules within the system and concurrent sample conditioning occurs to accelerate real-time / dynamic analysis.

[0010] Systems and methods of the invention address the bottleneck of sample processing by providing real-time / dynamic quality control measurement that is highly sensitive to secreted biomarkers with complex biochemical signatures. Systems and methods of the invention enable broad biomolecular detection such that unanticipated or unidentified biomarkers may be characterized for better process understanding.

[0011] In exemplary embodiments, systems and methods of the invention provide solutions to the current sample preparation bottleneck when utilizing modem mass spectrometry for sample analysis. Accordingly, the invention provides for accelerated mass spectrometry workflows applicable to measuring cell-state, (e.g. proliferation, confluence, differentiation, activation), Critical Process Parameters (CPPs), and to Critical Quality Attributes (CQAs) identification and Monitoring.

[0012] Systems of the invention include one or more microfluidic chip devices, and are capable of both bioreactor-agnostic integration and direct / indirect interfacing with analysis systems. Accordingly, systems and methods of the invention provide a simple, cost-effective, and fast direct-from-sample-to-analysis process. Thus, the systems and methods of the invention enable real-time and / or dynamic, in-process monitoring and feedback control, and cell state characterization.

[0013] In one aspect, the invention provides systems for sample processing. The system comprises a sample inlet configured to receive a fluidic sample. The inlet is in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter. The system includes a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter, such that, when the sample inlet receives a fluidic sample, molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter into the carrier fluid of the carrier channel. The flow may be, for example, parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. Further, the system includes a second filter in communication with the carrier channel downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter. The communication of the second filter with the carrier channel may be, for example, by one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. Here, when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into a conditioning fluid. In some embodiments, the sample inlet is in communication with the first filter via a well.

[0014] In some embodiments, the second filter is in communication with a conditioning channel via flow of the conditioning fluid along the second filter, such that, when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel. The flow may be, for example, one or more of parallel flow, cross-flow, co-flow, and / or tangential flow. In particular embodiments, the flow of the carrier channel and the conditioning channel are in parallel planes to one another along the second filter. In further embodiments, the system includes a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. In some embodiments, a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel is controlled via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

[0015] In some embodiments, the fluidic sample comprises one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample. In some embodiments, the first molecular weight threshold and the second molecular weight threshold are different thresholds. Accordingly, in particular embodiments, the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

[0016] In some embodiments, the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure. As such, the well comprises one or more integrated electrodes configured for electrical lysis of one or more cells within the sample. In some embodiments, the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well. Thus, a voltage and an electric pulse parameter are adjustable according to an applicable lysis procedure, in some embodiments. Other methods of cell lysis, including mechanical and chemical lysis, are also possible in the sample inlet and / or well.

[0017] In particular embodiments, the first filter and / or the second filter comprise one or more capture features. In some embodiments, the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel. In some embodiments, the one or more capture features comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, positioned within the well and / or the sample inlet and / or the carrier channel, such that when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier. The capture features of the first filter are different from the capture features of the second filter, in some embodiments. In some embodiments, the first filter and / or the second filter comprise a membrane configured to capture the molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality.

[0018] In some embodiments, a degree of communication between the carrier channel and the first filter is different from a degree of communication between the carrier channel and the second filter. For example, the degree of communication may be a contact area between the carrier channel and the first and / or second filter. The degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer across the respective filter that takes place.

[0019] A carrier fluid contact time with the first and / or second filters, also referred to as residence time, may be defined by varying, for example, the carrier channel dimensions. The carrier fluid contact time with the first and / or second filter, and thus the fluidic sample as the sample flows through the device, may be defined by the amount of time the carrier fluid is in contact with the first and / or second filter. Thus, in some embodiments, the contact time, i.e. residence time, is defined by a degree of communication between the carrier channel and the first and / or second filter. For example, the degree of communication may be a contact area between the carrier channel and the first and / or second filter. The degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer and / or flux across the respective filter that takes place. Accordingly, the contact time of the carrier fluid may be controlled and / or defined by varying characteristics of the portion of the carrier channel in communication with the first and / or second filter.

[0020] In some embodiments, the carrier fluid comprises one or more reagents, such that, when the carrier fluid flows to the first filter and / or the second filter, then the one or more reagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel thereby facilitating a chemical reaction in the well and / or the conditioning channel, system calibration, and / or analyte quantification. Further, the one or more reagents are reagents for one or more of organic solvents, enzymatic molecules, standard reagents, chemical lysis reagents, and / or additive molecules to form molecular complexes, including ionically charged complexes.

[0021] In some embodiments, the conditioning fluid comprises reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, wherein when the conditioning fluid flows along the second filter then the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel. The specified molecule comprises one or more of a fluorescent molecular tag, an analytical standard, an organic acid, an organic solvent, and a tandem mass tag (TMT), in some embodiments.

[0022] In some embodiments, the system further includes one or more analysis modules for molecular analysis of fluid from the first reservoir and / or the second reservoir. For example, the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays. Accordingly, the molecular analysis includes detecting and / or analyzing one or more properties of an analyte. In some embodiments, the one or more analysis modules are in communication with the first or second reservoir such that fluid from the carrier channel or the conditioning channel flows directly into the one or more analysis modules. The sample inlet is configured to receive a sample collected from a microenvironment of a cell system. In some embodiments, the system is configured to detect and / or analyze an analyte that is an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action.

[0023] Accordingly, the sample inlet may be configured to receive a sample collected from a microenvironment of a cell system. The system may be configured, in some embodiments, to detect and analyze an analyte associated with cell state characterization during product development and manufacture of advanced therapy products.

[0024] In some embodiments, a sample with one or more molecules of interest is captured via localized sampling and introduced into the system for subsequent analysis via mass spectrometry. The mass spectrometry data may be analyzed via, for example, a machine learning algorithm. The machine learning algorithm may comprise, for example, a multivariant analysis to provide deep insights into cell state characterization, such as cell phenotype, cell differentiation, cell disease state, cell healthy state, cell therapy product efficacy, cell therapy product safety, cell therapy product potency and / or a correlation with a mechanism of cell action.

[0025] In particular embodiments, the system can continuously monitor a cell culture process to optimize a cell culture process parameter comprising one or more of a feed rate, a cell seeding density, cell growth nutrients, and a media exchange rate. For example, the system can detect and / or analyze an analyte that is an indicator of a temporal change in a secretome of a cell such that the system provides a direct representation of a real-time cell state, in some embodiments.

[0026] Further, in some embodiments, the system includes one or more flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules.

[0027] In some embodiments, the fluidic sample is received by the inlet manually via pipette. In other embodiments, the fluidic sample is received by the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet or a sample delivery robot.

[0028] In particular embodiments, the system includes a bypass autosampler in fluidic communication with the carrier channel upstream of the first filter, such that when the bypass autosampler is provided the fluidic sample, then the bypass autosampler periodically and automatically introduces the fluidic sample directly into the carrier channel, thereby bypassing the inlet.

[0029] In various embodiments, the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time along each filter. For example, the residence time along the first filter is different from the residence time along the second filter, in some embodiments. In some embodiments, the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter. Further, in some embodiments, the cross-section of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter.

[0030] In another aspect, the invention provides methods for sample processing. The method comprises providing a system for sample processing comprising a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter, and a second filter in communication with the carrier channel via flow downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter. A carrier fluid contact time, also referred to as residence time, may be defined by varying, for example, the carrier channel dimensions. The carrier fluid contact time with the first and / or second filter, and thus the fluidic sample as the sample flows through the device, may be defined by the amount of time the carrier fluid is in contact with the first and / or second filter. Thus, in some embodiments, the contact time is defined by a degree of communication between the carrier channel and the first and / or second filter, such that the degree of communication may be a contact area between the carrier channel and the first and / or second filter. Accordingly, the contact time of the carrier fluid may be controlled and / or defined by varying characteristics of the portion of the carrier channel in communication with the first and / or second filter. In some embodiments, the contact time, i.e. residence time, of the carrier fluid along the first filter is different from the residence time along the second filter.

[0031] For example, the degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer and / or flux across the respective filter that takes place. The flow may be, for example, one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. Further, the method includes inserting a fluidic sample within the sample inlet, and flowing carrier fluid through the carrier channel past the first filter and second filter, such that molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter into the carrier fluid of the carrier channel and molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into a conditioning fluid. In some embodiments, the sample inlet is in communication with the first filter via a well.

[0032] In some embodiments of the method, the second filter is in communication with a conditioning channel via flow of the conditioning fluid along the second filter, whereby molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel.

[0033] In some embodiments of the method, the flow of the carrier channel and the conditioning channel are in parallel planes to one another along the second filter. In some embodiments, the flow of the carrier channel and the conditioning channel is a cross-flow orientation.

[0034] In some embodiments of the method, the method further comprises controlling a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

[0035] In some embodiments of the method, the system further comprises a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Thus, the method further includes the step(s) of collecting conditioning fluid from the conditioning channel that has flowed along the second filter and / or collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. In some embodiments, the sample collected and / or captured is analyzed via orthogonal methods.

[0036] In some embodiments of the method, the fluidic sample comprises one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample. In some embodiments, the first molecular weight threshold and the second molecular weight threshold are different thresholds. Accordingly, in particular embodiments, the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

[0037] In some embodiments, the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure. As such, the well comprises one or more integrated electrodes configured for electrical lysis of one or more cells within the sample. In some embodiments, the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well. Thus, a voltage and an electric pulse parameter are adjustable according to an applicable lysis procedure, in some embodiments.

[0038] In particular embodiments, the first filter and / or the second filter comprise one or more capture features. In some embodiments, the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel. In some embodiments, the one or more capture features comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, positioned within the well and / or sample inlet and / or the carrier channel, such that when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier. The capture features of the first filter are different from the capture features of the second filter, in some embodiments.

[0039] In some embodiments, the first filter and / or the second filter comprise a membrane configured to capture the molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality.

[0040] In some embodiments of the method, a degree of communication between the carrier channel and the first filter is different from a degree of communication between the carrier channel and the second filter.

[0041] In particular embodiments of the method, the carrier fluid includes one or more reagents, whereby during the flowing step one or more reagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel thereby facilitating a chemical reaction in the well and / or the conditioning channel, system calibration, and / or analyte quantification. The one or more reagents are reagents for one or more of organic solvents, enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules, in some embodiments.

[0042] In various embodiments, the conditioning fluid comprises reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, whereby during the flowing step the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel. For example, the specified molecule includes one or more of a fluorescent molecular tag, an analytical standard, an organic acid, an organic solvent, and a tandem mass tag (TMT), in some embodiments.

[0043] In some embodiments of the method, the system further includes one or more analysis modules for molecular analysis of fluid from the first reservoir or the second reservoir. The method therefore further includes the step of analyzing molecular of fluid from the first reservoir and / or the second reservoir. In some embodiments, the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays.

[0044] The molecular analysis includes detecting and / or analyzing one or more properties of an analyte, in some embodiments. The one or more analysis modules are in communication with the first or second reservoir, in some embodiments. Thus, the method comprises the step of flowing fluid from the carrier channel or the conditioning channel directly into the one or more analysis modules. Further, in some embodiments, the sample inlet is configured to receive a sample collected from a microenvironment of a cell system. As such, the method includes detecting and / or analyzing an analyte that is an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action. The method also includes continuously monitoring a cell culture process to optimize a cell culture process parameter comprising one or more of a feed rate, a cell seeding density, cell growth nutrients, and a media exchange rate, in some embodiments. In other embodiments, the method includes detecting and / or analyzing an analyte that is an indicator of a temporal change in a secretome of a cell such that the method provides a direct representation of a dynamic cell state.

[0045] In some embodiments, a sample with one or more molecules of interest is captured via localized sampling and introduced into the system for subsequent analysis via mass spectrometry. The mass spectrometry data may be analyzed via, for example, a machine learning algorithm. The machine learning algorithm may comprise, for example, a multivariant analysis to provide deep insights into cell state characterization, such as cell phenotype, cell differentiation, cell disease state, cell healthy state, cell therapy product efficacy, cell therapy product safety, cell therapy product potency and / or a correlation with a mechanism of cell action.

[0046] In some embodiments of the method, the system further includes one or more flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules, and the flowing step comprises using the flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules.

[0047] In some embodiments of the method, the inserting step comprises inserting the fluidic sample into the inlet manually via pipette. In other embodiments, the inserting step comprises inserting the fluidic sample into the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet.

[0048] In some embodiments of the method, the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time along each filter. Further, the residence time along the first filter may be different from the residence time along the second filter, in some embodiments. The length of the carrier channel along the first filter may be different from the length of the carrier channel along the second filter, in some embodiments. In other embodiments, the cross-section of the carrier channel along the first filter may be different from the cross-section of the carrier channel along the second filter. For example, the degree of communication may be a contact area between the carrier channel and the first and / or second filter. The degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer and / or flux across the respective filter that takes place.

[0049] In another aspect, the invention provides a method for sample processing comprising providing a system for sample processing comprising a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter, and a bypass autosampler in fluidic communication with the carrier channel upstream of the first filter; a second filter in communication with the carrier channel downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter. In non-limiting examples, the flow of the carrier fluid along the first filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. In non-limiting examples, the flow of the carrier fluid along the second filter may be one or more of parallel flow, crossflow, co-flow, counterflow, and / or tangential flow.

