Real-time monitoring of chromatographic purification of biological substances

Abstract

A chromatography unit (200) and system (400) for use in real-time monitoring of the purification of biological substances. The unit (200) comprises a housing (110), conductive support structures (101, 101a-101n) disposed within the housing (110), and a counter electrode (102), and optionally also comprises a chromatography column (100) including a reference electrode (103) disposed within the housing. The support structures (101, 101a-101n) are provided with a polymer electrolyte coating (111) that can be switched between a first state in which the biological substance to be purified is captured and a second state in which the captured biological substance is released. A voltage generator (300) is connected to the support structures (101, 101a-101n) and the counter electrode (102), and optionally to a reference electrode, and provides an AC voltage signal in the bias between the support structures (101, 101a-101n) and the counter electrode (102) / reference electrode (103). A recorder (300) is arranged to record the generated current signal and / or potential signal.

Description

[Technical Field]

[0001] This disclosure relates to a chromatography unit, chromatography system, and chromatography method for real-time monitoring of the purification of biological substances.

[0002] Background technology Many biological products, particularly biopharmaceuticals, must meet extremely high requirements for purity and potency. Downstream processing involves concentrating the biological product and separating it from impurities. This requires a lengthy series of separation steps. Chromatography is a central technique used for purifying biopharmaceutical products. The cost of purification by chromatography is very high due to the cumulative loss of product in each iteration. Another major cost factor is the time and resources required to perform frequent, labor-intensive analytical techniques offline to assess whether acceptable purity and potency of the product have been achieved.

[0003] The main strategy in process analysis techniques is to place different types of sensors in the process flow, both on the chromatography column and in-line, to measure physicochemical properties. A common drawback of in-line sensors is that the information acquisition location is far from where the actual purification on the surface of the chromatography medium takes place. An alternative strategy is to place very miniaturized sensors in-situ on the chromatography column. However, these sensors can only sense within a very small volume of the column, and the data is not surface-specific, meaning it is difficult to subtract background signals from the bulk process liquid, and the quality of information acquired in-situ is very similar to that acquired in-line.

[0004] Commercial chromatography systems are equipped with inline sensors that measure the refractive index, absorbance, conductivity, and pH of the process flow. Standard inline sensors are non-product specific and, for example, detect all biomolecules that absorb UV light (even impurities). The primary purpose of these sensors is to qualitatively monitor the purification process and track the progress of the chromatography cycle, binding, rinsing, and elution.

[0005] One strategy for specifically detecting the product involves combining an in-line localized surface plasmon resonance (LSPR) sensor functionalized with an affinity ligand with a UV photosensor (International Publication No. 2020009642). In this way, the product is efficiently detected in the eluate despite the presence of by-products. One problem with this method is that it only indirectly reports column saturation with a time delay. Once saturated, no new information about the product is provided until the next binding-elution cycle, and it cannot report the actual amount of product bound to the chromatographic support at a given time.

[0006] Computational modeling of chromatographic purification processes, also known as in-silico chromatography or digital twin modeling, has been explored for yield optimization and quality prediction (potency and purity) (U.S. Patent Application Publication No. 20210149361). The obvious advantage of modeling purification is that, with a minimal number of physical experiments, the results of purification can be predicted using computer models. However, the inputs to these models rely on data collected by existing inline sensors placed in the process flow at the inlet and outlet, which can limit the quality of predictions. Furthermore, column performance fluctuates between purification cycles, requiring recalibration of model data over time, and the use of incorrect parameters carries the risk of inaccurate predictions and product loss. Finally, while computational modeling is highly useful within specific domains, it fails under new circumstances, requiring different modalities, different types of chromatography, and new experimental data.

[0007] Today, critical quality attributes are measured offline only by techniques such as SPR, ELISA, and HPLC. HPLC and mass spectrometry are used to test for process impurities, and size exclusion chromatography is performed for the presence of aggregates and fragment impurities. Most importantly, the potency, i.e., the equilibrium binding constant Kd, is determined by ELISA or SPR immunoassay. Analytical validation is routinely used throughout every unit operation of purification, starting from cell culture harvesting to the final processing step, at which point the product is clarified, meaning it is of sufficient purity for the final formulation. Offline or at-line analysis is very resource-intensive and contributes substantially to product costs. Assay standards must be routinely controlled and maintained. Highly skilled and trained staff are required to execute protocols and interpret results.

[0008] Summary of the Invention The purpose of this disclosure is to provide a chromatography unit, chromatography system, and chromatography method for real-time monitoring of the purification of biological substances.

[0009] The present invention is defined by the appended independent claims. Non-limiting embodiments will become apparent from the independent claims, the appended drawings and the following description.

[0010] According to a first embodiment, a chromatography unit is provided for use in a chromatography system for real-time monitoring of the purification of biological substances. The chromatography unit comprises a chromatography column having a housing provided with a solution inlet and a solution outlet. A support structure is located within the housing in the space between the solution inlet and the solution outlet, and is arranged such that an electrolyte, which flows from the solution inlet to the solution outlet, is in contact with at least a portion of the support structure, and at least a portion of the surface of the support structure is conductive. The chromatography unit further comprises a) a counter electrode located within the housing and arranged to be electrically connected to the support structure via an electrolyte, which flows from the solution inlet to the solution outlet, or b) a counter electrode and a reference electrode located within the housing and arranged to be electrically connected to each other and to the support structure via an electrolyte, which flows from the solution inlet to the solution outlet. At least a portion of the surface of the support structure is provided with a polymer electrolyte coating arranged to be switchable between a first state in which the biological substance to be purified is trapped in the support structure and a second state in which the trapped biological substance is released from the support structure. The voltage generator is configured to be connected to the support structure and either a) connected to the counter electrode and configured to provide an AC voltage signal with a bias between the support structure and the counter electrode, or b) connected to both the counter electrode and the reference electrode and configured to provide an AC voltage signal with a bias between the support structure and the reference electrode. The recorder is configured to record the current signal and / or potential signal generated between the support structure and the counter electrode, or between the support structure and the reference electrode.

[0011] Therefore, the described chromatography unit may be a two-electrode configuration comprising a support structure / working electrode and a counter electrode, or a three-electrode configuration comprising a support structure / working electrode, a counter electrode and a reference electrode. In the tree electrode configuration, an AC voltage is applied between the support structure and the reference electrode, and the counter electrode acts as a current collector.

[0012] A polymer electrolyte coating is configured to be switchable between a first state in which the biological substance to be purified is trapped in a support structure, and a second state in which the trapped biological substance is released from the support structure. The polymer electrolyte coating can be brought into the first state by changing / switching the bias potential difference, such as the DC potential of the applied AC potential (by a voltage generator), and can be brought into the second state by changing / switching the bias potential difference of the applied AC potential. Thus, this is an electrochemical switch of the polymer electrolyte coating between the first and second states. Alternatively, the polymer electrolyte coating can be brought into the first state using an electrolytic running buffer with specific properties / conditions such as salt concentration, buffer species, and temperature, and can be brought into the second state by changing one or more properties / conditions of the running buffer, for example, by adding an elution buffer species to the electrolyte. Thus, this is a solution-induced non-electrochemical switch of the polymer electrolyte coating between the first and second states, as is customary in standard chromatography.

[0013] In this chromatography unit, the biological substance / biomolecular product to be purified can be captured / bound to and released from the polymer electrolyte coating with minimal interference from the AC voltage signal applied between the support structure and the counter or reference electrode. This is achieved by setting a low bias potential for the AC voltage signal between the support structure and the counter or reference electrode, and the recorder can then measure the degree of biological substance bound / captured to the support structure in real time at any given point in time without interfering with the binding of the biological substance, without interfering with the bound biological substance, and without interfering with the release / elution of the biological substance from the support structure.

[0014] By recording the current and / or potential signals generated between the support structure and the counter or reference electrode, the purification process of biological substances on the surface of the chromatography support structure can be measured and tracked in real time. In addition to investigating the surface binding process, in-situ information regarding the properties of the bulk solution and the binding of substances to the working electrode, as well as column packing density, potency, concentration, pH, and conductivity, can also be obtained.

[0015] The above should be compared to conventional chromatography, which relies on inline sensors to record limited information about product quality and further measures critical quality attributes offline.

[0016] Electrical signals used to investigate surface and in-bulk events can be electrochemical impedance signals or arbitrarily modulated electrical signals. This type of measurement is known as electrical impedance spectroscopy (EIS). In EIS, a small-amplitude perturbed sinusoidal voltage with a specific frequency centered on a fixed potential value is applied, and the resulting current is recorded. From this, the impedance Z(ω) = Z × ReZ(ω) + j × ImZ(ω) = Z0 × exp(jφ) is measured, where ω = 2πf, and the magnitude of the current Z0 and the phase shift φ are collected. Alternatively, the impedance is expressed in real and imaginary parts. EIS is an established technique for biosensing on an analytical scale.

[0017] The support structure of the chromatography unit may have a laminated structure comprising at least two separate layers physically separated from each other, wherein an electrolyte arranged to flow from a solution inlet to a solution outlet is arranged to be in contact with at least a portion of each separate layer, and at least one of the separate layers is provided with a polymer electrolyte coating, a voltage generator is arranged to be connected to each or at least a majority of the separate layers of the support structure, and a recorder is arranged to record current signals and / or potential signals generated between each separate layer of the support structure and the counter electrode or reference electrode, or between a majority of the separate layers.

[0018] This enables independent, and possibly simultaneous, electronic monitoring and control of individual layers of the support structure. Signals may be collected in parallel and simultaneously from all individual layers of the support structure, or from at least a majority of the individual layers. This allows for the measurement of concentrations of biological substances bound to the support structure in different individual layers. This allows for optimized elution concentration control based on characteristics such as the maximum eluate concentration. Information on when elution is complete can be obtained to reduce the overuse of chemicals for elution or the use of shifted bias potentials for elution.

[0019] The support structure may comprise at least first and second individual layers, wherein the polymer electrolyte coating disposed on the first individual layer has a different chemical composition from the polymer electrolyte coating disposed on the second individual layer.

[0020] Different chemical compositions of polymer electrolyte coatings mean that polymer electrolyte coatings can have different surface chemical properties, such as having affinity ligands, hydrophobic exchange chemistry, or ion exchange chemistry. One individual layer may be, for example, a polymer electrolyte coating functionalized with affinity ligands and act as a capture step, and a subsequent individual layer (in the flow direction) may have a polymer electrolyte coating having cationic ion exchange chemistry, and the next individual layer (in the flow direction) may have a multimodal anionic ion exchange chemistry coupled with hydrophobic interactions.