[0050] The method further includes introducing the fluidic sample directly into the carrier channel via the bypass autosampler, thereby bypassing the sample inlet; and flowing carrier fluid through the carrier channel past the first filter and second filter, such that molecules in the fluidic sample below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

[0051] Brief Description of the Drawings

[0052] FIG. 1 is a diagrammatic illustration of one embodiment of systems of the invention. FIG. 2 illustrates one embodiment of the systems of the invention in which the microfluidic system includes an inline ESI-MS analysis module.

[0053] FIG. 3 illustrates a top view of one embodiment of a microfluidic chip design of systems of the invention, in which the system includes a microwell for loading the sample.

[0054] FIG. 4 illustrates one embodiment of systems of the invention which includes an autosampler module.

[0055] FIG. 5 illustrates a method of the invention according to one embodiment.

[0056] FIG. 6 illustrates a method of the invention according to one embodiment.

[0057] Detailed Description

[0058] The present invention recognizes the limitations of current sample processing techniques for use in process analytical technologies. In particular, the invention provides systems and methods for improved sample processing in advanced therapy workflows. Systems and methods of the invention accelerate and improve sample clean-up and conditioning thus enabling realtime analysis for in-process monitoring and feedback control, and cell state characterization during product development and manufacture of advanced therapy products. Systems of the invention are capable of both bioreactor-agnostic integration and direct / indirect interfacing with analysis systems. In non-limiting examples, the analysis system may include one or more of an electrospray ionization device interfaced with a mass spectrometry system, an ion trapping system, an optical spectroscopy sensor, a Raman spectroscopy system, a FTIR spectrometer, a UV-VIS spectrometer, a nuclear magnetic resonance spectroscopy systems, an electrochemical redox and / or impedance sensor, a mass cytometry system, or a flow cytometry system, or a combination thereof.

[0059] Process analytical technology is a mechanism to design, analyze, and control pharmaceutical manufacturing processes through the measurement of critical process parameters which affect the critical quality attributes. Currently, predicting whether a therapeutic product will be potent, efficacious, and safe is challenging for advanced therapy workflows because the Critical Quality Attributes (CQAs) that indicate product quality remain relatively undefined. Some of the most reliable CQAs for predicting cell state (and thus product quality) are the secreted signaling and paracrine factors that cells release while developing in culture. Electrospray ionization mass spectrometry (ESI-MS) has emerged as a technique for the analysis of proteins and / or other macromolecules. ESI-MS is a highly sensitive label-free method useful to detect and identify biomolecules because it overcomes the propensity of these molecules to fragment when ionized. Specifically, ESI-MS takes advantage of liquid phase ionization amenable for direct infusion analysis of complex mixtures. ESI-MS offers broad molecular weight coverage, sensitivity, and the ability to preserve structure / folding and non-covalent interactions of biomolecular complexes through “soft-ionization.” ESI has broadened the application of mass spectrometry in the protein field by providing significantly improved sensitivity and accuracy in the analysis of complex analytical matrices.

[0060] However, ESI-MS requires extensive sample preparation before analysis, making online application of ESI-MS challenging. For example, a critical issue in quantitative ESI-MS is suppression of ionization due to matrix interference. The variability in matrix composition potentially means that the degree of ionization will vary from one sample to another with possible adverse effects on the analysis of target analytes. Therefore, it is essential that a target of interest is resolved from any endogenous materials that produce matrix effects in order to reduce or eliminate ion suppression within the mass spectrometer source. To overcome the matrix interference, extensive sample purification processes are required, such as liquid-liquid extraction and solid phase extraction by disposable columns. However, these procedures are time-consuming and can result in poor recovery. With conventional processing techniques, sample preparation requirements limit the capabilities of ESI-MS to offline applications and preclude its use for in-process monitoring. Thus, sample preparation is the bottleneck in modern mass spectrometry.

[0061] Systems and methods of the invention address this bottleneck by providing real-time, and / or dynamic, quality control measurement that is highly sensitive to secreted biomarkers with complex biochemical signatures. Measurement may be, in non-limiting examples, real-time measurement, continuous measurement, and / or a measurement s) taken at any desired interval. Systems and methods of the invention enable broad biomolecular detection such that unanticipated or unidentified biomarkers may be characterized for better process understanding.

[0062] In exemplary embodiments, systems and methods of the invention provide solutions to the sample preparation bottleneck when utilizing modem mass spectrometry for sample analysis. Accordingly, the invention provides for accelerated mass spectrometry workflows applicable to measuring cell-state, (e.g. proliferation, confluence, differentiation, activation), Critical Process Parameters (CPPs), and to Critical Quality Attributes (CQAs) identification and Monitoring.

[0063] Systems for sample processing

[0064] FIG. 1 illustrates one embodiment of systems of the invention. Systems of the invention may be embodied in a microfluidic chip design, such that the systems are capable of direct / indirect integration with cell culture systems, for example, well plates, culture dishes or flasks, stirred flask bioreactors, and direct / indirect interfacing with analysis systems. Systems of the invention provide for an integrated microfluidic sample processing and analysis workflow.

[0065] In one aspect, the system 100 includes a sample inlet 101 configured to receive a fluidic sample, such that the sample inlet 101 is in communication with a first filter 103. The first filter 103 allows for diffusion of molecules under a first molecular weight threshold across the first filter 103. The system includes a carrier channel 105 in communication with the first filter 103 via flow of a carrier fluid through the carrier channel 105 along the first filter 103. In nonlimiting examples, the flow of the carrier fluid along the first filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. In this way, when the sample inlet 101 receives a fluidic sample, molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter 103 into the carrier fluid of the carrier channel 105. Flow of the fluidic sample from the inlet 101 of the system to the first filter 103 is depicted in FIG. 1 as a gray striped arrow.

[0066] The system includes a second filter 107 in communication with the carrier channel 105 via flow of the carrier fluid and positioned downstream of the first filter 103 along the carrier channel 105, such that the second filter 107 allows for diffusion of molecules below a second molecular weight threshold across the second filter 107. Accordingly, when the carrier fluid flows along the second filter 107, molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter 107 into a conditioning fluid. In non-limiting examples, the flow of the carrier channel may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

[0067] In some embodiments, the second filter 107 is in communication with a conditioning channel 109 via flow of the conditioning fluid along the second filter 107. Accordingly, when the carrier fluid flows along the second filter 107, molecules from the fluidic sample in the carrier fluid that are below the second molecular weight threshold diffuse across the second filter 107 into the conditioning fluid of the conditioning channel 109. In non-limiting examples, the flow of the conditioning fluid along the second filter may be one or more of parallel flow, cross-flow, coflow, counterflow, and / or tangential flow.

[0068] In some embodiments, the flow of the carrier channel 105 and the conditioning channel 109 are parallel to one another along the second filter 107. In other embodiments the flow of the carrier fluid in the carrier channel and the conditioning fluid in the conditioning channel are counter to one another. In some embodiments, the flow of the carrier channel and the conditioning channel is a cross-flow orientation.

[0069] The carrier channel includes one or more inlets 111 and one or more outlets 113. The carrier channel is configured to transport molecules in the fluidic sample through the system via the carrier fluid. In some embodiments, the carrier channel is configured to transport molecules diffused into the carrier fluid from the sample inlet and through the first filter. As illustrated by solid arrows designating the direction of carrier fluid flow in FIG. 1, in some embodiments, the flow of the carrier fluid may be through the carrier channel inlet 111, along the first filter 103, i.e. parallel to the first filter, to the second filter 107, along the second filter, i.e. parallel to the second filter, and to the carrier channel outlet 113. Diffusion of molecules below a first molecular weight threshold from the fluidic sample across the first filter and into the carrier channel fluid is depicted by cross-hatched arrows. Likewise, diffusion of molecules below a second molecular weight threshold from the carrier fluid across the second filter and into the conditioning fluid are depicted by cross-hatched arrows. In some embodiments, the carrier channel is configured to transport molecules in a fluidic sample introduced directly into the carrier channel. In non-limiting examples, the flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

[0070] The conditioning channel 109 may include one or more inlets 115 and one or more outlets 117. Flow of conditioning fluid within the conditioning channel 109 may be from the conditioning channel inlet 115, across the second filter 107, i.e. substantially parallel to the second filter, to the conditioning channel outlet 117 as depicted by gray arrows in FIG. 1. In nonlimiting examples, the flow of the conditioning fluid along the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. In this way, in one embodiment, tangential flow filtration of the fluidic sample from the inlet across the first filter into the carrier channel, and from the carrier channel across the second filter into the conditioning channel is achieved. Accordingly, the molecules and / or material below a first molecular weight threshold that diffuse from the sample inlet across the first filter and into the carrier channel are transported toward the second filter. Where applicable, molecules and / or material below a second molecular weight threshold then diffuse from the carrier fluid across the second filter into the conditioning channel while the molecules from the conditioning fluid diffuse into the carrier fluid. As a result, the molecules / material remaining in the carrier channel are actively conditioned before analysis. In a non-limiting example, the molecules from the conditioning fluid that diffuse across the second filter into the carrier fluid may be mass spectrometry signal enhancing molecules.

[0071] In some embodiments, the system may include one or more reservoirs. For example, the system may include a carrier fluid reservoir, a conditioning fluid reservoir, a reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or a reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. In some embodiments, the sample collected and / or captured is analyzed via orthogonal methods.

[0072] The system is capable of monitoring and controlling the pressure within all channels and / or wells to achieve a desired sample processing or sample clean-up. In some embodiments, a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel is controlled via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

[0073] In particular, the pressure of the sample inlet / well, the conditioner well / inlet, and / or carrier channel may be modulated such that the pressure difference between each side of the first and / or second filter can be adjusted or tuned. This ensures that flow across the first and / or second filter, e g. trans-membrane flow, can be controlled such that the sample will flow from the sample inlet or well, to the carrier stream, from the carrier stream to the conditioning channel, and / or from the conditioning channel to the carrier stream. For example, controlling the pressure difference across the first filter or membrane, ensures that cytokines and low molecular weight contaminants flow into the carrier stream. Likewise, controlling the pressure difference across the second filter or membrane ensures that low molecular weight contaminants are removed.

[0074] The pressure controlling means may be any flow control mechanism as is known to persons skilled in the art. In non-limiting examples, the pressure controlling means may be one or more pinch valves positioned within any of the sample inlet, the carrier channel, and / or the conditioning channel. The pressure control mechanism may be any combination of valves and pumps, for example a centrifugal or volumetric pump, configured to adjust and control fluid flow pressure within the system. Further, the system may include a means, for example one or more pressure sensors, for monitoring the fluid pressure to / from the inlets / outlet, and within the carrier fluid channel and / or the conditioning channel. Thus, operation of the one or more flow control mechanism controls the various fluid flow through the system.

[0075] In some embodiments, one or more pinch valves are used to modulate the pressure on the conditioner channel to increase the pressure such that the pressure matches the pressure in an ESLMS sample flow loop. In some embodiments, the system operates in a pressure range from 2-400 kPa, as determined by a total pressure drop from the sample inlet to the ESLMS inlet.

[0076] In some embodiments, the system operates at an elevated pressure in the conditioning channel and in the carrier channel. In non-limiting examples, the system may operate at pressures in the range from sub-atmospheric down to 1 / 100 of an atmosphere (10A3 Pa) to above atmospheric, i.e. up 10 atmospheres (10A6 Pa) or higher, i.e. 10A7 Pa). Importantly, the system allows for controlling and balancing the pressure of the sample inlet, the carrier channel fluid, and / or the conditioning channel fluid, e.g. trans-membrane fluid flow, as necessary for stable operation of the system and efficient sample processing.

[0077] In some embodiments, the carrier fluid comprises one or more reagents. In some embodiments, when the carrier fluid flows along the first filter and / or the second filter, then the one or more reagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel. Thereby, a chemical reaction in the well and / or the conditioning channel may be facilitated. Further, system calibration and / or analyte quantification may be facilitated. In non-limiting examples, the reagents may be one or more of organic solvents, enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules. For example, the carrier fluid in the carrier channel may include enzymatic molecules which are able to diffuse across the first and / or second filter to facilitate one or more desired chemical reactions with the fluidic sample introduced into the sample inlet and / or with the conditioning fluid in the conditioning channel.

[0078] The conditioning fluid may include reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, such that when the conditioning fluid flows along the second filter then the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel. For example, the conditioning fluid may include analytical standards or additive molecules which may be used for calibration or quantitation. In specific examples, the additive molecules may be one or more of a fluorescent molecular tag, an analytical standard, an organic acid, such as ammonium acetate, an organic solvent, such as methanol, a tandem mass tag (TMT), and / or a supercharging reagent. The conditioning fluid may include the mass spectrometer signal enhancing components, such as 3- nitrobenzyle alcohol (m-NBA), organic solvents, organic acids, inorganic acids, volatile salts, ammonium acetate, biological standards, conjugated antibodies, fluorescent dyes, molecular tags, or a combination thereof), which may lower the detection limit, improve signal to noise ratio, shift charge distributions to mitigate effects of unwanted components, and the like. The specific molecules, e.g., fluorescent tags, analytical standards, organic acids, organic solvents, TMTs, and / or a supercharging reagent etc., may diffuse through the conditioning channel across the second filter to be introduced into the carrier fluid within the carrier channel before the carrier fluid flows through the carrier channel outlet.