[0021] Since the binding concentration can be measured in real time along each individual layer of the support structure in the flow direction, this provides local information in the flow direction, i.e., the axial direction of the chromatography unit, and can indicate with high accuracy when the chromatography unit has reached its full binding capacity.

[0022] The above should be compared to conventional chromatography, where several segmented layers of a resin or membrane having different surface chemistry (hydrophobic interactions, affinity, anionic / cationic ion exchange) are controlled by different eluting chemicals that do not match each other, and some chromatographic materials are, for example, incompatible with some eluting chemicals, making it impossible to monitor local information of binding and release in situ.

[0023] According to a second embodiment, a chromatography system including the above-described chromatography unit is provided.

[0024] The chromatography system may include, separately from the chromatography unit, a buffer pump, a sample pump, a mixer injection valve, and a column valve for administering the flow path through the column, followed by an outlet valve connected to the sample fractionator, a liquid management system. The chromatography columns are connected in-line.

[0025] According to a third aspect, a method for real-time monitoring of chromatographic purification of biological substances, comprising: i) obtaining an electrolyte volume containing the biological substance to be purified and contaminants; ii) providing the above-described chromatography system; iiia) applying an AC potential having a bias potential difference between the support structure and the counter electrode, the bias voltage being set with respect to the open-circuit potential measured between the support structure and the counter electrode or with respect to a reference electrode, the open-circuit potential being measured once at the start of the chromatography experiment or continuously before each AC potential signal is applied, or iiib) applying an AC potential having a fixed bias potential difference between the support structure and the reference electrode; iv) supplying an electrolytic running buffer to the housing at the solution inlet of the chromatography column and equilibrating the polyelectrolyte coating of the support structure in the electrolytic running buffer; v) setting the polyelectrolyte coating disposed on the support structure to its first state; vi) supplying the electrolyte volume containing the biological substance to be purified and contaminants to the housing at the solution inlet of the chromatography column, whereby the biological substance is captured in the polyelectrolyte coating on the support structure; vii) releasing the captured biological substance from the support structure by setting the polyelectrolyte coating disposed on the support structure to its second state; viii) A step of recording the current signal and associated impedance resulting from the AC potential signal applied between the support structure and the counter electrode, or between the support structure and the reference electrode, during steps iv) to vii). A method is provided that includes this.

[0026] The AC potential with a bias potential difference is a continuous, non-invasive AC voltage signal applied between the conductive support structure and the counter electrode, or between the conductive support structure and the reference electrode, and is applied throughout the chromatographic coupling elution cycle, i.e., steps vi) and vii).

[0027] Step viiii), recording the generated current and / or potential signals, may be continuous recording. Alternatively, recording may be performed at regular or irregular intervals over shorter or longer periods, such as recording every millisecond.

[0028] The voltage chromatography cycle can be monitored using the monitored open circuit.

[0029] Step iv) supplying an electrolytic running buffer to equilibrate the polymer electrolyte coating of the support structure can be evaluated by generated and recorded current and / or potential signals to confirm that the polymer electrolyte coating of the support structure is in equilibrium.

[0030] Step viiii), which involves recording the generated current and / or potential signals, can be used to measure the degree of binding / saturation of biological material to the polymer electrolyte coating of the support structure (step vi) and to detect the relative amount of biological material released from the support structure (step vii).

[0031] If the support structure has a laminated structure as described above, comprising at least two individual layers, in step iiia), an AC potential with a bias potential difference may be applied between each or at least a majority of the individual layers and the counter electrode, and optionally, an open-circuit potential may be measured between each or at least a majority of the individual layers and the counter electrode; or in step iiib), an AC potential with a bias potential difference may be applied between each or at least a majority of the individual layers and the reference electrode, and in step viiii), current signals and / or potential signals generated between each or at least a majority of the individual layers and the counter electrode, or between each or at least a majority of the individual layers and the reference electrode, during steps iv) to vii), are recorded.

[0032] Step v) setting the polymer electrolyte coating placed on the support structure to its first state may include changing / switching the bias potential difference of the applied AC potential.

[0033] Step vii) setting the polymer electrolyte coating placed on the support structure to its second state may include changing / switching the bias potential difference of the applied AC potential.

[0034] Compared to steps v) and vi), by changing the bias potential difference of the applied AC potential, the elution of bound biomolecules can be induced via an electrochemical reaction that changes the interface pH of the electrode by switching the polymer electrolyte coating to its second state.

[0035] Alternatively, step v) setting the polymer electrolyte coating placed on the support structure to its first state may be performed via the electrolytic running buffer used in step iv).

[0036] Alternatively, step vii) setting the polymer electrolyte coating placed on the support structure to its second state may include adding an elution buffer to the chromatography unit.

[0037] The elution buffer or running buffer to which the eluting components are added contains eluting chemicals that disrupt the interaction between the polymer electrolyte coating on the supporting structure and the biological material.

[0038] The method may further include a rinsing step after step vi).

[0039] Rinsing may be performed using the electrolytic running buffer or another running buffer, a rinsing buffer. Rinsing washes away unbound biological material and contaminants / impurities and equilibrates the binding of biological material to the polymer electrolyte coating on the support structure.

[0040] The rinsing step is evaluated by generated and recorded current and / or potential signals, allowing monitoring of the rinsing step and the removal of unbound impurities from the polymer electrolyte coating.

[0041] The method may further include a step of collecting biological material released from the support structure and exiting through the solution outlet.

[0042] The method may also include a final step of re-equilibriumizing the polymer electrolyte coating of the support structure by rinsing with an electrolytic buffer solution and / or by applying a clean-in-place treatment including chemicals to wash the chromatography unit. The method can then be restarted to purify the new biological material.

[0043] The applied bias AC potential may have a bias potential of + / -1.5V.

[0044] The applied bias AC potential may have a frequency of 1 μHz to 1 MHz.

[0045] Alternatively, the applied bias AC potential may have a frequency of 1 mHz to 1 MHz.

[0046] When the applied bias AC potential has a frequency of 1 Hz to 1 MHz, it is possible to measure the bilayer capacitance and the resistance of redox active species to charge transfer at the electrolyte electrode interface. On the support structure, the movement of ions and accumulation of charge across the polymer electrolyte coating surface are hindered by the presence of biological substances bound to the surface. This slows down the rate at which ions and redox active species move across the polymer electrolyte coating to the underlying electrode surface, resulting in changes in capacitance and resistance. This confirms the presence of biological substances bound to the surface, in contrast to the case where the polymer electrolyte coating is completely empty and free from biological substances. In this case, ions can move freely across the coating with relatively little obstruction.

[0047] When the applied bias AC potential has a frequency of 1 μHz to 100 Hz, diffusion, charge transfer resistance, and, in some cases, electrolyte conductivity information can be measured. This is because ions have sufficient time to move through the polymer electrolyte coating, regardless of the degree of biological material bound to the coating and the state of the polymer electrolyte coating on the supporting structure. The measured impedance signal precisely overlaps with the in-line measured conductivity signal measured by an in-line conductivity sensor.

[0048] The frequency spectrum from 1 mHz to 1 kHz has overlap, and a mixture of both bulk and surface effects can be present in the measured impedance signal.

[0049] At very high frequencies above 1 MHz, limitations in the electrochemical cell's inductance, connecting wires, and electrical hardware can substantially contribute to the impedance spectrum and cause flooding.

[0050] The amplitude of the AC potential may be between 1 μV and 100 mV.

[0051] The amplitude of the AC potential may be 1 to 50 mV, or optionally 5 mV to 20 mV, 5 mV to 15 mV, 8 mV to 12 mV, or approximately 10 mV.

[0052] The method may further include a step of providing in real time information regarding the bulk electrolyte properties, the degree of binding / saturation of the biological material captured by the polymer electrolyte coating, the degree of the column packed with the biological material, the potency of the biological material, the concentration of the biological material captured by the polymer electrolyte coating, the pH of the electrolyte, and / or the conductivity of the electrolyte, based on the current and / or potential signals recorded from step viiii).

[0053] To provide the concentration of biological substances captured by the polymer electrolyte coating, information is needed regarding the area of ​​the support structure having the polymer electrolyte coating and the bonding capacity of the coating.

[0054] The provided real-time information can be used in a feedback loop to control steps vi) and / or vii).

[0055] This allows the chromatography system to be programmed to stop the product supply before the chromatography unit's support structure becomes supersaturated, preventing excessive product loss due to sample loss. Furthermore, it is possible to precisely monitor when the chromatography unit's support structure becomes empty during elution and immediately return to pumping the running buffer electrolyte, thereby minimizing the exposure of biological substances to potentially harmful eluted chemicals. By precisely monitoring the degree of binding of the chromatography unit's support structure, it is possible to design an enhanced feedback loop for highly efficient cycle times for the purification process in the chromatography system.

[0056] The electrolyte volume, containing the electrolytic running buffer and the biological substance to be purified, may be supplied to the housing at a flow rate of 0.1 to 20 ml / min at the solution inlet.

[0057] In one example, the flow rate is 0.1 to 10 ml / min.

[0058] Preferably, the flow rate is kept constant throughout the chromatography cycle to prevent distortion of the impedance signal that does not result from sample application, binding of biomolecules to the support structure, elution of biomolecules from the support structure, and return of the empty support structure to its original baseline value. The flow rate can be adjusted stepwise and continuously, but with stepwise immediate changes of up to + / - 0.5 ml / min. [Brief explanation of the drawing]