[0079] For the application of the system and methods to analysis via mass spectrometry, the conditioning fluid may include, organic acids such as acetic acid, trifluoroacetic acid (TFA), formic acid, etc. (e.g., 0% to 100%), which can aid in protonation of biomolecules, for example. The conditioning fluid may also include one or more of ammonium acetate (e.g., 0% to 100%), m-NBA (e.g., 0% to 100%), propylene carbonate (e.g., 0% to 100%), ethylene carbonate (e.g., 0% to 100%), sulofane (e.g., 0% to 100%), and organic solvents such as methanol, acetonitrile, isopropyl alcohol (IP A), chloroform, acetone, N-methyl-2-pyrrolidone (NMP), etc. (e.g., 0% to 100%), chemical standards (i.e. to reduce instrument drift and enable quantitative analysis), and a combination thereof. Ammonium acetate can increase the acidity in the electrospray plume, enhance protonation, and / or reduce formation of salt adducts. m-NBA can increase the charge state in non-denaturing fluid samples. Methanol, and other organic solvents, may be used to selectively remove biomolecules with preferential solubility in the organic solvent and also to shift the charge state distribution of larger biomolecules like proteins through denaturing and unfolding effects on the molecule. In the case of in-line HPLC or direct ESI-MS analysis, chemical standards may be added to the conditioning flow to help with quantification of sample concentration and accurate mass identification.

[0080] A carrier fluid contact time with the first and / or second filters, also referred to as residence time, may be defined by varying, for example, the carrier channel dimensions. The carrier fluid contact time with the first and / or second filter, and thus the fluidic sample as the sample flows through the device, may be defined by the amount of time the carrier fluid is in contact with the first and / or second filter. Thus, in some embodiments, the contact time is defined by a degree of communication between the carrier channel and the first and / or second filter. For example, the degree of communication may be a contact area between the carrier channel and the first and / or second filter. The degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer and / or flux across the respective filter that takes place. Accordingly, the contact time of the carrier fluid may be controlled and / or defined by varying characteristics of the portion of the carrier channel in communication with the first and / or second filter. In some embodiments, the contact time, i.e. residence time, of the carrier fluid along the first filter is different from the residence time along the second filter.

[0081] In some embodiments, the carrier fluid contact time with the first and / or second filter may be defined by the length of the carrier channel along the first and / or second filter. In some embodiments, the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter. In some embodiments, the length of the carrier channel along the first filter is the same as the length of the carrier channel along the second filter. In other embodiments, the contact time of the carrier fluid and the first and / or second filter may be defined by changing the cross sectional area of the carrier channel where the channel is in communication with the first and / or second filter. Thus, in some embodiments, the crosssection of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter. In other embodiments, the cross-section of the carrier channel along the first filter is the same as the cross-section of the carrier channel along the second filter. As such, in non-limiting examples, the carrier channel may have varying lengths, and or varying cross-sectional areas, in relation to where the carrier channel is in communication with the first and / or second filter. However, the cross-sectional area and / or length of the carrier channel may be varied at any point in the channel to achieve a desired contact time between the carrier fluid and the first and / or second filter. Further, the variable length and / or cross sectional area may operate to increase and / or decrease flow of the carrier fluid to define the contact time between the carrier fluid, the first and / or second filter, and / or the fluidic sample.

[0082] In some embodiments, the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time of the carrier fluid along each filter. In specific embodiments, the residence time of the carrier fluid along the first filter is different from the residence time of the carrier fluid along the second filter.

[0083] As disclosed herein, the first filter and / or the second filter may be a size-selective filter. For example, the first filter may be a size-selective filter with a first molecular size separation threshold that allows diffusion of molecules with a size less than the size separation threshold from the well and / or sample inlet through the first filter into the carrier channel. The second filter may be a size-selective filter with a second size separation threshold that allows diffusion of molecules with a size less than the size separation threshold from the carrier fluid through the second filter into the conditioning channel. In non-limiting examples, the first filter may have a first molecular weight threshold of greater than 35 kDa such that molecules below 35 kDa diffuse across the first filter to the carrier fluid within the carrier channel. Likewise, in some embodiments, the second filter may have a second molecular weight threshold of 5kDa such that molecules below 5kDa diffuse from the carrier fluid in the carrier channel across the second filter into the conditioning fluid within the conditioning fluid channel.

[0084] The system provides for optimizing the filters based on a desired molecular separation. For example, the first filter and / or the second filter may be changeable and / or optimized to achieve separation based on a particular molecular weight threshold. As disclosed herein, the filters may be semi-permeable membranes modified for separation of molecules within the fluidic sample based on, for example, chemical, chemical structure, size, bioaffinity, and / or chirality. For example, the first filter may be configured such that cells and cell debris do not permeate across the first filter, and only smaller size-specific biomolecules permeate through the first filter into the carrier channel before being transported towards the second filter. The first filter may be configured to capture a range of cells shapes, cell sizes, and / or a range of cell rigidity, for example, thus removing high molecular weight, high abundance immunoproteins and cellular debris. The system could also be applied to vesicles and other biomolecule carriers.

[0085] The second filter may be configured to remove smaller interfering molecules. For example, the second filter may be configured to remove unwanted components present in the fluidic sample (e.g., remove or reduce the amount of components that can interfere with detection of the desired signal such as salts which can inhibit mass spectrometry signals), while retaining relatively larger components such as biomolecules as compared to smaller components (e g., small organic molecules). The second filter may be used for active sample clean up as well as active sample conditioning for analysis. For example, the second filter may remove smaller molecular weight components such as interfering salts and metabolites. In some embodiments, the conditioning channel may provide for infusing into the carrier channel, via diffusion through the second filter, supercharging agents to enhance ESI-MS signal in ESI-MS analysis.

[0086] The size-selective filter may be one or more semi-permeable membranes which allow one or more size-specific molecules to pass through. The size-selective filter may be one or more selectively permeable membranes. As disclosed herein, the selectively permeable membrane may function to separate unwanted components in the fluidic sample from those of interest and / or to cause the introduction of components to the fluidic sample to enhance detectability. For example, the permeable membrane may made of materials such as aluminum oxide, for example anodized porous alumina, etched silicon, perforated polymer (e.g., track etch membranes), cellulose, and zeolite, porous metal (e.g., nanoporous copper), porous graphene and graphene oxide, or a packed bed of various materials.

[0087] The membrane may be made of or coated with a material that aids the formation of a conditioned fluid sample. For example, the selectively permeable membrane may be made of or coated with a hydrophobic material / hydrophilic material, lipophilic material / lipophobic material, inert material, comprising selectively (positively or negatively) charged chemical compounds, electrically conducting, semiconducting or insulating material, and / or combinations thereof. The selectively permeable membrane may have a porosity of about 5% to 95%. As described in more detail herein, the selectively permeable membrane may have a thickness of about 1 nm to 10 pm, a length of about 10 pm to 50 mm, and a width of about 10 pm to 10 mm. The first filter and the second filter may have the same or different separation thresholds. In some embodiments, the threshold of the first filter is the same as the threshold of the second filter. For example, the first filter and the second filter may have a molecular weight threshold, such that the molecular weight threshold of the first filter is different than the molecular weight threshold of the second filter. In some embodiments, the threshold of the first filter, for example the maximum size of molecules which could diffuse through the filter, may be equal to or greater than the maximum size of molecules that could diffuse through the second filter.

[0088] In some embodiments, the sample inlet is in communication with the first filter via a well. Accordingly, a fluidic sample may flow through the sample inlet to the well. As described in more detail below, the sample may be manually introduced into the well, for example via a pipette. Thus, in a non-limiting example, cell-laden media may be pipetted into the well such that biomolecules below a particular molecular weight threshold will diffuse through the filter, e.g. membrane, based on selectivity of the membrane, and into the carrier channel positioned beneath the well. Alternatively and / or additionally, the sample may be introduced into the system via an auto sampler. The fluidic sample may be flowed into the well, and / or transferred into the system via an automated sampler. In some embodiments, the system is coupled to an autosampler with a sample loop for serial analysis. In such an embodiment, the sample may be loaded onto the sample loop and periodically transferred into the well and / or directly into the carrier fluid. Accordingly, the system provides for direct interfacing with an auto sampler to enable high- throughput analysis.

[0089] The fluidic sample may be any sample. In non-limiting examples, the fluidic sample may be one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample. In some embodiments, the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure. Accordingly, in some embodiments, the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

[0090] In some embodiments, the well comprises one or more integrated electrodes configured for electrical lysis of the one or more cells within the sample, where the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well. Further, as disclosed in more detail herein, in some embodiments, the voltage and electric pulse parameters are adjustable according to an applicable lysis procedure. In non-limiting examples, gold may be used as the electrode material as it will not corrode when exposed to the fluidic sample has favorable electromigration properties to withstand the repetitive application of high voltages applied across the thin layer.

[0091] For example, lysis pulses may be applied between electrodes lining the well, in some embodiments. Prior to the lysis sequence, leads of the lysis power supply may be held at the same potential until a high voltage insulated gate bipolar transistor (IGBT) gate is closed according to the chosen pulse parameters. The pulse parameters may be one or more of voltage, duration, and frequency. Closing the gate completes the circuit internal to the lysis system, effectively draining the current across the high resistance network formed by the resistor and microfluidic channel. With electric leads on each side of the network, one electrode becomes the “source” and the other, the “drain”. This results in an electric potential difference between the electrodes sufficient for lysis. Once the gate is re-opened, there is no current flow and thus no voltage drop across the resistance network, allowing the “drain” to again float to an equal potential as the “source”.

[0092] As is known to persons skilled in the art, the lysis voltage may be applied via a high voltage power supply, such as a Stanford Research Systems PS350 High Voltage Power Supply (Stanford Research Systems, Sunnyvale, CA). The pulse duration, shape, and frequency may be controlled by a waveform generator, such as an Agilent 33250A waveform generator (Keysight Technologies, Santa Rosa, CA), as is known to persons skilled in the art. Further, a 5 Vpp, +2.5 V DC offset output signal may be supplied to fully open and close the IGBT gate. An IXYS IXYL60N450 IGBT (Littelfuse, Chicago, IL) enables high voltage, high power control with nanosecond switching times (MHz switching frequency). The lysis efficiency depends on an optimal combination of pulse amplitude, duration, and number for a given electrode configuration. For example, an applied voltage of 60 V, corresponding to an electric field across a 100 pm channel of 6 kV / cm, is within the electric field strength required for irreversible electroporation of mammalian cells.

[0093] For the application to analysis via ESLMS, when not in operation, the entire lysis circuit may be electrically isolated from the ESI electrical circuit by a switch to prevent uncontrolled electrolysis in the system, ensuring stability of the ESLMS signal. During the lysis sequence, the leads may be held at the same potential until the high voltage transistor (i.e., MOSFET or IGBT) completes the circuit according to the chosen pulse parameters, allowing the voltage to drain across the high resistance (e.g., lOMohm) resistor. To enable complete lysis agnostic to cell type, the voltage and pulse duration may be set as high as can be accommodated by the device. This limit is set by the electromigration properties of the electrode and electrochemical breakdown of the flow solution.

[0094] An alternative electrode configuration in which a thin dielectric layer separates the electrode from the well prevents both electrolysis and Joule heating by inhibiting current flow between the electrodes, allowing for high voltages and long pulse durations to be implemented. For analysis of cell-like objects (e g., extracellular vesicles, organelles, bacteria, and viruses), the relevant lysis procedure (e.g., voltage and electric pulse parameters) may be adjusted accordingly to disrupt the membrane of the given objects.

[0095] In some embodiments, the first filter and / or the second filter may comprise one or more capture features. In some embodiments, the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel. In some embodiments, the well may comprise one or more capture features. In non-limiting embodiments, the one or more capture features may comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, positioned within the well and / or the carrier channel, such that when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier. In a nonlimiting example, the microcarrier may be a spherical support matrix such that the capture features of the well are configured to capture and immobilize the cell microcarrier in preparation for lysing. The microcarrier may be nano / micro-particle or a biomolecule-loaded vesicle.

[0096] As disclosed herein, in some embodiments, the first filter and / or the second filter comprise a membrane configured to capture molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality. In some embodiments, the capture features of the first filter are different than the capture features of the second filter. The dimensions and / or type of capture features, e.g. physical barriers, and / or membrane modification, may be selected based on the type of molecule targeted for capture.

[0097] In some embodiments, the system may further include one or more analysis modules for molecular analysis of fluid obtained from the first reservoir, e.g. the reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or the second reservoir, e.g. the reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Molecular analysis may comprise detecting and / or analyzing one or more properties of an analyte. For example, the analyte may be an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action. Accordingly, the sample inlet may be configured to receive a sample collected from a microenvironment of a cell system. The system may be configured, in some embodiments, to detect and analyze an analyte associated with cell state characterization during product development and manufacture of advanced therapy products. In some embodiments, the sample collected and / or captured is analyzed via orthogonal methods.

[0098] In some embodiments, a sample with one or more molecules of interest is captured via localized sampling and introduced into the system for subsequent analysis via mass spectrometry. The mass spectrometry data may be analyzed via, for example, a machine learning algorithm. The machine learning algorithm may comprise, for example, a multivariant analysis to provide deep insights into cell state characterization, such as cell phenotype, cell differentiation, cell disease state, cell healthy state, cell therapy product efficacy, cell therapy product safety, cell therapy product potency and / or a correlation with a mechanism of cell action.

[0099] In some embodiments, the system is configured to continuously detect and / or analyze an analyte that is an indicator of the cell culture process. Specifically, systems of the invention may be configured to monitor a cell culture process to optimize a cell culture process parameter. For example, the cell culture parameter may be one or more of a feed rate, a cell seeding density, cell growth nutrients, and a media exchange rate. In some embodiments, the system is configured to detect and / or analyze an analyte that is an indicator of a temporal change in a secretome of a cell such that the system provides a direct representation of a dynamic cell state.