[0059] [Figure 1a] This figure shows a chromatography column for use in a chromatography system. [Figure 1b]Figure 1a is a cross-sectional view of a chromatography column. The chromatography column has a conductive support structure, i.e., a working electrode, a counter electrode, and optionally a reference electrode, housed within it. At least a portion of the surface of the support structure is provided with an electrochemically compatible polymer electrolyte coating that can switch between a first state in which the biological substance to be purified is trapped in the support structure and a second state in which the trapped biological substance is released from the support structure. [Figure 2] This is a schematic diagram illustrating a chromatography system using a conventional chromatography column. [Figure 3] Figure 1b shows a chromatography system equipped with a chromatography column. A potentiostat is connected to a support structure, a counter electrode, and a reference electrode, and is positioned to provide a potential difference as a biased AC potential between the support structure and the reference electrode. The counter electrode acts as a current collector. The potentiostat is positioned to apply and record the current signal and / or potential signal generated between the support structure and the reference electrode. Thus, in this system, events occurring in and around the support structure can be recorded using electrochemical impedance spectroscopy (EIS). [Figure 4] This figure shows a conceptual comparison between data obtained when the chromatographic purification process on a conductive chromatography material was directly monitored in real time using EIS (upper graph) and data obtained from the chromatographic purification process indirectly monitored by downstream inline analysis tools such as UV sensors (lower graph). [Figure 5] This figure shows experimental data comparing the measurement output from direct real-time data of binding and elution of monoclonal antibody products to a protein A-coated electrode using electrochemical impedance spectroscopy with the measurement output from an inline UV sensor during the binding and elution cycle. [Figure 6]This diagram shows a conventional purification workflow for chromatographic purification of biopharmaceuticals, using a combination of inline sensors and offline analytical techniques. [Figure 7] This figure shows the purification workflow for biomolecular products using the chromatography system shown in Figure 3, which employs inline monitoring of critical quality attributes. [Figure 8] This is a Nyquist plot from EIS measurements, where the imaginary part is plotted as a function of the real part of the impedance during the purification process of binding of the bioproduct to the support structure of the chromatography column and elution of the bioproduct from the support structure in Figure 1b. [Figure 9] Figure 1b is an electrical circuit diagram used to analyze electrochemical impedance spectroscopy measurements of polymer electrolyte coatings, such as polymer brushes, coated on the working electrode of a chromatography unit. [Figure 10] This is a Bode plot of EIS measurements performed repeatedly throughout the purification process cycle, with the modulus and phase components of the measured impedance plotted as a function of frequency. [Figure 11] Figure 10 shows an analysis of the data for one selected frequency, 1258 Hz, where the modulus of the impedance signal (cross) is plotted as a function of time for comparison (solid line), and the data is shifted forward in time so that they are superimposed. [Figure 12] This figure shows elution (cross and ring shapes) directly monitored from the surface of the chromatography material by EIS. The corresponding UV signals indicating the elution peaks are acquired by in-line indirect measurement as the product reaches the UV sensor. The derivative of the elution peak will be a peak that closely resembles the recorded UV sensor output. [Figure 13]Figure 1b shows the normalized phase shift of the measurement impedance Z as a function of time during the immobilization of a bioproduct onto a polymer-coated electrode surface in the chromatography unit shown, with two different frequency values ​​shown. At a high frequency of 10 kHz, the surface sensitivity of the signal indicates the amount of product bound to the electrode surface, while the low-frequency signal measures the bulk solution characteristics, the flow of the bioproduct through the device. [Figure 14] This figure compares the measurement output of an inline conductivity sensor plotted as a function of time in a commercially available chromatography system with the real part of the EIS signal. [Figure 15] This figure shows the direct, real-time monitoring of surface-specific binding and elution of monoclonal antibody samples on a protein A-functionalized conductive chromatography material. [Figure 16] This diagram schematically illustrates the steps of a method for real-time monitoring of chromatographic purification of biological substances. [Figure 17] This diagram shows a system of an analytical scale EIS chromatography unit connected downstream of a preparative chromatography unit, which can measure important quality attributes in line. [Figure 18a] This is a cross-sectional view of one embodiment of a chromatography column. Here, the support structure and working electrode have separate, physically isolated layers. [Figure 18b] This is a cross-sectional view of one embodiment of a chromatography column. Here, the support structure and the working electrode have separate, physically separated layers. In Figure 18b, the counter electrode is formed as a hollow cylinder surrounding the support structure while being physically separated from it. [Figure 18c] This is a cross-sectional view of one embodiment of a chromatography column. Here, the support structure and working electrode have separate, physically isolated layers. [Figure 19]Figure 19a shows a chromatography unit in which each individual layer of the support structure of a chromatography column is connected to a potentiostat that can handle multiple channels in parallel. Figure 19b shows real-time monitoring by AC signals that can be used to monitor in real time the concentration of biological material bound to the support structure and the presence of the sample in bulk for each individual layer of the support structure / working electrode support structure throughout the experiment. Figure 19c shows information from each individual layer of the support structure that can be used to control product binding and elution in a highly optimized and efficient manner, i.e., by eliminating product waste, solvent use, exposure to elution buffer (if used), and the desired output concentration. [Figure 20] The vertical diagram below shows a time series illustrating how elution can be regulated using a support structure with multiple distinct layers to optimize elution conditions and provide real-time in-situ images of column packing density (0-100%) / n, where n is the number of distinct layers in the support structure. [Figure 21] This is a schematic diagram of a support structure with n distinct layers of binding, where in-situ monitoring is used to track the degree of concentration of bound biological material throughout the entire support structure with axial resolution along the direction of flow.

[0060] Modes for carrying out the invention Figures 1a and 1b show a chromatography column 100 for use in chromatography unit 200 and chromatography system 400 (see Figure 3). The chromatography column 100 comprises a housing 110 having a multi-electrode configuration, which also includes a support structure / working electrode 101, a counter electrode 102, and optionally a reference electrode 103. The column has external electrical connections 104, 105, and 106 for the working electrode 101, the counter electrode 102, and the optional reference electrode 103. The support structure 101 is located within the housing 110 in the space between the solution inlet 107 and the solution outlet 108. The working electrode 101 is configured such that the electrolyte F, which is arranged to flow from the solution inlet to the solution outlet, is in contact with at least a portion of the support structure 101. The electrolyte is a mobile phase containing a buffer and an electrolyte. At least a portion of the surface of the support structure 101 is conductive. The counter electrode 102 is located within the housing 110 and is electrically connected to the support structure 101 via an electrolyte that flows from the solution inlet 107 to the solution outlet 108. In a three-electrode configuration, the counter electrode 102 and the reference electrode 103 are located within the housing 110 and are electrically connected to each other and to the support structure 101 via an electrolyte.

[0061] Figures 18a to 18c show alternative embodiments of the chromatography column 100, where the support structure 101 has separate, physically isolated layers 101a to 101n. In Figure 18b, the counter electrode 102 is formed as a hollow cylinder surrounding the support structure 101, while being physically separated from it. As can be seen from the figures, the electrolyte F, which is arranged to flow from the solution inlet 107 to the solution outlet 108, is in contact with at least a portion of each of the individual layers 101a to 101n.

[0062] The chromatography unit 200 also includes a voltage generator 300 arranged to be connected to the electrodes via external electrical connections 104, 105, and 106.

[0063] In a two-electrode configuration, the voltage generator 300 may be connected to the support structure 101 and the counter electrode 102, and is arranged to provide an AC voltage signal in the bias between the support structure 101 and the counter electrode 102. In a three-electrode configuration, the voltage generator 300 may be connected to the support structure 101, the counter electrode 102, and the reference electrode 103, and is arranged to provide an AC voltage signal in the bias between the support structure 101 and the reference electrode 103, with the counter electrode 102 functioning as a current collector. Preferably, the voltage generator 300 is located outside the housing 110.

[0064] The recorder 300 is positioned to record current and / or potential signals generated between the support structure 101 and the counter electrode 102, or between the support structure 101 and the reference electrode 103. The voltage generator / recorder 300 may be, for example, a potentiostat, or any other device capable of generating oscillating AC currents across the electrode configuration. The recorder may be equipped with a display for showing direct real-time information about the purification process obtained from the recorded current and / or potential signals. The recorder may be connected to equipment for controlling flow and fraction collection, enabling automation of the purification process based on direct real-time information obtained from the analysis of the recorded AC current signals.

[0065] In the embodiments shown in Figures 18a to 18c, the recorder 300 is positioned to record current signals and / or potential signals generated between each individual layer 101a to 101n of the support structure, or a large portion of the individual layers 101a to 101n, and the counter electrode 102 or the reference electrode 103.

[0066] This enables independent, and possibly simultaneous, electronic monitoring and control of individual layers of the support structure. Signals may be collected in parallel and simultaneously from all individual layers of the support structure, or from at least a majority of the individual layers. This allows for the measurement of concentrations of biological substances bound to the support structure in different individual layers. This allows for optimized elution concentration control based on characteristics such as the maximum eluate concentration. Information on when elution is complete can be obtained to reduce the overuse of chemicals for elution or the use of shifted bias potentials for elution.

[0067] A polymer electrolyte coating 111 is provided on at least a portion of the surface of the support structure 101 (see Figure 3). The polymer electrolyte coating 111 is configured to be switchable between a first state and a second state. In the first state, the biological substance to be purified is trapped in the support structure, and in the second state, the trapped biological substance is released from the support structure. The molecular characteristics of the interaction between the polymer electrolyte coating and the biological substance determine which characteristics determine the first binding state and the second release state. The first state may be a charged state, and the second state may be a neutral state. Alternatively, the first state may be a neutral state, and the second state may be a charged state.

[0068] In the embodiments shown in Figures 18a to 18c, a polymer electrolyte coating can be placed on at least one of the individual layers 101a to 101n of the support structure 101. In one example, the polymer electrolyte coating on the first individual layer 101a has a different chemical composition from the polymer electrolyte coating placed on the second individual layer 101b. They may have different surface chemical properties, such as having affinity ligands, hydrophobic exchange chemistry, or ion exchange chemistry.

[0069] A polymer electrolyte coating can be brought to a first state by using an electrolytic running buffer with specific characteristics / conditions and exposing the coating to the running buffer for equilibration. The polymer electrolyte coating can be brought to a second state by changing the characteristics / conditions of the running buffer, for example, by changing the pH, salt concentration, adding chemicals, changing the temperature, or adding an elution buffer.

[0070] Alternatively, the polymer electrolyte coating 111 may be brought to a first state by changing / switching the bias potential difference of the applied AC potential, and the polymer electrolyte coating may be brought to a second state by changing / switching the bias potential difference of the applied AC potential. Thus, this is an active electrochemical switching of the polymer electrolyte coating between a first state and a second state that causes substantial chemical changes on the surface of the support structure and within the polymer electrolyte coating.

[0071] Figure 16 is a schematic diagram illustrating a method for real-time monitoring of chromatographic purification of biological substances. In step i), the electrolyte volume containing the biological substances to be purified and contaminants is obtained. In step ii), the chromatography system 400 described above is obtained. Next, there is a step with two options: in option iiia), an AC potential with a bias potential difference is applied between the support structure 101 and the counter electrode 102, optionally, the open-circuit potential is first measured between the support structure 101 and the counter electrode 102, and the bias potential is applied to the measured open-circuit potential; and in option iiib), an AC potential with a bias potential difference is applied between the support structure 101 and the reference electrode 103. In step iv), an electrolytic running buffer is supplied to the housing 110 at the solution inlet 107 of the chromatography column 100 to equilibrate the polymer electrolyte coating 111 of the support structure 101 in the electrolytic running buffer. In step v), the polymer electrolyte coating 111 placed on the support structure is set to its first state. In step vi), the electrolyte volume containing the biological material to be purified and contaminants is supplied to the housing 110 at the solution inlet 107 of the chromatography column 100, thereby trapping the biological material on the polymer electrolyte coating on the support structure 101. In step vii), the polymer electrolyte coating 111 placed on the support structure is set to its second state, thereby releasing the trapped biological material from the support structure 101, and in step viiii), the current signal and / or potential signal generated between the support structure 101 and the counter electrode 102 or between the support structure 101 and the reference electrode 103 during steps iv) to vii) is recorded.