[0100] Further, systems of the invention may be configured to directly interface with the analysis module to measure, in real-time and / or dynamically, critical process parameters which affect the critical quality attributes such as secreted signaling and paracrine factors that cells release while developing in culture. Accordingly, in some embodiments, the one or more analysis modules are in communication with the first or second reservoir such that fluid from the carrier channel and / or the conditioning channel flows directly into a respective reservoir, and then to the analysis module. In some embodiments, the fluid from the carrier channel and / or the conditioning channel flows directly to the one or more analysis modules for analysis via ESI-MS, Raman, NMR, antibody assays, and the like. Thus, in some embodiments, the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays.

[0101] In some embodiments, the analysis module is a mass spectrometry system and the ion trapping system can include, but are not limited to, a time-of-flight (TOF) mass spectrometry system, an ion trap mass spectrometry system (IT-MS), a quadrapole (Q) mass spectrometry system, a magnetic sector mass spectrometry system, and an ion cyclotron resonance (ICR) mass spectrometry system, and combinations thereof. The mass spectrometry system and the ion trapping system can include an ion detector for recording the number of ions that are subjected to an arrival time or position in a mass spectrometry system, as is known by one skilled in the art. Ion detectors can include, for example, a microchannel plate multiplier detector, an electron multiplier detector, or a combination thereof. In addition, the mass spectrometry system may include vacuum system components and electric system components, as are known by one skilled in the art. In particular embodiments, the analysis module is an ESI-MS system, such that the system of the invention is directly coupled to inline an ESI-MS system for continuous ESI- MS sensing.

[0102] FIG. 2 illustrates one embodiment of the systems of the invention in which the microfluidic system includes an inline ESI-MS analysis module. The system 200 includes a sample inlet 101, with which to receive the fluidic sample. For example, the sample may contain biomarkers of a particular molecular weight, salts, and metabolites as constituents of the sample. The sample is received by the sample inlet of the system where a first filter 103 may remove high abundance immunoproteins and cell debris. The molecules below a first molecular weight threshold diffuse across the first filter 103 into the carrier fluid within the carrier channel 105. The carrier fluid may flow through the carrier channel at a pre-defined rate. In non-limiting examples, the carrier fluid may include a buffered solution, such as phosphate buffered saline (PBS) or water flowing at a rate of 10-50pL / hr. The carrier channel 105 is in communication with a second filter 107 downstream of the first filter 103, and in communication with a conditioning channel 109. In this way, active sample clean up and conditioning may take place via the second filter and the conditioning channel. For example, molecules below a second molecular weight threshold may diffuse from the carrier fluid across the second filter and into the conditioning fluid. For example, molecules less than 5kDa, such as interfering salts and metabolites, may diffuse across the second filter into the conditioning channel. Likewise, supercharging reagents in the conditioning fluid may diffuse across the second filter into the carrier fluid. The system may include or more carrier channel inlets 111 and one or more carrier channel outlets 113. The one or more outlets of the carrier channel may be in fluid communication with an analysis module, such as an inline ESLMS system. Likewise, the conditioning channel may also include one or more inlets 115 and one or more outlets 117.

[0103] FIG. 3 illustrates a top view of one embodiment of a microfluidic chip design of systems 300 of the invention, in which the system includes a microwell for loading the sample, for example via a pipette.

[0104] The system of the invention is capable of very small sample volumes. For example, in non-limiting embodiments, the sample volume received by the sample inlet is ~1 pL of liquid from a reactor microenvironment, which is then processed through the system for use in realtime and / or dynamic, direct-from-culture ESLMS analysis of, for example, CQA content of the bioreactor characterizing the status of cell development. Exploiting the concurrent sample cleanup and active conditioning capabilities of the system, the resultant processed sample provides for increased MS signal intensity and signaLto-noise ratio. As a result, sensitivity for low- concentration biomolecules is significantly improved, and multiple proteins may be detected from chemically complex samples.

[0105] Accordingly, systems of the invention are able to continuously capture the dynamic and heterogeneous nature of in vitro cell growth, especially internal metabolic processes, with a time scale on the order of minutes, using a reduced sample size. Thus, the systems of the invention allow for dynamic characterization of the cell state directly from the growth environment with untargeted detection, via ESLMS, of a sufficiently broad range of relevant biomarkers, including intracellular metabolites. Thus, as disclosed herein, systems of the invention include a microfluidic device capable of processing ultra-small sample volumes for direct infusion into analysis systems such as ESLMS. While systems of the invention may be optimized for ESI-MS analysis, it is important to note that, while the system may be configured for improved ESI-MS analysis, the system is capable of interfacing with any analysis system appropriate for a specific application.

[0106] In some embodiments, the system further comprises one or more flow valves and / or one or more pumps to control flow along one or more of the sample inlet, the well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules. Thus the valves are in fluid communication with the components of the system, i.e. the one or more reservoirs, the conditioning channel, the carrier channel, the sample inlet and the one or more pumps to thereby control a type, a rate, and a direction of fluid flow through the system.

[0107] In some embodiments, the fluidic sample is received by the inlet manually via a pipette or syringe. In other embodiments, the fluidic sample is received by the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet. In some embodiments, the system further comprises a bypass autosampler in fluidic communication with the carrier flow channel upstream of the first filter, wherein when the bypass autosampler is provided the fluidic sample, then the bypass autosampler periodically and automatically introduces the fluidic sample directly into the carrier flow channel, thereby bypassing the inlet.

[0108] FIG. 4 illustrates one embodiment of systems of the invention which includes an autosampler module. In non-limiting examples, the system includes a liquid handling module. The liquid handing module may include a sample loop and an autosampler robot. The module may load samples and then serially inject samples into the system. The autosampler may include a needle port, pump, and metering device for loading and injection of the sample into the microfluidic system of the invention. In some embodiments, the sample is injected directly into the carrier channel inlet as opposed to the sample inlet. Thus, the system may be integrated into a mass spectrometry workflow for improved sample clean-up and conditioning for analysis. The microfluidic chip of systems of the invention are thus reusable for multiple sequential measurements with no detectable carry-over between the runs.

[0109] Methods for sample processing

[0110] FIG. 5 illustrates one embodiment of methods of the invention. In one aspect, the method 500 includes providing 501 a system for sample processing as described herein. For example, the system may include a sample inlet configured to receive a fluidic sample and in communication with a first filter, such that the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter. The system may include a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter. As described herein, the system may further include a second filter in communication with the carrier channel via flow of the carrier fluid and downstream of the first filter along the carrier channel, such that the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter. In non-limiting examples, the flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

[0111] Systems of the invention may be embodied in a microfluidic chip design, such that the systems are capable of direct / indirect integration with cell culture systems, for example, well plates, culture dishes or flasks, stirred flask bioreactors, and direct / indirect interfacing with analysis systems. Systems of the invention provide for an integrated microfluidic sample processing and analysis workflow.

[0112] The method further includes inserting 503 a fluidic sample within the sample inlet, and flowing 505 carrier fluid through the carrier channel past the first filter and second filter, such that molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter into the carrier fluid of the carrier channel and molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

[0113] As disclosed herein, the system may include a conditioning channel in fluidic communication with the second filter, for example via parallel flow of the conditioning fluid along the second filter. Accordingly, when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid that are below the second molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel. Likewise, and as discussed in more detail herein, conditioning agents / molecules may flow from the conditioning fluid, across the second filter, and into the carrier fluid.

[0114] In some embodiments of the method, the flow of the carrier channel and the flow of the conditioning channel are in parallel planes to one another along the second filter. In other embodiments the flow of the carrier fluid in the carrier channel and the flow of the conditioning fluid in the conditioning channel are counter to one another. In some embodiments, the flow of the carrier channel and the conditioning channel is a cross-flow orientation.

[0115] As disclosed herein, the carrier channel may include one or more inlets and one or more outlets. The carrier channel is configured to transport molecules in the fluidic sample through the system via the carrier fluid. In some embodiments, the carrier channel is configured to transport molecules diffused into the carrier fluid from the sample inlet and through the first filter. In some embodiments, the flow of the carrier fluid may be through the carrier channel inlet, along the first filter, i.e. parallel to the first filter, to the second filter, along the second filter, i.e. parallel to the second filter, and to the carrier channel outlet. In some embodiments, the carrier channel is configured to transport molecules in a fluidic sample introduced directly into the carrier channel. In non-limiting examples, the flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

[0116] The conditioning channel may also include one or more inlets and one or more outlets. Flow of conditioning fluid within the conditioning channel may be from the conditioning channel inlet, across the second filter, i.e. substantially parallel to the second filter, to the conditioning channel outlet.

[0117] In this way, in some embodiments, tangential flow filtration of the fluidic sample from the inlet across the first filter into the carrier channel, and from the carrier channel across the second filter into the conditioning channel is achieved. Accordingly, the molecules and / or material below a first molecular weight threshold that diffuse from the sample inlet across the first filter and into the carrier channel are transported toward the second filter. Where applicable, molecules and / or material below a second molecular weight threshold then diffuse from the carrier fluid across the second filter into the conditioning channel. Conditioning reagents from the conditioning fluid may diffuse across the second filter into the carrier fluid. As a result, the molecules / material remaining in the carrier channel are actively conditioned before analysis. In a non-limiting example, the molecules from the conditioning fluid that diffuse across the second filter into the carrier fluid may be mass spectrometry signal enhancing molecules.

[0118] In some embodiments of the method, the system further comprises a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Accordingly, the method may further comprise the steps of collecting conditioning fluid from the conditioning channel that has flowed along the second filter and / or collecting the carrier fluid 507 from the carrier channel that has flowed along the first and second filters. In some embodiments, the method includes collecting conditioning fluid 509. The collected carrier fluid and / or the collected conditioning fluid may be analyzed. In some embodiments, the biomolecular content of both the collected carrier fluid and the conditioning fluid is analyzed.

[0119] In some embodiments of the method, the system may include one or more reservoirs. For example, the system may include a carrier fluid reservoir, a conditioning fluid reservoir, a reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or a reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters.

[0120] The system is capable of monitoring and controlling the pressure within all channels and / or wells to achieve a desired sample processing or sample clean-up. In some embodiments, the method includes controlling a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

[0121] In some embodiments, the method includes controlling the pressure of the sample well, the conditioner well / inlet, and / or carrier channel may such that the pressure difference between each side of the first and / or second filter can be adjusted or tuned. This ensures that flow across the first and / or second filter, e.g. trans-membrane flow, can be controlled such that the sample will flow from the sample inlet or well, to the carrier stream, or from the carrier stream to the conditioning channel, or from the conditioning channel to the carrier stream. For example, controlling the pressure difference across the first filter or membrane, ensures that cytokines and low molecular weight contaminants flow into the carrier stream. Likewise, controlling the pressure difference across the second filter or membrane ensures that low molecular weight contaminants are removed.

[0122] The pressure controlling means may be any flow control mechanism as is known to persons skilled in the art. In non-limiting embodiments, the pressure controlling means comprises one or more pinch valves positioned within any of the sample inlet, the carrier channel, and / or the conditioning channel. The pressure control mechanism may be any combination of valves and pumps, for example a centrifugal or volumetric pump, configured to adjust and control fluid flow pressure within the system. Further, the system may include a means, for example one or more pressure sensors, for monitoring and controlling the fluid pressure to / from the inlets / outlet, and within the carrier fluid channel and / or the conditioning channel. Thus, operation of the one or more flow control mechanism controls the various fluid flow through the system.

[0123] In some embodiments, one or more pinch valves are used to modulate the pressure on the conditioner channel to increase the pressure such that the pressure matches the pressure in an ESI-MS sample flow loop. In some embodiments, the system operates in a pressure range from 2-400 kPa, as determined by a total pressure drop from the sample inlet to the ESI-MS inlet.

[0124] In some embodiments, the system operates at an elevated pressure in the conditioning channel and in the carrier channel. In non-limiting examples, the system may operate at pressures in the range from sub-atmospheric down to 1 / 100 of an atmosphere (10A3 Pa) to above atmospheric, i.e. up 10 atmospheres (10A6 Pa) or higher, i.e. 10A7 Pa). Importantly, the system allows for controlling and balancing the pressure of the sample inlet, the carrier channel fluid, and / or the conditioning channel fluid, e.g. trans-membrane fluid flow, as necessary for stable operation of the system and efficient sample processing.

[0125] As disclosed in detail herein, in some embodiments of the method, the carrier fluid comprises one or more reagents, such that during the flowing step one or more reagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel. Thereby, a chemical reaction in the well and / or the conditioning channel may be facilitated. Further, system calibration and / or analyte quantification may be facilitated. In non-limiting examples, the reagents may be one or more of organic solvents, enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules. For example, the carrier fluid in the carrier channel may include enzymatic molecules which are able to diffuse across the first and / or second filter to facilitate one or more desired chemical reactions with the fluidic sample introduced into the sample inlet and / or with the conditioning fluid in the conditioning channel. The conditioning fluid may include reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, such that, during the flowing step, the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel. For example, the conditioning fluid may include analytical standards or additive molecules which may be used for calibration or quantitation. In specific examples, the additive molecules may be one or more of a fluorescent molecular tag, an analytical standard, an organic acid, such as ammonium acetate, an organic solvent, such as methanol, a tandem mass tag (TMT), and / or a supercharging reagent. The conditioning fluid may include signal enhancing components, such as 3 -nitrob enzyle alcohol (m-NBA), organic solvents, organic acids, inorganic acids, volatile salts, ammonium acetate, biological standards, conjugated antibodies, fluorescent dyes, molecular tags, or a combination thereof), which may lower the detection limit, improve signal to noise ratio, shift charge distributions to mitigate effects of unwanted components, and the like. The specific molecules, e.g., fluorescent tags, analytical standards, organic acids, organic solvents, TMTs, and / or a supercharging reagent etc., may diffuse through the conditioning channel across the second filter to be introduced into the carrier fluid within the carrier channel before the carrier fluid flows through the carrier channel outlet.