[0072] The method may further include a rinsing step after step vi) and a step of collecting biological material released from the support structure 101 and exiting through the solution outlet 108.

[0073] The method may further include a step of providing in real time, based on the recorded current and / or potential signals from step viiii), the bulk electrolyte properties, the degree of binding / saturation of the biological material captured by the polymer electrolyte coating, the degree of the column packed with the biological material, the potency of the biological material, the concentration of the biological material captured by the polymer electrolyte coating, the pH of the electrolyte, and / or the conductivity of the electrolyte. Such information can then be displayed on a display on the recorder 300 or on a display directly or indirectly connected to the recorder 300. Such information may also be used as decision input to enhance the manufacturing process by providing real-time commands to equipment such as pumps, pump valves, and fraction collectors for automation of the manufacturing process.

[0074] Electrical signals used to investigate surface and in-bulk events can be electrochemical impedance signals or arbitrary modulated electrical signals (AC) where an AC potential is applied across a two-electrode / three-electrode system and an AC current is measured. This type of measurement is known as electrical impedance spectroscopy (EIS). In EIS, an AC current or voltage is applied around a fixed potential value, and the resulting AC current is recorded. The magnitude and phase shift of the current are collected. EIS is an established technique for biosensing at the analytical scale. In analytical applications of EIS, increasing sensitivity is crucial. To achieve this, the strategy has been to construct electrodes and devices using nanofabrication techniques with extremely miniaturized features such as interlocking electrode patterns and interfaces with nanoscale separation between electrodes. Challenges in using EIS for biosensing include the fact that the signal transmission mechanism in EIS is not well understood from a fundamental perspective, and the associated analysis for extremely sensitive detection is very complex. A key materials problem that has prevented EIS from becoming a process analysis technique is that most surface chemistry is incompatible with electrochemistry. For example, thiol-immobilized molecules on the surface, a standard surface chemical functionalization for sensors, are electrically unstable and are easily oxidized even at small voltages, separating from the electrode surface.

[0075] In the chromatography system 400 shown in Figure 3, using the chromatography unit 200 described above eliminates the need for inline or offline sensors to evaluate whether acceptable purity and potency of the biological substance have been achieved.

[0076] The primary strategy in conventional chromatography processes is to construct a system like the one shown in Figure 2, where different types of sensors, such as UV sensors, are placed inline with the chromatography column to measure physicochemical properties of the process flow, such as refractive index, absorbance, conductivity, and pH. A common drawback of inline sensors is that the information collection location is far from where the actual purification takes place on the surface of the chromatography medium. The main purpose of these sensors is to qualitatively monitor the purification process and track the progress of the chromatography cycle, binding, rinsing, and elution.

[0077] An alternative strategy is to place extremely small sensors in-situ on the chromatography column. However, these sensors can only sense within a very small volume of the column, the data is not surface-specific, which means it is difficult to subtract background signals from the bulk process liquid, and the quality of information obtained in-situ is very similar to data obtained in-line.

[0078] The chromatography unit 200 described above can be implemented as a completely sensor-free system for purification, or as a system that uses a minimum amount of inline sensors to at least monitor the purification process. The main purification information is acquired in real time directly on the surface of the chromatography support structure, rather than indirectly by sensors or systems of sensors placed inline or in-situ on the chromatography column.

[0079] In conventional chromatography systems, critical quality attributes are measured only offline by techniques such as SPR, ELISA, and HPLC. Analytical validation is routinely used throughout every unit operation of the purification process, starting from cell culture collection to the final processing step, at which point the product is clarified, meaning it is of sufficient purity for the final formulation.

[0080] Offline analysis is highly resource-intensive and contributes significantly to product costs. Assay standards must be routinely controlled and maintained. Highly skilled and trained staff are required to execute protocols and interpret results.

[0081] The chromatography system 400 described herein can be implemented as a purification solution, and the decision to clarify the product can be made based on purification data measured directly on the surface of the chromatography support structure by EIS measurement. Thus, the chromatography system described herein can be used to minimize or replace the need to perform offline analytical techniques to determine the concentration, purity, and potency of bioproducts.

[0082] The described chromatography unit 200 can be used to separate a wide range of analytes (μg to kg) in a scalable and repeatable manner by adjusting the size of electrodes 101 and 102 (and the area coated with the polymer electrolyte coating 111).

[0083] The system can measure both surface-specific information and bulk solution properties. At certain frequency values, the EIS spectrum measures surface-specific information, such as charge transfer through the polymer coating, pH of the material surface, and conductivity of the material surface, which can be used to detect, but are not limited to, target binding to the surface, and the presence of impurities in the coating of process samples and chromatography materials. At other frequencies, the EIS signal monitors bulk solution properties, such as pH of the column solution, its conductivity, the presence of process solution, or the presence of background buffer.

[0084] The chromatography systems described herein can be used to measure real-time data representing an entire batch of biopharmaceuticals, which can be used to determine important quality attributes such as binding and dissolution performance, behavior, characteristics, and the overall equilibrium binding constant Kd.

[0085] Furthermore, using this system, the concentration of the product can be measured in real time, based on knowledge of the electrode surface area, its binding capacity which can be used for improved control of elution, and enhancements to administration and fraction collection.

[0086] The chromatography systems described herein can determine the flow of the product through a process that directly reduces the current time it takes for the product to reach the inline sensor, i.e., eliminates the column-sensor delay tc2s shown in Figure 4.

[0087] The chromatography system described herein provides accurate information on the time and volume at which the column is fully saturated by eliminating delayed tc2s, thereby preventing excessive product loss during breakthrough. Breakthrough is a point in the chromatography cycle where the sample and / or product are registered by an in-line sensor located behind the chromatography column.

[0088] The chromatography system 400 described herein provides calibration-free binding capacity determination of the chromatography support material and does not require post-analysis of elution peaks, offline complementary analysis, and multiple cycles to characterize performance with respect to binding capacity.

[0089] Furthermore, the system reports when rinsing is complete by reporting a combination of stabilized dissociation curves and surface-sensitive EIS signals, while the bulk solution-sensitive signal returns to the background buffer baseline.

[0090] The chromatography system described herein reports the elution of chemical substances as soon as they reach the surface of the chromatography material.

[0091] Furthermore, the system provides precise measurements of column removal from the initial 100% packing to the final 0% remaining product binding. This system makes it possible to completely reduce the unnecessary or excessive use of eluted chemicals.

[0092] By using the chromatography system described herein, the physicochemical properties of the bulk process solution and the binding of the product to the surface can be simultaneously tracked, thereby minimizing the total volume of buffer and water required for the purification cycle and providing clear cutoffs for each step of the purification cycle: binding, rinsing, elution, in-situ washing, and equilibration.

[0093] Figure 3 shows a chromatography system 400 for real-time monitoring of the purification process. The system may include a liquid control system including a buffer pump 29, a sample pump 30, a mixer 31, an injection valve 32, and a column valve 33 for controlling the flow path through the device chromatography column 100, followed by an outlet valve 34 connected to a sample fractionation device 35. The device column 100 is connected inline and also includes a working electrode 101, a counter electrode 102, and optionally a reference electrode 103. At least a portion of the working electrode 101, which is a supporting structure, is provided with a polymer electrolyte coating 111. The polymer electrolyte coating 111 binds to and elutes biological substances by some intermolecular bonding mechanism (affinity, ion exchange, size, hydrophobic interaction). The working electrode 101, the counter electrode 102, and the optionally reference electrode 103 are connected to a potentiostat 300, thereby enabling real-time direct monitoring of the purification process.

[0094] A conventional chromatography system 500 is shown in Figure 2, and Figure 6 shows an example of the steps involved in purifying a biological substance using such a conventional chromatography system 500. The chromatography column 600 contains non-conductive polymer beads 601 or a fibrous membrane. The conventional chromatography system 500 monitors the purification process with a series of in-line sensor devices 602 that measure the physicochemical properties of the eluate by pH, UV / Vis, ATR-FTIR, conductivity, fluorescence, refractive index, and light scattering downstream of the chromatography column 600. In addition to the need for many in-line sensors, it is also necessary to perform large-scale offline or at-line analysis of the biological substance to understand whether the purification meets the required purity and whether the product can be clarified.

[0095] The chromatography system 400 shown in Figure 3 reduces the need for in-line sensor equipment to monitor the purification process by providing in-situ surface-specific information of the biological substance bound to the polymer electrolyte coating 111 of the working electrode 101. An example of the steps involved in purifying a biological substance using such a chromatography system 400 is shown in Figure 7. This chromatography system 400 can directly quantify the binding of the product in the bulk solution within the capsule, column packing, potency concentration, conductivity, pH, and the presence of the supernatant in real time. As shown in Figure 7, the system 400 measures key quality attributes in-line, enabling in-line determination of whether satisfactory purity of the product has been achieved, reducing the need for offline analysis and significantly decreasing the number and frequency of manual process steps in the downstream purification process.

[0096] The conductive chromatography support structure 101 is ideally composed of a highly conductive material such as carbon, a metal alloy, a precious metal, or a semiconductor surface. Alternatively, the underlying support structure may be non-conductive, as long as it has a coating that makes its surface conductive. The conductive chromatography support needs to be physically interconnected to enable EIS monitoring across the entire support surface, and is ideally a single piece.

[0097] Figure 4 shows a comparison of data acquired using EIS on a conductive chromatography support structure (upper graph) and data acquired using an inline UV sensor placed downstream of a conventional chromatography column 600 (lower graph). During binding, the biological substance AB is directly detected across the chromatographic surface using EIS, whereas the UV sensor cannot be used to detect the product because the signal is heavily contaminated with UV absorption process impurities. The EIS signal can be used for immediate identification of the complete binding curve (upper graph). However, the UV sensor signal (lower graph) is still saturated at this point and cannot detect saturated binding of the product to the column, making it likely that the product is lost. Point C in Figure 4 indicates the point at which product elution begins, i.e., when the elution buffer reaches the chromatographic material, resulting in the release of the product / biological substance from the chromatographic surface. True real-time onset of elution cannot be obtained using a conventional inline sensor (lower graph), which carries the risk of using an excess amount of elution buffer. This is undesirable because the buffer can harm the product during prolonged exposure, and many elution buffers are also harmful to health and unenvironmentally friendly (e.g., imidazole). At point D in Figure 4, the end of elution is again recorded in real time, and the chromatography cycle is accelerated by immediate feedback on the state of the polymer electrolyte coating on the working electrode 101 at any given time. For a standard workflow for downstream processing, the graph at the bottom of Figure 4 shows an inline process in which the conventional coupled elution chromatography unit step is performed, and process stability is monitored and ensured using standard inline analytical sensors. To assess the purity after operation of the chromatography unit, different offline assays and analytical techniques are performed to measure key quality attributes: purity, potency, and concentration. If sufficient purity is achieved, the product is clarified; otherwise, another purification step by chromatography is used.

[0098] The workflow proposed using the system described herein is simpler compared to conventional downstream processes, as the purification process monitoring is measured directly within the chromatography column, the in-line sensor is redundant, and ideally, offline analysis is not required.

[0099] The support structure 101 used is made of a conductive or semiconducting material. Alternatively, the support structure has a conductive and electrically continuous coating. The support structures are interconnected, i.e., they do not contain a bed of beads such as agarose, as in conventional chromatography columns. The support structure or its surface can be made from, for example, glassy carbon, stainless steel, or gold.

[0100] The working electrode 101 may be porous and may be placed within the housing so that the electrolyte can flow through the working electrode 101 from the inlet 107 through at least a portion of the working electrode to the outlet 108. Using a porous electrode, the solution can be filtered through a micrometer opening. This makes it possible to achieve the separation of larger objects such as impurities or aggregates in the solution. The micrometer opening also allows for a high surface area, which promotes a high binding capacity of biological substances to the working electrode 101.

[0101] Alternatively, the working electrode 101 may be solid, but it may have a microstructured surface to increase the coupling capacity, thereby allowing the flow to pass tangentially to the electrode surface.

[0102] The main direction of extension of the working electrode 101 may be substantially perpendicular to the flow direction F from the solution inlet 107 to the solution outlet 108.

[0103] The working electrode 101 may have a porosity of 40% to 99%, and the electroactive surface area of ​​the working electrode may be 100 to 10,000 m². 2 / m 3Porosity may be 40% to 99%, or 50% to 99%, or 60% to 99%, or 70% to 99%, or 80% to 99%, or 50% to 90%, or 50% to 80%, or 50% to 70%, or 50% to 60%, or 60% to 80%.

[0104] For example, by using foam or sponge material for the electrode, a high porosity of up to 99% can be obtained, which may have 10 to 100 pores per inch. A porosity of 40% or more can be obtained, for example, by using a mesh electrode. The pore size of the mesh may be 0.01 to 10 μm. The pores or channels of the working electrode are preferably open ends only and have no dead ends.

[0105] The electroactive surface area of ​​the working electrode 101 is 100 to 10,000 m². 2 / m 3 , or 500-10,000m 2 / m 3 , or 1,000~10,000m 2 / m 3 That's fine.

[0106] The counter electrode 102 may be made of a porous material capable of high current / charge transfer. The counter electrode 102 may be made of a different material than the working electrode 101. Alternatively, the working electrode 101 and the counter electrode 102 may be made of the same material, in which case it is preferable that the effective surface area of ​​the counter electrode 102 is at least twice as large as the effective surface area of ​​the working electrode 101 in order to enable sufficient current capacity of the counter electrode to supply current that enables setting a controlled specified voltage on the working electrode within the total aqueous potential range of ±1.5V.

[0107] The working electrode 101 and the counter electrode 102 are preferably made of inert materials that do not undergo permanent chemical changes during exposure to the electrolyte or during the application of an electrochemical signal.

[0108] The working electrode 101 and the counter electrode 102 can be arranged within the housing 110 such that the electrolyte, which is configured to flow from the inlet 107 to the outlet 108, first passes through the working electrode 101 and then through or past the counter electrode 102.

[0109] The working electrode 101 and counter electrode 102 may be manufactured to minimize voids / dead volume within the housing, i.e., to minimize the internal volume of the housing that does not capture biological material, and may be incorporated into the housing 110. By minimizing voids / dead volume within the housing 110, the electrochemical properties of the chromatography column 100 are simultaneously optimized, and conditions are facilitated under which electrochemical release of biological material at high concentrations can be achieved.

[0110] 70-100% of the working electrode 101 may overlap with the counter electrode 102 when viewed in a plane perpendicular to the direction of electrolyte flow from the solution inlet 107 to the solution outlet 108. This allows for a more efficient electrochemical reaction on the working electrode. The average distance between electrodes 101 and 102 may be 1 μm to 200 mm. In one embodiment, the average distance is smaller, between 1 μm and 20 μm. In another embodiment, the distance may be between 1 μm and 200 mm. Electrodes 101 and 102 can be physically separated by spacers that may have a thickness between 0.01 mm and 0.6 mm, ensuring that the electrodes do not come into contact with each other while simultaneously reducing the thickness and thus the total column volume.

[0111] The support structure / working electrode 101 may have a thickness of 1 mm to 100 cm (when measured in the flow direction F). If the support structure comprises only one individual layer, a thickness of 1 mm to 1 cm is preferably used. A thickness of 1 cm to 100 cm can be used when the electrode is divided into one or more separate, physically isolated individual layers 101a to 101n. Each individual layer may have a thickness of 0.05 mm to 10 cm. The thickness and number of individual layers may depend on the height / thickness of the column used. The distance between two adjacent individual layers may be 0.01 mm to 1 cm. If the distance between two adjacent individual layers is greater, an empty space is formed between the layers that may dilute the product and consume the liquid. Therefore, the individual layers should preferably be placed as close together as possible, within a range where they do not come into contact with each other and begin to conduct current.

[0112] The chromatography unit 100 may further include a reference electrode 103 located within a housing 110 and arranged to be electrically connected to the working electrode 101 and counter electrode 102 via an electrolyte. The reference electrode 103 may have a stable, well-known electrode potential and is used as a reference point for potential control and measurement. The working electrode and counter electrode may be located in the same electrolyte, and the reference electrode may be located in a separate tube containing a reference solution. The reference electrode may be made of silver wire with a silver chloride coating (AgCl), or an electrode coated with silver particles with an AgCl coating, such as a carbon electrode, and the reference electrode may be directly exposed to the analyte solution or separated by a semipermeable membrane that allows ions to be transported but does not transport the analyte or other molecules present in the analyte solution. For reference electrodes shielded by a semipermeable membrane, the reference electrode solution used was 3M potassium chloride (KCl), which was also the solution used to store the AgCl reference electrode. The reference electrode may, in principle, be any electrode having a stable and well-known reference electrode potential, such as a standard hydrogen electrode, a saturated calomel electrode, or a copper sulfate electrode. The working electrode, counter electrode, and reference electrode are preferably manufactured and constructed from materials that do not actively or passively leach elements and compounds harmful to downstream processes, such as metal ions, radicals, and other compounds that can react with the analyte.

[0113] The reference electrode 103 may be positioned at an average distance of 1 to 50 mm from the counter electrode 102, or at an average distance of 1 to 50 mm from the working electrode 101.

[0114] The average distances from the counter electrode and the working electrode can be 1-50 mm, 1-300 mm, 1-20 mm, 1-10 mm, 1-5 mm, 5-30 mm, 10-20 mm, or 5-10 mm, respectively. The purpose of the reference electrode 103 is to have an inert and well-known reference point through which the minimum current passes, while reliably determining the potential difference between the working electrode 101 and the counter electrode 102.

[0115] The chromatography support structure 101 or at least its surface is provided / coated / functionalized with a polymer coating 111 fixed to the support structure (see Figure 3). The coating is an electrochemically compatible polymer electrolyte coating. If the working electrode 101 is porous, the polymer electrolyte coating 111 may also extend into the pores of the electrode. The coating can withstand electrochemical potentials of + / - 2V without degradation, allowing the working electrode to be used multiple times / multiple chromatography cycles.

[0116] The average thickness of the polymer electrolyte coating 111 provided on the working electrode 101 may be 1 nm to 1 μm, but is preferably 10 to 50 nm, 10 to 40 nm, 10 to 30 nm, 20 to 50 nm, 20 to 40 nm, or 20 to 30 nm. The polymer electrolyte coating may be in the form of a polymer electrolyte brush, film, gel, or layer.

[0117] The polymer electrolyte coating may be covalently bonded to the working electrode, and may include a pH-responsive polymer covalently bonded to the electrode surface through an electrochemically insensitive monolayer of aryl bonds, for example, by surface functionalization of a diazonium salt.

[0118] Electrochemically stable chemical anchors, i.e., electrochemically insensitive bonds containing aryls, enable monitoring by EIS and controllable release of electrochemically captured substances over a wide bias potential range of + / - 2V. These electrochemically stable aryl bonds allow for multiple reuses of the polymer electrolyte coatings on the chromatography column 100 and working electrode 101. Furthermore, the polymer coating 111 of the chromatography support material 101 can have different functionalizations to enable different modes of chromatography. For example, it may be an anionic polymer electrolyte coating such as polymethacrylic acid, in which case column 100 can be used for cation exchange chromatography. It can also be a cationic polymer electrolyte coating such as poly(dimethylaminomethacrylate), in which case column 100 can be used for anion exchange chromatography.

[0119] pH-responsive polymers may include polymers containing carboxylic acid groups that have the ability to dissociate or incorporate protons as a result of an increase or decrease in pH at the electrode interface. pH-responsive polymers may include, for example, poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA). pH-responsive polymers may also be polymers functionalized with pH-responsive and analyte-specific ligands.

[0120] Polymers, such as those containing the carboxylic acid groups mentioned above, or other types of polymers, such as poly(glycidyl methacrylate), poly(2-hydroxyethyl methacrylate), heparin, hyaluronic acid, and dextran, can be modified / functionalized to contain functional groups that are pH-responsive and have affinity for the analyte of interest. In some cases, polymers are functionalized with molecules having several functional groups that generate "handles" for gripping specific substances / analytes in the electrolyte. Such handles may be analyte-binding at one pH and analyte-repellent at another.

[0121] The side groups of the monomer (the repeating groups of the polymer) may include functional groups that can be used as linkers, such as carboxylic acids, epoxy groups, glycidyl functional groups, or 2-hydroxyethyl groups, to which enzymes, nitrilotriacetate-metal ion 2+ (NTA-Me2+), protein A, protein G, calmodulin, streptavidin, etc., can be immobilized. Thus, the polymer can be functionalized with analyte-specific ligands, which are enzymes, NTA-Me2+, protein A, protein G, calmodulin, or streptavidin.

[0122] The polyelectrolytic coating 111 may have and may be manufactured to have the features and properties described in International Publication No. 2021 / 107836, which is incorporated herein by reference.