[0126] For the application of the system and methods to analysis via mass spectrometry, the conditioning fluid may include, organic acids such as acetic acid, trifluoroacetic acid (TFA), formic acid, etc. (e.g., 0% to 100%), which can aid in protonation of biomolecules, for example. The conditioning fluid may also include one or more of ammonium acetate (e.g., 0% to 100%), m-NBA (e.g., 0% to 100%), propylene carbonate (e.g., 0% to 100%), ethylene carbonate (e.g., 0% to 100%), sulofane (e.g., 0% to 100%), and organic solvents such as methanol, acetonitrile, isopropyl alcohol (IP A), chloroform, acetone, N-methyl-2-pyrrolidone (NMP), etc. (e.g., 0% to 100%), chemical standards (i.e. to reduce instrument drift and enable quantitative analysis), and a combination thereof. Ammonium acetate can increase the acidity in the electrospray plume, enhance protonation, and / or reduce formation of salt adducts. m-NBA can increase the charge state in non-denaturing fluid samples. Methanol, and other organic solvents, may be used to selectively remove biomolecules with preferential solubility in the organic solvent and also to shift the charge state distribution of larger biomolecules like proteins through denaturing and unfolding effects on the molecule. In the case of in-line HPLC or direct ESI-MS analysis, chemical standards may be added to the conditioning flow to help with quantification of sample concentration and accurate mass identification.

[0127] During the flowing step, a carrier fluid contact time, also referred to as residence time, may be defined by varying, for example, the carrier channel dimensions. The carrier fluid contact time with the first and / or second filter, and thus the fluidic sample as the sample flows through the device, may be defined by the amount of time the carrier fluid is in contact with the first and / or second filter. Thus, in some embodiments, the contact time is defined by a degree of communication between the carrier channel and the first and / or second filter. Accordingly, the contact time of the carrier fluid may be controlled and / or defined by varying characteristics of the portion of the carrier channel in communication with the first and / or second filter. In some embodiments, the contact time, i.e. residence time, of the carrier fluid along the first filter is different from the residence time along the second filter.

[0128] In some embodiments of the method, the carrier fluid contact time with the first and / or second filter may be defined by the length of the carrier channel along the first and / or second filter. In some embodiments, the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter. In some embodiments, the length of the carrier channel along the first filter is the same as the length of the carrier channel along the second filter. In other embodiments, the contact time of the carrier fluid and the first and / or second filter may be defined by changing the cross sectional area of the carrier channel where the channel is in communication with the first and / or second filter. Thus, in some embodiments, the cross-section of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter. In other embodiments, the cross-section of the carrier channel along the first filter is the same as the cross-section of the carrier channel along the second filter.

[0129] As such, in non-limiting examples, the carrier channel may have varying lengths, and or varying cross-sectional areas, in relation to where the carrier channel is in communication with the first and / or second filter. However, the cross-sectional area and / or length of the carrier channel may be varied at any point in the channel to achieve a desired contact time between the carrier fluid and the first and / or second filter. Further, the variable length and / or cross sectional area may operate to increase and / or decrease flow of the carrier fluid to define the contact time between the carrier fluid, the first and / or second filter, and / or the fluidic sample. In some embodiments, the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time of the carrier fluid along each filter. In specific embodiments, the residence time of the carrier fluid along the first filter is different from the residence time of the carrier fluid along the second filter.

[0130] As disclosed herein, the first filter and / or the second filter may be a size-selective filter. For example, the first filter may be a size-selective filter with a first molecular size separation threshold that allows diffusion of molecules with a size less than the size separation threshold from the well and / or sample inlet through the first filter into the carrier channel. The second filter may be a size-selective filter with a second size separation threshold that allows diffusion of molecules with a size less than the size separation threshold from the carrier fluid through the second filter into the conditioning channel. In non-limiting examples, the first filter may have a first molecular weight threshold of greater than 35 kDa such that molecules below 35 kDa diffuse across the first filter to the carrier fluid within the carrier channel. Likewise, in some embodiments, the second filter may have a second molecular weight threshold of 5kDa such that molecules below 5kDa diffuse from the carrier fluid in the carrier channel across the second filter into the conditioning fluid within the conditioning fluid channel.

[0131] The system provides for optimizing the filters based on a desired molecular separation. For example, the first filter and / or the second filter may be changeable and / or optimized to achieve separation based on a particular molecular weight threshold. As disclosed herein, the filters may be semi-permeable membranes modified for separation of molecules within the fluidic sample based on, for example, chemical, chemical structure, size, bioaffinity, and / or chirality. For example, the first filter may be configured such that cells and cell debris do not permeate across the first filter, and only smaller size-specific biomolecules permeate through the first filter into the carrier channel before being transported towards the second filter. The first filter may be configured to capture a range of cells shapes, cell sizes, and / or a range of cell rigidity, for example, thus removing high molecular weight, high abundance immunoproteins and cellular debris.

[0132] The second filter may be configured to remove smaller interfering molecules. For example, the second filter may be configured to remove unwanted components present in the fluidic sample (e.g., remove or reduce the amount of components that can interfere with detection of the desired signal such as salts which can inhibit mass spectrometry signals), while retaining relatively larger components such as biomolecules as compared to smaller components (e.g., small organic molecules). The second filter may be used for active sample clean up as well as active sample conditioning for analysis. For example, the second filter may remove smaller molecular weight components such as interfering salts and metabolites. In some embodiments, the conditioning channel may provide for infusing into the carrier channel, via diffusion through the second filter, supercharging agents to enhance ESI-MS signal in ESI-MS analysis.

[0133] The size-selective filter may be one or more semi-permeable membranes which allow one or more size-specific molecules to pass through. The size-selective filter may be one or more selectively permeable membranes. As disclosed herein, the selectively permeable membrane may function to separate unwanted components in the fluidic sample from those of interest and / or to cause the introduction of components to the fluidic sample to enhance detectability. For example, the permeable membrane may made of materials such as aluminum oxide, for example anodized porous alumina, etched silicon, perforated polymer (e.g., track etch membranes), cellulose, and zeolite, porous metal (e.g., nanoporous copper), porous graphene and graphene oxide, or a packed bed of various materials.

[0134] The membrane may be made of or coated with a material that aids the formation of a conditioned fluid sample. For example, the selectively permeable membrane may be made of or coated with a hydrophobic material / hydrophilic material, lipophilic material / lipophobic material, inert material, comprising selectively (positively or negatively) charged chemical compounds, electrically conducting, semiconducting or insulating material, and / or combinations thereof. The selectively permeable membrane may have a porosity of about 5% to 95%. As describe in more detail herein, the selectively permeable membrane may have a thickness of about 1 nm to 10 pm, a length of about 10 pm to 50 mm, and a width of about 10 pm to 10 mm.

[0135] The first filter and the second filter may have the same or different separation thresholds. In some embodiments, the threshold of the first filter is the same as the threshold of the second filter. For example, the first filter and the second filter may have a molecular weight threshold, such that the molecular weight threshold of the first filter is different than the molecular weight threshold of the second filter. In some embodiments, the threshold of the first filter, for example the maximum size of molecules which could diffuse through the filter, may be equal to or greater than the maximum size of molecules that could diffuse through the second filter. In some embodiments of the method, the sample inlet is in communication with the first filter via a well. Accordingly, a fluidic sample may flow through the sample inlet to the well. As described in detail herein, the sample may be manually introduced into the well, for example via a pipette. Thus, in a non-limiting example, cell-laden media may be pipetted into the well such that biomolecules below a particular molecular weight threshold will diffuse through the filter, e.g. membrane, based on selectivity of the membrane, and into the carrier channel positioned beneath the well. Alternatively and / or additionally, the sample may be introduced into the system via an auto sampler. The fluidic sample may be flowed into the well, and / or transferred into the system via an automated sampler. In some embodiments, the system is coupled to an autosampler with a sample loop for serial analysis. In such an embodiment, the method includes loading the sample may onto the sample loop and periodically transferring a fluidic sample into the well and / or directly into the carrier fluid. Accordingly, the method provides for direct interfacing with an auto sampler to enable high-throughput analysis.

[0136] The fluidic sample may be any sample. In non-limiting examples, the fluidic sample may be one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample. In some embodiments, the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure. Accordingly, in some embodiments of the method, the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

[0137] In some embodiments of the method, the well comprises one or more integrated electrodes configured for electrical lysis of the one or more cells within the sample, where the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well. Further, as disclosed in more detail herein, in some embodiments, the voltage and electric pulse parameters are adjustable according to an applicable lysis procedure. In nonlimiting examples, gold may be used as the electrode material as it will not corrode when exposed to the fluidic sample has favorable electromigration properties to withstand the repetitive application of high voltages applied across the thin layer. For example, lysis pulses may be applied between electrodes lining the well, in some embodiments. Prior to the lysis sequence, leads of the lysis power supply may be held at the same potential until a high voltage insulated gate bipolar transistor (IGBT) gate is closed according to the chosen pulse parameters. The pulse parameters may be one or more of voltage, duration, and frequency. Closing the gate completes the circuit internal to the lysis system, effectively draining the current across the high resistance network formed by the resistor and microfluidic channel. With electric leads on each side of the network, one electrode becomes the “source” and the other, the “drain”. This results in an electric potential difference between the electrodes sufficient for lysis. Once the gate is reopened, there is no current flow and thus no voltage drop across the resistance network, allowing the “drain” to again float to an equal potential as the “source”.

[0138] As is known to persons skilled in the art, the lysis voltage may be applied via a high voltage power supply, such as a Stanford Research Systems PS350 High Voltage Power Supply (Stanford Research Systems, Sunnyvale, CA). The pulse duration, shape, and frequency may be controlled by a waveform generator, such as an Agilent 33250A waveform generator (Keysight Technologies, Santa Rosa, CA), as is known to persons skilled in the art. Further, a 5 Vpp, +2.5 V DC offset output signal may be supplied to fully open and close the IGBT gate. An IXYS IXYL60N450 IGBT (Littelfuse, Chicago, IL) enables high voltage, high power control with nanosecond switching times (MHz switching frequency). The lysis efficiency depends on an optimal combination of pulse amplitude, duration, and number for a given electrode configuration. For example, an applied voltage of 60 V, corresponding to an electric field across a 100 pm channel of 6 kV / cm, is within the electric field strength required for irreversible electroporation of mammalian cells.

[0139] For the application to analysis via ESLMS, when not in operation, the entire lysis circuit may be electrically isolated from the ESI electrical circuit by a switch to prevent uncontrolled electrolysis in the system, ensuring stability of the ESI-MS signal. During the lysis sequence, the leads may be held at the same potential until the high voltage transistor (i.e., MOSFET or IGBT) completes the circuit according to the chosen pulse parameters, allowing the voltage to drain across the high resistance (e.g., lOMohm) resistor. To enable complete lysis agnostic to cell type, the voltage and pulse duration may be set as high as can be accommodated by the device. This limit is set by the electromigration properties of the electrode and electrochemical breakdown of the flow solution.

[0140] An alternative electrode configuration in which a thin dielectric layer separates the electrode from the well prevents both electrolysis and Joule heating by inhibiting current flow between the electrodes, allowing for high voltages and long pulse durations to be implemented. For analysis of cell-like objects (e.g., extracellular vesicles, organelles, bacteria, and viruses), the relevant lysis procedure (e.g., voltage and electric pulse parameters) may be adjusted accordingly to disrupt the membrane of the given objects.

[0141] In some embodiments of the method, the first filter and / or the second filter may comprise one or more capture features. In some embodiments, the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel. In some embodiments, the well may comprise one or more capture features. In non-limiting embodiments, the one or more capture features may comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, positioned within the well and / or the carrier channel, such that when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier. In a non-limiting example, the microcarrier may be a spherical support matrix such that the capture features of the well are configured to capture and immobilize the cell microcarrier in preparation for lysing.

[0142] As disclosed herein, in some embodiments of the method, the first filter and / or the second filter comprise a membrane configured to capture molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality. In some embodiments, the capture features of the first filter are different than the capture features of the second filter. The dimensions and / or type of capture features, e.g. physical barriers, and / or membrane modification, may be selected based on the type of molecule targeted for capture.

[0143] In some embodiments of the method, the system may further include one or more analysis modules for molecular analysis of fluid obtained from the first reservoir, e.g. the reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or the second reservoir, e.g. the reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Molecular analysis may comprise detecting and / or analyzing one or more properties of an analyte. For example, the analyte may be an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action. Accordingly, the sample inlet may be configured to receive a sample collected from a microenvironment of a cell system. The system may be configured, in some embodiments, to detect and analyze an analyte associated with cell state characterization during product development and manufacture of advanced therapy products. In some embodiments, the sample collected and / or captured is analyzed via orthogonal methods.

[0144] In some embodiments, a sample with one or more molecules of interest is captured via localized sampling and introduced into the system for subsequent analysis via mass spectrometry. The mass spectrometry data may be analyzed via, for example, machine learning algorithm. The machine learning algorithm may comprise, for example, a multivariant analysis to provide deep insights into cell state characterization, such as cell phenotype, cell differentiation, cell disease state, cell healthy state, cell therapy product efficacy, cell therapy product safety, cell therapy product potency and / or a correlation with a mechanism of cell action.

[0145] In some embodiments of the method, the method includes continuously detecting and / or analyzing an analyte that is an indicator of the cell culture process. Specifically, systems of the invention may be configured to monitor a cell culture process to optimize a cell culture process parameter. For example, the cell culture parameter may be one or more of a feed rate, a cell seeding density, and a media exchange rate. In some embodiments, the system is configured to detect and / or analyze an analyte that is an indicator of a temporal change in a secretome of a cell such that the system provides a direct representation of a dynamic cell state.

[0146] Further, systems of the invention may be configured to directly interface with the analysis module such that the method measures, in real-time and / or dynamically, critical process parameters which affect the critical quality attributes such as secreted signaling and paracrine factors that cells release while developing in culture. Accordingly, in some embodiments of the method, the one or more analysis modules are in communication with the first or second reservoir such that fluid from the carrier channel and / or the conditioning channel flows directly into a respective reservoir, and then to the analysis module. In some embodiments, the fluid from the carrier channel and / or the conditioning channel flows directly to the one or more analysis modules for analysis via ESI-MS, Raman, NMR, antibody assays, and the like. Thus, in some embodiments, the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays. In some embodiments of the method, the analysis module is a mass spectrometry system and the ion trapping system can include, but are not limited to, a time-of-flight (TOF) mass spectrometry system, an ion trap mass spectrometry system (IT -MS), a quadrapole (Q) mass spectrometry system, a magnetic sector mass spectrometry system, and an ion cyclotron resonance (ICR) mass spectrometry system, and combinations thereof. The mass spectrometry system and the ion trapping system can include an ion detector for recording the number of ions that are subjected to an arrival time or position in a mass spectrometry system, as is known by one skilled in the art. Ion detectors can include, for example, a microchannel plate multiplier detector, an electron multiplier detector, or a combination thereof. In addition, the mass spectrometry system may include vacuum system components and electric system components, as are known by one skilled in the art. In particular embodiments, the analysis module is an ESIMS system, such that the system of the invention is directly coupled to inline an ESI-MS system for continuous ESI-MS sensing.

[0147] In some embodiments of the method, the sample may contain biomarkers of a particular molecular weight, salts, and metabolites as constituents of the sample. The sample may be received by the sample inlet of the system where a first filter may remove high abundance immunoproteins and cell debris. The molecules below a first molecular weight threshold then diffuse across the first filter into the carrier fluid within the carrier channel. The carrier fluid may flow through the carrier channel at a pre-defined rate. In non-limiting examples of the method, the carrier fluid may include a buffered solution, such as phosphate buffered saline (PBS) or water flowing at a rate of 10-50pL / hr. As described herein, the carrier channel is in communication with a second filter downstream of the first filter, and in communication with a conditioning channel. In this way, the method includes active sample clean up and conditioning via the second filter and the conditioning channel. For example, in some embodiments of the method, molecules below a second molecular weight threshold may diffuse from the carrier fluid across the second filter and into the conditioning fluid. For example, molecules less than 5kDa, such as interfering salts and metabolites, may diffuse across the second filter into the conditioning channel. Likewise, supercharging reagents in the conditioning fluid may diffuse across the second filter into the carrier fluid.

[0148] The method is capable of processing very small sample volumes. For example, in nonlimiting embodiments, the sample volume received by the sample inlet is ~1 pL of liquid from a reactor microenvironment, which is then processed through the system for use in real-time and / or dynamic, direct-from-culture ESI-MS analysis of, for example, CQA content of the bioreactor characterizing the status of cell development. Exploiting the concurrent sample clean-up and active conditioning capabilities of the system, the method provides a resultant processed sample for increased MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low- concentration biomolecules is significantly improved, and multiple proteins may be detected from chemically complex samples.

[0149] Accordingly, methods of the invention are able to continuously capture the dynamic and heterogeneous nature of in vitro cell growth, especially internal metabolic processes, with a time scale on the order of minutes, using a reduced sample size. Thus, the systems of the invention allow for dynamic characterization of the cell state directly from the growth environment with untargeted detection, via ESI-MS, of a sufficiently broad range of relevant biomarkers, including intracellular metabolites. Thus, as disclosed herein, systems of the invention include a microfluidic device capable of processing ultra-small sample volumes for direct infusion into analysis systems such as ESI-MS.

[0150] While methods of the invention may be optimized for ESI-MS analysis, it is important to note that, while the system may be configured for improved ESI-MS analysis, the system is capable of interfacing with any analysis system appropriate for a specific application.

[0151] In some embodiments of the method, the system further comprises one or more flow valves and / or one or more pumps to control flow along one or more of the sample inlet, the well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules. Thus the valves are in fluid communication with the components of the system, i.e. the one or more reservoirs, the conditioning channel, the carrier channel, the sample inlet and the one or more pumps to thereby control a type, a rate, and a direction of fluid flow through the system.

[0152] In some embodiments of the method, the fluidic sample is received by the inlet manually via a pipette or syringe. In other embodiments, the fluidic sample is received by the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet. In some embodiments, the system further comprises a bypass autosampler in fluidic communication with the carrier flow channel upstream of the first filter, wherein when the bypass autosampler is provided the fluidic sample, then the bypass autosampler periodically and automatically introduces the fluidic sample directly into the carrier flow channel, thereby bypassing the inlet. In non-limiting examples, the system includes a liquid handling module. The liquid handing module may include a sample loop and an autosampler robot. The module may load samples and then serially inject samples into the system. The autosampler may include a needle port, pump, and metering device for loading and injection of the sample into the microfluidic system of the invention. Thus, the system may be integrated into a mass spectrometry workflow for improved sample clean-up and conditioning for analysis. The microfluidic chip of systems of the invention are thus reusable for multiple sequential measurements with no detectable carry-over between the runs.

[0153] FIG. 6 illustrates a method of the invention according to one embodiment.

[0154] In another aspect, the invention provides a method 600 for sample processing. The method includes providing 601 a system for sample processing as disclosed herein. Specifically, the system includes a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter, and a bypass autosampler in fluidic communication with the carrier flow channel upstream of the first filter. Further, the system includes a second filter in communication with the carrier channel via flow of the carrier fluid and downstream of the first filter along the carrier channel, such that the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter. The method further includes introducing 603 the fluidic sample directly into the carrier flow channel via the bypass autosampler, thereby bypassing the sample inlet, and flowing 605 carrier fluid through the carrier channel past the first filter and second filter. In this way, molecules in the fluidic sample below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

[0155] In non-limiting examples, bypass autosampler is a liquid handling module. The liquid handing module may include a sample loop and an autosampler robot. The module may load samples and then serially inject samples into the system. The autosampler may include a needle port, pump, and metering device for loading and injection of the sample into the microfluidic system of the invention. As described in detail herein, the method may include analyzing 607 carrier fluid from the carrier channel. In some embodiments, the method includes collecting conditioning fluid, and analyzing the conditioning fluid 609. The collected carrier fluid and / or the collected conditioning fluid may be analyzed. In some embodiments, the biomolecular content of both the collected carrier fluid and the conditioning fluid is analyzed.

[0156] Systems of the invention may be embodied in a microfluidic chip design as disclosed herein, such that the systems are capable of direct / indirect integration with cell culture systems, for example, well plates, culture dishes or flasks, stirred flask bioreactors, and direct / indirect interfacing with analysis systems. Systems of the invention provide for an integrated microfluidic sample processing and analysis workflow.

[0157] As disclosed herein, the system may include a conditioning channel in fluidic communication with the second filter, for example via parallel flow of the conditioning fluid along the second filter. Accordingly, when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid that are below the second molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel. Likewise, and as discussed in more detail herein, conditioning agents / molecules may flow from the conditioning fluid, across the second filter, and into the carrier fluid.

[0158] In some embodiments of the method, the flow of the carrier channel and the flow of the conditioning channel are in parallel planes to one another along the second filter. In other embodiments the flow of the carrier fluid in the carrier channel and the flow of the conditioning fluid in the conditioning channel are counter to one another or in a cross-flow orientation.

[0159] As disclosed herein, the carrier channel may include one or more inlets and one or more outlets. The carrier channel is configured to transport molecules in the fluidic sample through the system via the carrier fluid. In some embodiments, the carrier channel is configured to transport molecules in a fluidic sample introduced directly into the carrier channel. The conditioning channel may also include one or more inlets and one or more outlets. Flow of conditioning fluid within the conditioning channel may be from the conditioning channel inlet, across the second filter, i.e. substantially parallel to the second filter, to the conditioning channel outlet. In nonlimiting examples, the flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow. In some embodiments of the method, the system further comprises a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Accordingly, the method may further comprise the steps of collecting conditioning fluid from the conditioning channel that has flowed along the second filter and / or collecting the carrier fluid 507 from the carrier channel that has flowed along the first and second filters.

[0160] In some embodiments of the method, the system may include one or more reservoirs. For example, the system may include a carrier fluid reservoir, a conditioning fluid reservoir, a reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or a reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters.

[0161] The system is capable of monitoring and controlling the pressure within all channels and / or wells to achieve a desired sample processing or sample clean-up. In some embodiments of the method, the method includes controlling a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

[0162] Thus, in some embodiments, the method includes controlling the pressure of the sample well, the conditioner well / inlet, and / or carrier channel such that the pressure difference between each side of the first and / or second filter can be adjusted or tuned. This ensures that flow across the first and / or second filter, e.g. trans-membrane flow, can be controlled such that the sample will flow from the sample inlet or well, to the carrier stream, or from the carrier stream to the conditioning channel, or from the conditioning channel to the carrier stream. For example, controlling the pressure difference across the first filter or membrane, ensures that cytokines and low molecular weight contaminants flow into the carrier stream. Likewise, controlling the pressure difference across the second filter or membrane ensures that low molecular weight contaminants are removed. The pressure controlling means may be any flow control mechanism as is known to persons skilled in the art. In non-limiting embodiments, the pressure controlling means comprises one or more pinch valves positioned within any of the sample inlet, the carrier channel, and / or the conditioning channel. The pressure control mechanism may be any combination of valves and pumps, for example a centrifugal or volumetric pump, configured to adjust and control fluid flow pressure within the system. Further, the system may include a means, for example one or more pressure sensors, for monitoring and controlling the fluid pressure to / from the inlets / outlet, and within the carrier fluid channel and / or the conditioning channel. Thus, operation of the one or more flow control mechanism controls the various fluid flow through the system.

[0163] In some embodiments, one or more pinch valves are used to modulate the pressure on the conditioner channel to increase the pressure such that the pressure matches the pressure in an ESI-MS sample flow loop. In some embodiments, the system operates in a pressure range from 2-400 kPa, as determined by a total pressure drop from the sample inlet to the ESI-MS inlet.

[0164] In some embodiments, the system operates at an elevated pressure in the conditioning channel and in the carrier channel. In non-limiting examples, the system may operate at pressures in the range from sub-atmospheric down to 1 / 100 of an atmosphere (10A3 Pa) to above atmospheric, i.e. up 10 atmospheres (10A6 Pa) or higher, i.e. 10A7 Pa). Importantly, the system allows for controlling and balancing the pressure of the sample inlet, the carrier channel fluid, and / or the conditioning channel fluid, e.g. trans-membrane fluid flow, as necessary for stable operation of the system and efficient sample processing.

[0165] As disclosed in detail herein, in some embodiments of the method, the carrier fluid comprises one or more reagents. Thereby, a chemical reaction in the conditioning channel may be facilitated. Further, system calibration and / or analyte quantification may be facilitated. In non-limiting examples, the reagents may be one or more of organic solvents, enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules. For example, the carrier fluid in the carrier channel may include enzymatic molecules which are able to diffuse across the first and / or second filter to facilitate one or more desired chemical reactions with the fluidic sample and / or with the conditioning fluid in the conditioning channel.

[0166] The conditioning fluid may include reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, such that, during the flowing step, the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel. For example, the conditioning fluid may include analytical standards or additive molecules which may be used for calibration or quantitation. In specific examples, the additive molecules may be one or more of a fluorescent molecular tag, an analytical standard, an organic acid, such as ammonium acetate, an organic solvent, such as methanol, a tandem mass tag (TMT), and / or a supercharging reagent. The conditioning fluid may include signal enhancing components, such as 3 -nitrob enzyle alcohol (m-NBA), organic solvents, organic acids, inorganic acids, volatile salts, ammonium acetate, biological standards, conjugated antibodies, fluorescent dyes, molecular tags, or a combination thereof), which may lower the detection limit, improve signal to noise ratio, shift charge distributions to mitigate effects of unwanted components, and the like. The specific molecules, e.g., fluorescent tags, analytical standards, organic acids, organic solvents, TMTs, and / or a supercharging reagent etc., may diffuse through the conditioning channel across the second filter to be introduced into the carrier fluid within the carrier channel before the carrier fluid flows through the carrier channel outlet.