[0123] In electrochemically activated elution, applying a bias potential difference between the working electrode 101 and the counter electrode 102 generates a local microscale pH gradient extending from the surface of the working electrode. The pH-sensitive / responsive polymer switches its state as a result of the local pH difference on the surface. The switching of the pH-sensitive / responsive polymer results in either the capture or release of the substance / analyte from the electrode surface, causing separation of the substance / analyte from other components in the electrolyte. Separation occurs because the analyte has a different affinity for the electrode compared to other components in the electrolyte. The difference in affinity includes non-electrostatic intermolecular attractive forces, such as hydrogen bonds between the analyte and the polymer-coated electrode. Furthermore, it may be due to electrostatic attraction or repulsion. This allows for the separation and elution of the purified substance from the chromatography column 100.

[0124] Polymer coatings provide chromatography columns that possess the same capabilities as current chromatography materials in terms of immobilization of biological substances / products through different mechanisms, affinity, ion exchange, ion pair formation, hydrophobic interactions, and size exclusion.

[0125] The biological substances, analytes to be purified from other substances and / or contaminants in the electrolyte may be larger biological entities such as oligonucleotides, proteins, gene vectors, lipid nanoparticles, liposomes, carbohydrates, glycosylated biomolecules, protein pharmaceuticals, therapeutic proteins, vesicles, oligonucleotides, glycans, cells or viral capsids, or combination products such as protein-DNA conjugates, or other components such as synthetic or biologically derived hydrogen-bonded polymers provided in the electrolyte containing other components such as other biomolecules and / or chemicals.

[0126] If the substance / analyte is a viral particle or a structure primarily composed of proteins or lipids, such as an exosome, the analyte is captured by the polymer electrolyte coating in a neutral state via non-electrostatic bonding, e.g., hydrogen bonding, and in a charged state, the captured analyte is released from the polymer electrolyte coating by electrostatic repulsion. Conversely, if the analyte is a carbohydrate or oligonucleotide, the analyte is captured by the polymer electrolyte coating in a first charged state and then released upon switching to a second neutral state. If the analyte is a fusion of two different types of biomolecules, e.g., an antibody conjugated to an oligonucleotide, both capture and release modes are possible, and which is more practical depends on which part of the molecule dominates the interaction. Finally, if the polymer electrolyte coating is post-functionalized with biological ligand molecules, the physicochemical bonding properties of the ligand-biomolecule pair as a function of pH determine the conditions for capture and release.

[0127] Large (multilayer) proteins, when in their neutral, protonated state, can spontaneously immobilize in their native state to a polymer electrolyte coating through non-electrostatic intramolecular attractive interactions, such as hydrogen bonding. The proteins are irreversibly bound to the polymer electrolyte coating, provided that the coating remains in its neutral state with conserved structure and catalytic function. Upon conversion to a second charged state, the biological substance / analyte can be repelled / eluted from the coating and collected.

[0128] An electrolyte is generally a conductive solution containing ions, atoms, or molecules that have lost or gained electrons. Preferably, the electrolyte is completely free of chemicals that would cause the elution of substances / analytes from the supporting structure. Such electrolytes may be, for example, cell culture media or buffer solutions. Electrolytes consist of all buffer species ranging from 1 mM (very low) to 100 mM and up to 1 M physiological buffer concentrations. Salts, ions, must be present in the electrolyte as charge carriers. Total salt concentration, ionic strength, affects the pKa of the polymer electrolyte coating. Higher salt concentrations result in higher pKa, and lower salt concentrations result in lower pKa, changing the pivot point between the first (neutral) and second (charged) states, which means the pH at which the polymer electrolyte coating is binding and repelling the analyte.

[0129] To enable electrochemical reactions, the electrolyte contains redox-active species. Such redox-active species may be essentially present in the electrolyte, for example, oxygen or glucose, or may be added to the electrolyte, and include, for example, hydroquinone, hydrogen peroxide, dopamine hydrochloride (DOPA), ascorbic acid, 4-aminophenethyl alcohol (tyrosol), 3,4-dihydroxyphenethylacetic acid (DOPAC), β-nicotinamide adenine dinucleotide, oxygen, and reduced disodium salt hydrate (NADH).

[0130] Redox-active species should be present in such a way that they can switch the polymer electrolyte coating from a charged, high-surface-pH state to a neutral, low-surface-pH state. To switch between the neutral and charged states, the redox-active species are present in the solution in the form of oxygen.

[0131] The working electrode 101 has the ability to generate a pH gradient extending at least 5 micrometers away from the working electrode surface by an electrocatalytic reaction with a reducing agent, and the concentration of the reducing agent is in the range of 1 nM to 100 mM.

[0132] The voltage generator 300 is connected to the support structure 101 and the counter electrode 102 and is positioned to provide a potential difference between the support structure and the counter electrode, thereby bringing the polymer electrolyte coating to a first state and allowing the working electrode to capture the biological substance to be purified. When the voltage generator switches the potential between the electrodes, the captured substance is released / eluted from the working electrode as the polymer electrolyte coating changes to a second state, and can be collected by exiting the chromatography column 100 through the solution outlet 108.

[0133] The elution and release of the analyte bound to the working electrode 101 can be achieved by applying a potential difference, an electrochemical signal, which changes the surface pH and thereby alters the intermolecular interactions between the polymer electrolyte coating 111 on the working electrode and the bound analyte / substance.

[0134] Electrochemical potential generates a local pH gradient that disrupts specific interactions, leading to elution without changing the device's solution pH.

[0135] In one method, the introduction of the analyte into a functionalized polymer electrolyte coating achieves preferred bonding conditions at a neutral to slightly basic pH, for example, pH 7-8, where immediate bonding between highly specific ligands occurs.

[0136] By changing the ion concentration of the electrolyte, the pKa of the polymer electrolyte coating can be altered, thereby changing the conditions for attractive and repulsive interactions between the analyte and the polymer electrolyte coating. This can be used to change the pH at which the analyte spontaneously binds to the polymer electrolyte coating. For example, at lower total salt concentrations, the pKa of a polyacid coating composed of carboxylic acids rises to a neutral pH, allowing the capture of biomolecules to occur at a neutral pH rather than a slightly acidic pH.

[0137] One method involves using an electrolyte with a low total salt concentration and buffer capacity, enabling highly sensitive switching of the interfacial pH by applying very small currents (<100 μA) and potentials (±100 mV).

[0138] This method may include a step of running a buffer through the column before supplying the electrolyte containing the biological substance / analyte to the column 100, wherein the running buffer has a pH between pH 5 and pH 7.5.

[0139] The pH is selected based on a preferred selection of buffer for the analyte, in combination with the specific pH and solution composition that allows the analyte to spontaneously bind to the electrode.

[0140] The buffer is used as a background buffer to equilibrate the system at the selected pH and salt concentration at which the separation is performed. The running buffer does not bind to the working electrode 101. Analyte interaction is facilitated, and the analyte binds to the working electrode of the device.

[0141] The running buffer used is one that does not contain chemical species that are harmful to the analyte, cause degradation, pose environmental concerns, or significantly increase process costs. Examples of such running buffers and their concentrations include 500 mM imidazole, highly acidic buffers such as 100 mM acetate buffer or 0.1 M glycine × HCl pH 2-3, 0.5 M sodium hydroxide, organic surfactants, and organic solvents such as ethylene glycol, glycerol, PEG, amino acids, and sodium alkyl sulfate.

[0142] The electrolyte containing the substance can be supplied at a flow rate of 0 mL / min to 10 L / min at the solution inlet. This allows the entire substance sample to flow through the column 100, maximizing uptake while providing sufficient residence time for efficient binding of the substance to the polymer electrolyte coating 111 of the working electrode 101.

[0143] After the step of flowing the electrolyte from the inlet 107 to the outlet 108 so that the substance / analyte is captured by the polymer electrolyte coating 111 placed on the working electrode 101, the column 100 can be rinsed to remove unbound analytes and other components in the solution from the internal volume of the column.

[0144] The following details a method for actively generating an electrochemically induced pH gradient at the interface of a support structure using a modified bias potential of the EIS measurement during the elution step. The step of setting the polymer electrolyte coating placed on the support structure to its second state by changing / switching the bias potential difference of the applied AC potential, thereby eluting the biological substance / analyte from the working electrode, may include applying a constant potential difference over time, the duration of which may be 1 to 3600 seconds. It may be 1 to 10 seconds, 10 to 30 seconds, 30 to 60 seconds, 60 to 120 seconds, or 120 to 300 seconds, or 300 to 600 seconds, and may be 600 to 3600 seconds.

[0145] This establishes a pH gradient. The degree of the pH gradient is determined primarily by (i) the buffer capacity of the solution that counteracts the electrochemical reactions that change the surface pH, (ii) the magnitude of the electrochemical potential that determines the rate of the electrochemical reactions on the surface, (iii) the concentration of electroactive species, i.e., proton-receiving or proton-donating species, and (iv) the buffer flow rate renewal and the diffusion and convection of mass transfer characteristics through the device.

[0146] The applied potential difference can be positive or negative, with a magnitude between 0V and 1.5V.

[0147] The potential difference is applied in the presence of redox species that can change the pH by generating or consuming protons.

[0148] If the intention is to lower the pH of the electrode surface, the potential is positive; if the intention is to raise the pH of the surface, the potential is negative.

[0149] Applying a potential difference between the working electrode 101 and the counter electrode 102, thereby eluting a biological substance / analyte from the working electrode, may involve continuously changing the potential difference between the two potential values ​​over time, with a duration of 1 to 600 seconds. It may also be 1 to 10 seconds, 10 to 30 seconds, 30 to 60 seconds, 60 to 120 seconds, or 120 to 300 seconds, or 300 to 600 seconds, and may even be 600 to 3600 seconds.

[0150] The application of a variable electrochemical potential establishes a variable pH gradient. In addition to the effects described above, the rate of potential change affects the extension of the pH gradient, resulting in temporal variations in the surface pH. The variable potential may also be a stepwise increasing potential difference, resulting in a stepwise increase in pH that produces a net electrostatic repulsion between the electrode and the analyte. The applied potential difference can be continuously varied between two positive or negative voltage values ​​within the range of 0V to 1.5V for a duration of 1 to 3600 seconds.

[0151] The duration may be between 1 second and 600 seconds. It may be between 1 and 10 seconds, 10 and 30 seconds, 30 and 60 seconds, 60 and 120 seconds, or 120 and 300 seconds, or 300 and 600 seconds, and may be between 600 and 3600 seconds.

[0152] The selected potential window for changing the voltage may vary depending on which redox active species are present in the electrolyte that alter the electrode surface pH. The local pH on the surface can be measured using analytical techniques to connect the potential window and the voltage value used with the actual pH generated on the surface.

[0153] To elute the analyte, a combination of providing a variable potential difference and providing a constant potential difference may be used. For example, a variable potential may be used first, followed by a constant potential difference.