[0167] For the application of the system and methods to analysis via mass spectrometry, the conditioning fluid may include, organic acids such as acetic acid, trifluoroacetic acid (TFA), formic acid, etc. (e.g., 0% to 100%), which can aid in protonation of biomolecules, for example. The conditioning fluid may also include one or more of ammonium acetate (e.g., 0% to 100%), m-NBA (e.g., 0% to 100%), propylene carbonate (e.g., 0% to 100%), ethylene carbonate (e.g., 0% to 100%), sulofane (e.g., 0% to 100%), and organic solvents such as methanol, acetonitrile, isopropyl alcohol (IP A), chloroform, acetone, N-methyl-2-pyrrolidone (NMP), etc. (e.g., 0% to 100%), chemical standards (i.e. to reduce instrument drift and enable quantitative analysis), and a combination thereof. Ammonium acetate can increase the acidity in the electrospray plume, enhance protonation, and / or reduce formation of salt adducts. m-NBA can increase the charge state in non-denaturing fluid samples. Methanol, and other organic solvents, may be used to selectively remove biomolecules with preferential solubility in the organic solvent and also to shift the charge state distribution of larger biomolecules like proteins through denaturing and unfolding effects on the molecule. In the case of in-line HPLC or direct ESI-MS analysis, chemical standards may be added to the conditioning flow to help with quantification of sample concentration and accurate mass identification. During the flowing step, a carrier fluid contact time, also referred to as residence time, may be defined by varying, for example, the carrier channel dimensions. The carrier fluid contact time with the first and / or second filter, and thus the fluidic sample as the sample flows through the device, may be defined by the amount of time the carrier fluid is in contact, with the first and / or second filter. Thus, in some embodiments, the contact time is defined by a degree of communication between the carrier channel and the first and / or second filter. For example, the degree of communication may be a contact area between the carrier channel and the first and / or second filter. The degree of communication may be a length and cross sectional area of the carrier channel, and / or the extent of mass transfer and / or flux across the respective filter that takes place. Accordingly, the contact time of the carrier fluid may be controlled and / or defined by varying characteristics of the portion of the carrier channel in communication with the first and / or second filter. In some embodiments, the contact time, i.e. residence time, of the carrier fluid along the first filter is different from the residence time along the second filter.

[0168] In some embodiments of the method, the carrier fluid contact time with the first and / or second filter may be defined by the length of the carrier channel along the first and / or second filter. In some embodiments, the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter. In some embodiments, the length of the carrier channel along the first filter is the same as the length of the carrier channel along the second filter. In other embodiments, the contact time of the carrier fluid and the first and / or second filter may be defined by changing the cross sectional area of the carrier channel where the channel is in communication with the first and / or second filter. Thus, in some embodiments, the cross-section of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter. In other embodiments, the cross-section of the carrier channel along the first filter is the same as the cross-section of the carrier channel along the second filter.

[0169] As such, in non-limiting examples, the carrier channel may have varying lengths, and or varying cross-sectional areas, in relation to where the carrier channel is in communication with the first and / or second filter. However, the cross-sectional area and / or length of the carrier channel may be varied at any point in the channel to achieve a desired contact time between the carrier fluid and the first and / or second filter. Further, the variable length and / or cross sectional area may operate to increase and / or decrease flow of the carrier fluid to define the contact time between the carrier fluid, the first and / or second filter, and / or the fluidic sample.

[0170] In some embodiments, the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time of the carrier fluid along each filter. In specific embodiments, the residence time of the carrier fluid along the first filter is different from the residence time of the carrier fluid along the second filter.

[0171] As disclosed herein, the first filter and / or the second filter may be a size-selective filter. For example, the first filter may be a size-selective filter with a first molecular size separation threshold that allows diffusion of molecules with a size less than the size separation threshold through the first filter. The second filter may be a size-selective filter with a second size separation threshold that allows diffusion of molecules with a size less than the size separation threshold from the carrier fluid through the second filter into the conditioning channel. In nonlimiting examples, the second filter may have a second molecular weight threshold of 5kDa such that molecules below 5kDa diffuse from the carrier fluid in the carrier channel across the second filter into the conditioning fluid within the conditioning fluid channel.

[0172] The system provides for optimizing the filters based on a desired molecular separation. For example, the first filter and / or the second filter may be changeable and / or optimized to achieve separation based on a particular molecular weight threshold. As disclosed herein, the filters may be semi-permeable membranes modified for separation of molecules within the fluidic sample based on, for example, chemical, chemical structure, size, bioaffinity, and / or chirality. For example, the first filter may be configured such that cells and cell debris do not permeate across the first filter, and only smaller size-specific biomolecules permeate through the first filter into the carrier channel before being transported towards the second filter. The first filter may be configured to capture a range of cells shapes, cell sizes, and / or a range of cell rigidity, for example, thus removing high molecular weight, high abundance immunoproteins and cellular debris.

[0173] The second filter may be configured to remove smaller interfering molecules. For example, the second filter may be configured to remove unwanted components present in the fluidic sample (e.g., remove or reduce the amount of components that can interfere with detection of the desired signal such as salts which can inhibit mass spectrometry signals), while retaining relatively larger components such as biomolecules as compared to smaller components (e.g., small organic molecules). The second filter may be used for active sample clean up as well as active sample conditioning for analysis. For example, the second filter may remove smaller molecular weight components such as interfering salts and metabolites. In some embodiments, the conditioning channel may provide for infusing into the carrier channel, via diffusion through the second filter, supercharging agents to enhance ESI-MS signal in ESI-MS analysis.

[0174] The size-selective filter may be one or more semi-permeable membranes which allow one or more size-specific molecules to pass through. The size-selective filter may be one or more selectively permeable membranes. As disclosed herein, the selectively permeable membrane may function to separate unwanted components in the fluidic sample from those of interest and / or to cause the introduction of components to the fluidic sample to enhance detectability. For example, the permeable membrane may made of materials such as aluminum oxide, for example anodized porous alumina, etched silicon, perforated polymer (e.g., track etch membranes), cellulose, and zeolite, porous metal (e.g., nanoporous copper), porous graphene and graphene oxide, or a packed bed of various materials.

[0175] The membrane may be made of or coated with a material that aids the formation of a conditioned fluid sample. For example, the selectively permeable membrane may be made of or coated with a hydrophobic material / hydrophilic material, lipophilic material / lipophobic material, inert material, comprising selectively (positively or negatively) charged chemical compounds, electrically conducting, semiconducting or insulating material, and / or combinations thereof. The selectively permeable membrane may have a porosity of about 5% to 95%. As describe in more detail herein, the selectively permeable membrane may have a thickness of about 1 nm to 10 pm, a length of about 10 pm to 50 mm, and a width of about 10 pm to 10 mm.

[0176] The first filter and the second filter may have the same or different separation thresholds. In some embodiments, the threshold of the first filter is the same as the threshold of the second filter. For example, the first filter and the second filter may have a molecular weight threshold, such that the molecular weight threshold of the first filter is different than the molecular weight threshold of the second filter. In some embodiments, the threshold of the first filter, for example the maximum size of molecules which could diffuse through the filter, may be equal to or greater than the maximum size of molecules that could diffuse through the second filter. The fluidic sample may be any sample. In non-limiting examples, the fluidic sample may be one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample.

[0177] In some embodiments of the method, the first filter and / or the second filter may comprise one or more capture features. In some embodiments, the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel. In some embodiments, the well may comprise one or more capture features. In non-limiting embodiments, the one or more capture features may comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, positioned within the well and / or the carrier channel, such that when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier. In a non-limiting example, the microcarrier may be a spherical support matrix such that the capture features of the well are configured to capture and immobilize the cell microcarrier in preparation for lysing.

[0178] As disclosed herein, in some embodiments of the method, the first filter and / or the second filter comprise a membrane configured to capture molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality. In some embodiments, the capture features of the first filter are different than the capture features of the second filter. The dimensions and / or type of capture features, e.g. physical barriers, and / or membrane modification, may be selected based on the type of molecule targeted for capture.

[0179] In some embodiments of the method, the system may further include one or more analysis modules for molecular analysis of fluid obtained from the first reservoir, e.g. the reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter, and / or the second reservoir, e.g. the reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters. Molecular analysis may comprise detecting and / or analyzing one or more properties of an analyte. For example, the analyte may be an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action. Accordingly, the sample inlet may be configured to receive a sample collected from a microenvironment of a cell system. The system may be configured, in some embodiments, to detect and analyze an analyte associated with cell state characterization during product development and manufacture of advanced therapy products.

[0180] In some embodiments, a sample with one or more molecules of interest is captured via localized sampling and introduced into the system for subsequent analysis via mass spectrometry. The mass spectrometry data may be analyzed via, for example, a machine learning algorithm. The machine learning algorithm may comprise, for example, a multivariant analysis to provide deep insights into cell state characterization, such as cell phenotype, cell differentiation, cell disease state, cell healthy state, cell therapy product efficacy, cell therapy product safety, cell therapy product potency and / or a correlation with a mechanism of cell action.

[0181] In some embodiments of the method, the method includes continuously detecting and / or analyzing an analyte that is an indicator of the cell culture process. Specifically, systems of the invention may be configured to monitor a cell culture process to optimize a cell culture process parameter. For example, the cell culture parameter may be one or more of a feed rate, a cell seeding density, and a media exchange rate. In some embodiments, the system is configured to detect and / or analyze an analyte that is an indicator of a temporal change in a secretome of a cell such that the system provides a direct representation of a dynamic cell state.

[0182] Further, systems of the invention may be configured to directly interface with the analysis module such that the method measures, in real-time and / or dynamically, critical process parameters which affect the critical quality attributes such as secreted signaling and paracrine factors that cells release while developing in culture. Accordingly, in some embodiments of the method, the one or more analysis modules are in communication with the first or second reservoir such that fluid from the carrier channel and / or the conditioning channel flows directly into a respective reservoir, and then to the analysis module. In some embodiments, the fluid from the carrier channel and / or the conditioning channel flows directly to the one or more analysis modules for analysis via ESI-MS, Raman, NMR, antibody assays, and the like. Thus, in some embodiments, the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays. In some embodiments of the method, the analysis module is a mass spectrometry system and the ion trapping system can include, but are not limited to, a time-of-flight (TOF) mass spectrometry system, an ion trap mass spectrometry system (IT -MS), a quadrapole (Q) mass spectrometry system, a magnetic sector mass spectrometry system, and an ion cyclotron resonance (ICR) mass spectrometry system, and combinations thereof. The mass spectrometry system and the ion trapping system can include an ion detector for recording the number of ions that are subjected to an arrival time or position in a mass spectrometry system, as is known by one skilled in the art. Ion detectors can include, for example, a microchannel plate multiplier detector, an electron multiplier detector, or a combination thereof. In addition, the mass spectrometry system may include vacuum system components and electric system components, as are known by one skilled in the art. In particular embodiments, the analysis module is an ESIMS system, such that the system of the invention is directly coupled to inline an ESI-MS system for continuous ESI-MS sensing.

[0183] In some embodiments of the method, the sample may contain biomarkers of a particular molecular weight, salts, and metabolites as constituents of the sample. The sample may be received by the sample inlet of the system where a first filter may remove high abundance immunoproteins and cell debris. The molecules below a first molecular weight threshold then diffuse across the first filter into the carrier fluid within the carrier channel. The carrier fluid may flow through the carrier channel at a pre-defined rate. In non-limiting examples of the method, the carrier fluid may include a buffered solution, such as phosphate buffered saline (PBS) or water flowing at a rate of 10-50pL / hr. As described herein, the carrier channel is in communication with a second filter downstream of the first filter, and in communication with a conditioning channel. In this way, the method includes active sample clean up and conditioning via the second filter and the conditioning channel. For example, in some embodiments of the method, molecules below a second molecular weight threshold may diffuse from the carrier fluid across the second filter and into the conditioning fluid. For example, molecules less than 5kDa, such as interfering salts and metabolites, may diffuse across the second filter into the conditioning channel. Likewise, supercharging reagents in the conditioning fluid may diffuse across the second filter into the carrier fluid.

[0184] The method is capable of processing very small sample volumes. For example, in nonlimiting embodiments, the sample volume received by the sample inlet is ~1 pL of liquid from a reactor microenvironment, which is then processed through the system for use in real-time and / or dynamic, direct-from-culture ESI-MS analysis of, for example, CQA content of the bioreactor characterizing the status of cell development. Exploiting the concurrent sample clean-up and active conditioning capabilities of the system, the method provides a resultant processed sample for increased MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low- concentration biomolecules is significantly improved, and multiple proteins may be detected from chemically complex samples.

[0185] Accordingly, methods of the invention are able to continuously capture the dynamic and heterogeneous nature of in vitro cell growth, especially internal metabolic processes, with a time scale on the order of minutes, using a reduced sample size. Thus, the systems of the invention allow for dynamic characterization of the cell state directly from the growth environment with untargeted detection, via ESI-MS, of a sufficiently broad range of relevant biomarkers, including intracellular metabolites. Thus, as disclosed herein, systems of the invention include a microfluidic device capable of processing ultra-small sample volumes for direct infusion into analysis systems such as ESI-MS.

[0186] While methods of the invention may be optimized for ESI-MS analysis, it is important to note that, while the system may be configured for improved ESI-MS analysis, the system is capable of interfacing with any analysis system appropriate for a specific application.

[0187] In some embodiments of the method, the system further comprises one or more flow valves and / or one or more pumps to control flow along one or more of the sample inlet, the well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules. Thus the valves are in fluid communication with the components of the system, i.e. the one or more reservoirs, the conditioning channel, the carrier channel, the sample inlet and the one or more pumps to thereby control a type, a rate, and a direction of fluid flow through the system.