[0154] By continuously changing the potential difference at different rates, separation with higher resolution can be achieved compared to simply applying a potential abruptly, and the analytes can be gradually separated as the interaction with the polymer electrolyte coating changes.

[0155] By altering the electrical potential, it is possible to selectively desorb viral capsids filled with genetic material, empty viral capsids, or partially empty viral capsids, as well as specific analytes such as host cell proteins.

[0156] The resolution of analytes separated by the difference in isoelectric points can be as low as 0.4 pH units, which is the isoelectric point difference between a filled viral capsid and an empty viral capsid.

[0157] By applying a potential at which biomolecules with specific isoelectric points cease to bind to the polymer surface, the remaining analyte molecules are separated from any biomolecular impurities and other impurities that may be present in the electrolyte.

[0158] By using electrochemical signals, it is possible to instantaneously release all / most of the analytes bound to the working electrode, thereby increasing the concentration of the pure analyte sample separated from the diluted sample.

[0159] Subsequently, a single electrochemical signal can increase the concentration of the analyte sample by at least 20 times.

[0160] The amount of bound analyte recovered by sample retention and measured electrochemical signals may be, but is not limited to, up to 94%.

[0161] The solution exiting the outlet can be collected into fractions. These fractions contain different components. Depending on the applied voltage, different analytes are eluted from the chromatography column.

[0162] The system may further include a solution analysis device positioned to analyze the contents of the solution collected at the solution outlet. The solution analysis device may include, for example, a UV analyzer, a fluorescence detection analyzer, or the analysis may be performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (qPCR), or other analytical assays.

[0163] The following experimental section describes specific examples of chromatography columns 100 and chromatography units 200 having a working electrode provided with a particular polymer electrolyte coating 111. Other polymer electrolyte coatings that can be used with chromatography column 100 may have and be manufactured with characteristics such as those described in International Publication No. 2021 / 107836, which is incorporated herein by reference.

[0164] experiment chemicals Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich. H2O2 (30%) and NH4OH (28-30%) were obtained from ACROS, and H2SO4 (98%) and ethanol (99.5%) were obtained from SOLVECO. Water was ASTM research grade 1 ultrafiltered water (Milli-Q water). The chemicals used in the synthesis of diazonium salt 1 were 4-aminophenethyl alcohol, tetrafluoroboric acid (48% aqueous solution), acetonitrile, tert-butyl nitrate, and diethyl ether. L-ascorbic acid was used in water to adhere the diazonium salt to the gold. Dichloromethane, triethylamine, and α-bromoisobutyryl bromide were used to convert the diazonium monolayer into a polymerization initiator layer. The chemicals used for polymerization were tert-butyl acrylate, tert-butyl methacrylate, dimethyl sulfoxide, dichloromethane, methanesulfonic acid, N,N,N',N''-pentamethyldiethylenetriamine (PMDTA), CuBr2, and L-ascorbic acid. For post-modification of the brush after synthesis, 1-ethyl-3-8(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used.

[0165] The buffers used in this study were based on phosphate-buffered saline (PBS) tablets (0.01 M phosphate, 0.13 M NaCl, pH 7.4), disodium hydrogen phosphate and NaCl, or tris(hydroxymethyl)aminomethane (TRIS) titrated to a specific pH with HCl (1 M aqueous solution) or NaOH (1 M aqueous solution).

[0166] The biological compounds used were protein A (Medicago), monoclonal antibody, and immunoglobulin (Sigma Aldrich).

[0167] material Stainless steel (316L) mesh with apertures of 1, 5, and 10 micrometers was used as the starting material for the working electrode and counter electrode. A thin gold coating was prepared on the working electrode by electron beam physical deposition (Lesker PVD225) of gold (50 nm). A 1 nm titanium dioxide layer was used as the bonding layer. Before deposition, the mesh was rinsed in isopropanol and dried in N2. A reference electrode was prepared using silver wire. The AgCl reference electrode was prepared by immersing the silver wire in HCl (37%) diluted 10-fold with ultrapure water.

[0168] Preparation of diazonium salts Diazonium salts were synthesized using a modified procedure from a previous report (S. Gam-Derouich et al., Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surface and Interface Analysis 42, 1050-1056 (2010)). Under an inert atmosphere, 4-aminophenethyl alcohol (2.94 g, 20 mmol) and tetrafluoroboric acid (9.94 g, 113 mmol) were dissolved in acetonitrile (20 mL). In a separate flask, tert-butyl nitrate (2.269 g, 22 mmol) was dissolved in acetonitrile (12 mL). Both solutions were degassed and cooled to -20°C with 200 mL of diethyl ether.

[0169] After 20 minutes, the solution was warmed to 0°C, and then tert-butyl nitrate was added dropwise to the 4-aminophenethyl alcohol solution with stirring. The reaction was then stirred for a further 1 hour. The reaction was terminated by adding the dark yellow solution dropwise to 200 mL of rapidly stirring diethyl ether. After stirring for another 1 hour, the supernatant was decanted. The brown precipitate was dried to obtain 3.69 g of impure diazonium salt.

[0170] To verify the product, the ¹H NMR spectrum was recorded at ambient temperature using a Varian 400 MHz NMR spectrometer. The spectrum was analyzed against external TMS, and the residual solvent resonance at the lowest magnetic field (CDCl3:δH 7.26 ppm) was used as the reference. The ¹H NMR resonance of the diazonium salt was consistent with previously reported findings (S. Gam-Derouich et al., Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surface and Interface Analysis 42, 1050-1056 (2010)), and the analysis revealed a purity of 80%.

[0171] Polymerization initiator for electrode surface A stainless steel mesh was placed in a septum-sealed glass jar containing diazonium salt (0.301 g, 1.28 mmol), and the jar was purged with N2. In a separate flask, ascorbic acid (0.028 g, 0.16 mmol) was dissolved in water (40 mL), and the solution was degassed for 1 hour. The ascorbic acid solution was then transferred to the sealed glass jar, and the diazonium salt was dissolved. The gold surface was stirred in the solution for 1 hour using a platform shaker (nitrogen bubbles appearing on the surface after 15 minutes indicate successful formation of a diazonium salt monolayer), and then the gold surface was thoroughly rinsed in water, and then in ethanol, and dried.

[0172] To convert the diazonium monolayer into a polymerization initiator layer as shown in Figure 3, the gold surface was exposed to α-bromoisobutyryl bromide (0.222 mL, 1.80 mmol) and triethylamine (0.302 mL, 2.17 mmol) in dichloromethane (20 mL) for 10 minutes, after which the surface was rinsed with ethanol and dried under N2 conditions.

[0173] Synthesis of polymer coatings on electrodes Using SI-ATRP (surface initiated activator-regenerated atom transfer radical polymerization), poly(acrylic acid) (PAA) polymer brushes (see Figure 3), i.e., polymer electrolyte coating 111, were prepared in a manner similar to the published procedure (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)).

[0174] After removing the inhibitor from the monomer tert-butyl acrylate (TBA) using an alumina column, the solution was stored at -20°C and warmed to room temperature immediately before use. The reaction was carried out using standard Schlenklein technique under an inert atmosphere of N2. CuBr2 (0.006 g, 0.03 mmol) and pentamethyldiethylenetriamine (PMDTA) (0.056 mL, 0.276 mmol) were dissolved in dimethyl sulfoxide (20 mL) and deoxygenated for 30 minutes by vigorous bubbling of N2 together with a separate flask of tert-butyl acrylate (20 mL, 0.1378 mol).

[0175] Next, the reaction solution and monomers were transferred via a cannula to a screw-top jar (with a rubber septum lid) containing a gold surface prepared with the initiator. The reaction was initiated by adding ascorbic acid (0.049 g, 0.276 mmol). The final concentrations of each component in the reaction medium were [monomer]=3.4 M, [CuBr2]=1.1 mM, [PMDTA]=11.0 mM, and [ascorbic acid]=11.0 mM. The reactants were placed under magnetic stirring. The reaction was quenched by immersing the sample in pure ethanol. The poly(tert-butyl acrylate) (PTBA) brush was then converted to PAA by exposure to 0.2 mM methanesulfonic acid in dichloromethane (10 mL) for 15 minutes, followed by rinsing in dichloromethane and ethanol. The electrode was dried in N2 and stored under dry conditions until further use.

[0176] Post-functionalization of polymer coatings with biological ligands To allow protein A to adhere to the polymer brush, PAA was post-modified using EDC / NHS coupling technology. The electrode surface was exposed to an aqueous solution containing 50 mM EDC and 50 mM NHS for 30 minutes, and then rinsed with water. Protein A was immobilized on the electrode surface for 1 hour using a 0.3 g / L solution of protein A at pH 7.4, followed by rinsing with PBS and water. After post-functionalization, the electrodes were stored in water at 2–8°C.

[0177] Manufacturing and assembly of capsule devices including electrodes A three-dimensional model of the chromatography column (see Figures 1a and 1b) was designed using CAD software (Autodesk Fusion360). The prototype was sliced ​​using PrusaSlicer (Prusa3D), and the device was made leak-resistant by printing it on an MK3S 3D printer (Prusa3D) using poly(ethylene terephthalate glycol) (PETG) or polypropylene (PP) filament with the following print settings: layer height 0.1 mm, 5-6 wall layers, 5% extrusion ratio, 100% packing density.

[0178] The column was assembled as shown in Figure 1b by screwing the central body component into the inlet body component. The reference electrode 103 was connected to the inlet body component. The counter electrode 102 was placed on the inner surface of the inlet body component, and then the spacer and O-ring were placed above the counter electrode 102 so that the O-ring was in full contact with the inlet body component to form a fluid-resistant seal. The working electrode 101 and outlet body component, placed on the spacer element disk, were connected by screwing them into the central component. Connector pins 104 and 105 for the working and counter electrodes, including stainless steel metal threads, were screwed into their respective inlet ports until they were in contact with their respective electrodes. This was verified under dry conditions using a multimeter before being installed in the chromatography system.

[0179] Chromatography system settings Pure IgG, 0.5 mg / mL, and a sample solution with a total sample volume of 1 mL were injected into the AKTA Explorer chromatography system. The flow rate used throughout the experiment was 0.25 ml / min, and the injection loop was emptied with a total volume of 6 mL of running buffer. The running buffer used (A) was 1 mM phosphate and 10 mM NaCl at pH 7.0. The elution buffer used (B) was 1 mM phosphate and 10 mM NaCl at pH 2.5. Bounded IgG was eluted from the electrode surface using a step-by-step injection of a total of 10 mL of elution buffer.