[0188] Microfluidic chip manufacture

[0189] Systems of the invention provide a micro-fabricated microfluidic chip for highly repeatable sample preparation and / or processing. The microfluidic chip aspect of systems of the invention is manufactured using advanced microfabrication techniques known to those skilled in the art. The microfluidic chip nature of the systems enable integration of numerous features in a single system with opportunities for scaled production via batch processing. The various fluidic components of the system were designed to balance sample transit time, dead volume, pressure drop, and diffusion / dispersion effects. Incorporation by Reference

[0190] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0191] Equivalents

[0192] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims1. A system for sample processing, the system comprising: a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter, wherein when the sample inlet receives a fluidic sample, molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter into the carrier fluid of the carrier channel; and a second filter in communication with the carrier channel via flow of the carrier fluid and downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules below a second molecular weight threshold across the second filter, wherein when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

2. The system of claim 1, wherein molecules from the conditioning fluid flow across the second filter into the carrier fluid.

3. The system of claim 1, wherein flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

4. The system of claim 1, wherein the sample inlet is in communication with the first filter via a well.

5. The system of claim 1, wherein the second filter is in communication with a conditioning channel via flow of the conditioning fluid along the second filter, wherein when the carrier fluid flows along the second filter, molecules from the fluidic sample in the carrier fluid below thesecond molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel.

6. The system of claim 5, wherein the flow of the carrier channel and the conditioning channel are in parallel planes to one another along the second filter.

7. The system of claim 5, wherein a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel is controlled via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

8. The system of claim 5, further comprising a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters.

9. The system of claim 1, wherein the fluidic sample comprises one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample.

10. The system of claim 9, wherein the first molecular weight threshold and the second molecular weight threshold are different thresholds.

11. The system of claim 10, wherein the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

12. The system of claim 4, wherein the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure.

13. The system of claim 12, wherein the well comprises one or more integrated electrodes configured for electrical lysis of the one or more cells within the sample, where the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well.

14. The system of claim 13, wherein a voltage and an electric pulse parameter are adjustable according to an applicable lysis procedure.

15. The system of claim 4, wherein the first filter and / or the second filter comprise one or more capture features.

16. The system of claim 15, wherein the one or more capture features comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, wherein the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel, and wherein when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier.

17. The system of claim 15, wherein the capture features of the first filter are different from the capture features of the second filter.

18. The system of claim 1, wherein the first filter and / or the second filter comprise a membrane configured to capture the molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality.

19. The system of claim 1, wherein a degree of communication between the carrier channel and the first filter is different from a degree of communication between the carrier channel and the second filter.

20. The system of claim 1, wherein the carrier fluid comprises one or more reagents, wherein when the carrier fluid flows to the first filter and / or the second filter, then the one or morereagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel thereby facilitating a chemical reaction in the well and / or the conditioning channel, system calibration, and / or analyte quantification.

21. The system of claim 20, wherein the one or more reagents are reagents for one or more of organic solvents, enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules.

22. The system of claim 1, wherein the conditioning fluid comprises reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, wherein when the conditioning fluid flows along the second filter then the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel.

23. The system of claim 22, wherein the specified molecule comprises one or more of a fluorescent molecular tag, an analytical standard, an organic acid, an organic solvent, and a tandem mass tag (TMT).

24. The system of claim 8, further comprising one or more analysis modules for molecular analysis of fluid from the first reservoir or the second reservoir.

25. The system of claim 24, wherein molecular analysis comprises detecting and / or analyzing one or more properties of an analyte.

26. The system of claim 24, wherein the one or more analysis modules are in communication with the first or second reservoir such that fluid from the carrier channel or the conditioning channel flows directly into the one or more analysis modules.

27. The system of claim 25, wherein the sample inlet is configured to receive a sample collected from a microenvironment of a cell system,28. The system of claim 27, wherein the system is configured to detect and / or analyze an analyte that is an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action.

29. The system of claim 27, wherein the system can continuously monitor a cell culture process to optimize a cell culture process parameter comprising one or more of a feed rate, a cell seeding density, and a media exchange rate.

30. The system of claim 27, wherein the system can detect and / or analyze an analyte that is an indicator of a temporal change in a secretome of a cell such that the system provides a direct representation of a dynamic cell state.

31. The system of claim 27, wherein the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays.

32. The system of claim 25, wherein analyzing comprises analyzing mass spectrometry data via a machine learning algorithm.

33. The system of claim 27, further comprising one or more flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules.

34. The system of claim 1, wherein the fluidic sample is received by the inlet manually via pipette.

35. The system of claim 1, wherein the fluidic sample is received by the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet.

36. The system of claim 1, further comprising a bypass autosampler in fluidic communication with the carrier channel upstream of the first filter, wherein when the bypass autosampler is provided the fluidic sample, then the bypass autosampler periodically and automatically introduces the fluidic sample directly into the carrier channel, thereby bypassing the inlet.

37. The system of claim 1, wherein the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time along each filter.

38. The system of claim 37, wherein the residence time along the first filter is different from the residence time along the second filter.

39. The system of claim 38, wherein the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter.

40. The system of claim 38, wherein the cross-section of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter.

41. A method for sample processing, the method comprising: providing a system for sample processing comprising: a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter; and a second filter in communication with the carrier channel via flow of the carrier fluid and downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter; inserting a fluidic sample within the sample inlet; flowing carrier fluid through the carrier channel past the first filter and second filter,whereby molecules in the fluidic sample below the first molecular weight threshold diffuse across the first filter into the carrier fluid of the carrier channel and molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

42. The method of claim 41, wherein molecules from the conditioning fluid flow across the second filter into the carrier fluid.

43. The method of claim 41, wherein the flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

44. The method of claim 43, wherein the sample inlet is in communication with the first filter via a well.

45. The method of claim 43, wherein the second filter is in communication with a conditioning channel via flow of the conditioning fluid along the second filter, whereby molecules from the fluidic sample in the carrier fluid below the second molecular weight threshold diffuse across the second filter into the conditioning fluid of the conditioning channel.

46. The method of claim 45, wherein the flow of the carrier channel and the conditioning channel are in parallel planes to one another along the second filter.

47. The method of claim 45, further comprising controlling a pressure in one or more of the sample inlet, the carrier channel, and the conditioning channel via one or more pressure controlling means to achieve a pressure difference across the first filter and / or the second filter, wherein controlling the pressure adjusts fluid flow of one or more of fluid flow from the sample inlet to the carrier stream, fluid flow from the carrier stream to the conditioning channel, and / or fluid flow from the conditioning channel to the carrier channel.

48. The method of claim 45, wherein the system further comprises a first reservoir for collecting the conditioning fluid from the conditioning channel that has flowed along the second filter and / or a second reservoir for collecting the carrier fluid from the carrier channel that has flowed along the first and second filters, and the method further comprises the step(s) of collecting conditioning fluid from the conditioning channel that has flowed along the second filter and / or collecting the carrier fluid from the carrier channel that has flowed along the first and second filters.

49. The method of claim 44, wherein the fluidic sample comprises one or more of a cell, cell debris, vesicles, cell components, cell-culture media, microcarriers, extracellular vesicles, one or more biomolecules, a biological fluid, whole blood, blood serum, sweat, urine, and a diagnostic sample.

50. The method of claim 49, wherein the first molecular weight threshold and the second molecular weight threshold are different thresholds.

51. The method of claim 50, wherein the first molecular weight threshold prevents cells and / or cellular debris from diffusing across the first filter.

52. The method of claim 45, wherein the well is configured for lysing cells within the sample fluid via one or more of an electrical, a mechanical, and / or a chemical lysis procedure.

53. The method of claim 52, wherein the well comprises one or more integrated electrodes configured for electrical lysis of the one or more cells within the sample, where the one or more integrated electrodes are arranged to line one or more walls of the sample inlet and / or well.

54. The method of claim 52, wherein a voltage and an electric pulse parameter are adjustable according to an applicable lysis procedure.

55. The method of claim 45, wherein the first filter and / or the second filter comprise one or more capture features.

56. The method of claim 55, wherein the one or more capture features comprise one or more of a plurality of individual pillars, a plurality of rows of pillars, and / or a plurality of groups of pillars, wherein the capture features are positioned within the well, and / or sample inlet, and / or the carrier channel, and wherein when the sample flows from the well to the carrier channel and / or from the carrier channel to the conditioning channel, then the spacing between pillars, pillars and the well, and / or pillars and the channel captures one or more of a cell, a cell component, and / or a microcarrier.

57. The method of claim 55, wherein the capture features of the first filter are different from the capture features of the second filter.

58. The method of claim 41, wherein the first filter and / or the second filter comprise a membrane configured to capture the molecules based on one or more of a chemical entity, a molecular structure, a biochemical affinity, and a chirality.

59. The method of claim 41, wherein a degree of communication between the carrier channel and the first filter is different from a degree of communication between the carrier channel and the second filter.

60. The method of claim 41, wherein the carrier fluid comprises one or more reagents, whereby during the flowing step one or more reagents diffuse from the carrier fluid through the first filter towards the sample inlet and / or through the second filter to the conditioning channel thereby facilitating a chemical reaction in the well and / or the conditioning channel, system calibration, and / or analyte quantification.

61. The method of claim 60, wherein the one or more reagents are reagents for one or more of enzymatic molecules, standard reagents, lysis reagents, and / or additive molecules,62. The method of claim 41, wherein the conditioning fluid comprises reagents for one or more of analyte calibration, quantification, ionization enhancement, or modification, whereby duringthe flowing step the one or more reagents diffuse from the conditioning fluid channel across the second filter to the carrier channel.

63. The method of claim 62, wherein the specified molecule comprises one or more of a fluorescent molecular tag, an analytical standard, an organic acid, an organic solvent, and a tandem mass tag (TMT).

64. The method of claim 41, wherein the system further comprises one or more analysis modules for molecular analysis of fluid from the first reservoir or the second reservoir, and wherein the method further comprises the step of analyzing molecular of fluid from the first reservoir and / or the second reservoir.

65. The method of claim 64, wherein molecular analysis comprises detecting and / or analyzing one or more properties of an analyte.

66. The method of claim 64, wherein the one or more analysis modules are in communication with the first or second reservoir and wherein the method comprises the step of flowing fluid from the carrier channel or the conditioning channel directly into the one or more analysis modules.

67. The method of claim 65, wherein the sample inlet is configured to receive a sample collected from a microenvironment of a cell system.

68. The method of claim 67, wherein the method comprises detecting and / or analyzing an analyte that is an indicator of one or more of a cell phenotype, a cell state, a cell differentiation, a cell disease state, a cell healthy state, a cell therapy product efficacy, a cell therapy product safety, a cell therapy product potency and / or a correlation with a mechanism of a cell action.

69. The method of claim 66, wherein the method comprises continuously monitoring a cell culture process to optimize a cell culture process parameter comprising one or more of a feed rate, a cell seeding density, cell growth nutrients, and a media exchange rate.

70. The method of claim 66, wherein the method comprises detecting and / or analyzing an analyte that is an indicator of a temporal change in a secretome of a cell such that the method provides a direct representation of a dynamic cell state.

71. The method of claim 66, wherein the one or more analysis modules comprise equipment for electrospray ionization mass spectroscopy (ESI-MS), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and / or antibody assays.

72. The system of claim 65, wherein analyzing comprises analyzing mass spectrometry data via a machine learning algorithm.

73. The method of claim 66, further comprising one or more flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules, and the flowing step comprises using the flow valves to control flow along one or more of the sample inlet, a well, the carrier channel, the conditioning channel, the first or second reservoir, and / or the one or more analysis modules.

74. The method of claim 41, wherein the inserting step comprises inserting the fluidic sample into the inlet manually via pipette.

75. The method of claim 41, wherein the inserting step comprises inserting the fluidic sample into the inlet automatically via an inlet autosampler that periodically introduces the fluidic sample into the inlet.

76. The method of claim 41, wherein the carrier channel has a first length and cross section along the first filter and second length and cross section along the second filter, thereby defining a residence time along each filter.

77. The method of claim 76, wherein the residence time along the first filter is different from the residence time along the second filter.

78. The method of claim 77, wherein the length of the carrier channel along the first filter is different from the length of the carrier channel along the second filter.

79. The method of claim 77, wherein the cross-section of the carrier channel along the first filter is different from the cross-section of the carrier channel along the second filter.

80. A method for sample processing, the method comprising: providing a system for sample processing comprising: a sample inlet configured to receive a fluidic sample and in communication with a first filter, wherein the first filter allows for diffusion of molecules under a first molecular weight threshold across the first filter; a carrier channel in communication with the first filter via flow of a carrier fluid through the carrier channel along the first filter; and a bypass autosampler in fluidic communication with the carrier channel upstream of the first filter; a second filter in communication with the carrier channel via flow of the carrier fluid and downstream of the first filter along the carrier channel, wherein the second filter allows for diffusion of molecules under a second molecular weight threshold across the second filter; introducing the fluidic sample directly into the carrier channel via the bypass autosampler, thereby bypassing the sample inlet; flowing carrier fluid through the carrier channel past the first filter and second filter, whereby molecules in the fluidic sample below the second molecular weight threshold diffuse across the second filter into a conditioning fluid.

81. The method of claim 80, wherein molecules from the conditioning fluid flow across the second filter into the carrier fluid.

82. The method of claim 80, wherein flow of the carrier fluid along the first filter and / or the second filter may be one or more of parallel flow, cross-flow, co-flow, counterflow, and / or tangential flow.

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