[0180] Electrochemical impedance spectroscopy (EIS) measurement Potentiostatic EIS measurements were performed using a Gamry 1010E potentiostat (Gamry Instruments) with EIS capability. A typical EIS setting used was a bias potential of -0.2V relative to either the E reference or the E open-circuit potential. The AC amplitude was 10mV. The complete frequency window investigated was 1e6–20Hz. Specific frequency windows were adjusted for different measurements. A complete measurement across a wide frequency window took approximately 1 minute. In some cases, smaller intervals or even single frequencies were measured to increase the measurement cadence. When the bias potential is measured relative to the open-circuit potential, a certain initial delay period of the open-circuit potential (OCP) is measured before the initialization of the EIS measurement.

[0181] For passive monitoring of the binding process, the bias potential was selected to a value where electrochemical reactions do not occur at a significant rate that alters the interfacial pH of the supporting structure. The absence of altered pH ensures that the EIS scan is passive and does not interfere with binding or release.

[0182] For active electrochemistry combined with EIS monitoring of the support structure. Electrochemical elution is generated by changing the bias potential to a value where a significant interfacial pH change can be expected, such as -1V relative to AgCl.

[0183] EIS measurements were performed before the start of the chromatography-coupled and elution cycles to verify steady-state conditions and equilibration signals while only the buffer solution was flowing. Once stable baseline conditions were met, the chromatography-coupled elution cycle was initiated in the chromatography system, and EIS scans were collected during the sample application, column washing, elution, and equilibration steps.

[0184] Surface-specific binding to the surface was demonstrated and quantified using different methods for electrically measuring the degree of column packing and removal. (1) In the simplest method, the floating open-circuit potential was used to track the coupling to the working electrode surface, and neither AC signals nor EIS measurements were applied. This measurement method was very sensitive to disturbances. (2) A second, more robust method involved measuring impedance signals in different frequency ranges (20 to 1e6 Hz), where the impedance Z(w), its real component Re(Z), imaginary component im(Z), modulus Mod(Z), phase angle Pha(Z), and Vdc were recorded for each frequency value. (3) A third option was to measure the binding process with a single or a small number of selected frequency values ​​at a very high data acquisition rate in order to capture the binding process of the biopharmaceutical at high resolution (ms).

[0185] Analysis of EIS measurements In OCP-based methods, column packing and removal are directly measured using the OCP signal as a function of time throughout the entire column purification cycle.

[0186] When using EIS, the impedance signal at any given time of the chromatography cycle was analyzed in either its real or imaginary part. Re(Z) was sensitive to the conductivity of the biomolecular product electrode, while Im(Z) was sensitive to the surface packing. Z(w) = Zre(w) + jZim(w) = Mod(Z) * e(jwt), where w = 2pif and j = sqrt(-1).

[0187] Another approach involved using the polar form representation of the EIS signal, studying the modulus Mod(Z) and phase angle Arg(Z) at specific frequencies as functions of time throughout the chromatographic experiment. For visualization purposes, where relative column packing is shown, the real and imaginary parts of the measured impedance signals were normalized between 0 and 1.

[0188] Another method is to plot the Nyquist diagram, where Re(Z) is plotted as a function of -Im(Z), and the relative position of the plot against the time of the entire purification cycle is used to indicate the degree of binding to the column. Figure 8 shows the Nyquist diagram from the EIS measurement, where the imaginary part is plotted as a function of the real part of the impedance during the purification process of the binding of the bioproduct to the support structure of the chromatography column and the elution of the bioproduct from the support structure in Figure 1b.

[0189] Figure 9 shows an electrical circuit diagram used to analyze electrochemical impedance spectroscopy measurements of polymer electrolyte coatings, such as polymer brushes, coated on the working electrode of the chromatography unit shown in Figure 1b.

[0190] The method for analyzing EIS measurements involves creating a model that is an electrical representation of the electrical circuit of the system shown in Figure 9, where the EIS data Z(w) is fitted with respect to the following model parameters: Rct is the charge transfer resistance, Zw is the Warburg impedance, Rs is the solution resistance, and Cdl is the capacitance between the working electrode surface and the target species of ions in the buffer electrolyte system. The capacitance of the electrical double layer reflects the relative presence of biomolecular products on the electrode surface. Studying the capacitance as a function of time provides the degree of filling and removal on the electrode surface throughout the chromatographic cycle.

[0191] Examples: How to use The following examples are provided for illustrative purposes only and should not be construed as limiting.

[0192] Example 1: Chromatographic purification process compared to conventional chromatography, with direct real-time monitoring of conductive chromatography materials using EIS. Figure 4 shows a conceptual comparison between data obtained by directly monitoring the chromatographic purification process on a conductive chromatography material in real time using EIS, and data obtained from a conventional chromatographic purification process indirectly monitored by downstream inline analysis tools such as a UV sensor.

[0193] Figure 4 shows (A) binding initiation, (B) binding saturation, (C) elution initiation, and (D) elution completion, which can be directly detected in real time using EIS when they occur on the surface of the chromatographic material before being recorded by an in-line downstream process analysis sensor (see graph below). Six characteristic features of EIS monitoring for in-line sensors such as UV detectors can be estimated. 1. High-precision product positioning: In binding-elution chromatography, the product binds to the chromatographic material with some specificity. In the case of highly specific interactions in affinity chromatography, such as IGG purification using protein A ligand-functionalized chromatographic materials, it can be almost certain that the equilibrium signal resulting from binding to the surface is 99% related to the product. 2. Elimination of delay time from column to sensor: While inline sensors record in real time, there is a delay from when the product was actually in the chromatography column. This means that EIS monitoring has the ability to acquire process information and transfer commands directly to the system's electronic hardware or operator, and at a faster rate than the methods currently used. 3. Minimizing and eliminating breakthrough: Today, a decision is made when the sensor signal increases by a specific value, e.g., +200%, at a UV wavelength of 280 nm. However, by that point, the material may have already been wasted. 4. Use of invasive elution buffers / reducing exposure: Knowing when elution begins and ends allows for minimizing the residence time of biological substances in potentially hazardous environments. 5. Process Enhancement and Efficiency Improvement: EIS monitoring directly captures information on when impurity-containing samples are removed from the column, when binding begins, when it is completed, when elution begins, and when the chromatography column is equilibrated. This results in opportunities for faster decision-making and a faster process overall. 6. Development of Chromatography Units: Ground truth data on the binding and release of biological substances cannot be obtained with non-conductive chromatographic materials. By directly measuring these processes, the design can be optimized, for example, in terms of mass transfer and binding capacity, using support structures and chromatographic unit optimization.

[0194] Example 2: Direct monitoring of binding and elution of biological substances using EIS. Figure 5 shows an experimental example similar to the conceptual image provided in Figure 4. Here, the biomolecular substance IGG 0.5 mg / mL is captured on a PAA polymer electrolyte coating functionalized with protein A using EDC / NHS. The inventors used impedance spectroscopy to investigate the binding, equilibration, and elution of the biomolecular substance while simultaneously monitoring the flow of uncaptured sample through a chromatography unit. The frequency range used was 10 Hz to 1 MHz with a bias potential of -0.2 V, a potential at which electrochemical reactions that change the interfacial pH of the supporting structure do not occur at a significant rate, meaning that the EIS scan is passive and does not interfere with binding or release. The bias potential was set to float relative to the open-circuit potential, meaning that -0.2 V was applied to the measured OCP, which was in the range of 76 mV to 300 mV throughout the duration of the experiment.

[0195] Figure 8 shows an example of raw data obtained by EIS represented by a so-called Nyquist plot, where the negative imaginary part of the measured impedance signal is plotted against the corresponding real part. Figure 10 is yet another example of a representation where each signal is plotted on a separate y-axis as a function of frequency. When the support structure binds or releases the biomolecular substance to / from the column, the spectrum shifts. The spectrum also shifts as a function of the sample present in the bulk solution, and the shift of the spectrum as a function of frequency is used to elucidate whether surface binding or flow of the sample through the column is occurring. However, the graphical displays of Figures 8 and 10 are not very suitable for visualizing and detecting real-time changes. Instead, selected frequency values are analyzed and plotted and analyzed throughout the course of the purification process as shown in Figure 5.

[0196] The resulting normalized impedance signal of Figure 5 indicates specific binding of IGG to the support structure on the left side (cross) by detecting a shift at a high frequency of 10 6 Hz. On the right axis (circle), at a low frequency (10 1The bulk conductivity of the sample flowing through the chromatography unit is detected by the UV sensor (Hz). The first thing to note is the column-sensor delay time (tc2s) for the UV sensor; sample binding is recorded immediately with the EIS, but later with the UV absorbance sensor. The end of binding is recorded in the EIS as the maximum value of the normalized imaginary EIS signal, although this event goes unnoticed with the UV sensor. During the rinsing stage (B), verification of complete equilibration of the biomolecular material to the support structure and that all unbound sample has left the chromatography unit is obtained immediately. The start of elution (C) when the elution buffer reaches the surface of the support structure is detected immediately, as is the complete removal of the sample from the support structure; both events are delayed information from the UV absorbance sensor, and furthermore, the end user cannot confirm that the entire amount of bound biomolecular material has been released without applying excess elution buffer. The EIS signal returned to baseline provides information on complete removal. Similar to SPR and surface sensitivity analysis, it is possible to simulate the elution of biomolecular substances from the chromatography unit support structure by fitting binding association curves during binding, dissociation curves during the rinsing phase, and even S-shaped curves and logistic functions. This allows for efficient modeling of chromatography materials and enables the optimization of the chromatography unit using the information and feedback directly.

[0197] Another experiment in which IgG (0.5 mg / mL) was purified using a polymer electrolyte coating coated with protein A is shown in Figures 14 and 15. Here, the impedance signal is collected at a fixed bias potential of -0.2 V, rather than at the floating open-circuit potential value. In Figure 15, the normalized phase angle of the impedance spectrum (dashed line) is plotted as a function of time at an intermediate frequency of 400 Hz (on the right axis). For comparison, the UV absorbance at 215 nm is plotted on the left axis. The UV absorbance is temporally shifted to compensate for the delay time from the column to the sensor. It is interesting to note that the phase angle begins to increase before breakthrough is recorded in the UV absorbance signal. During this stage, the EIS provides direct information about the progress of binding and packing to the supporting structure, while the UV absorbance signal does not record any changes due to the fact that unbound IgG has not yet escaped through the chromatography unit. Initially, if binding saturates and the product exits uncaptured, the breakthrough peak is recorded in the UV signal. The phase angle stabilizes at the equilibrium binding level, and the UV signal returns to the baseline. When IgG is eluted, the phase angle decreases and returns to its value as unbound. Unbound IgG is identified by an increase in offline UV absorbance. Meanwhile, in Figure 14, the bulk solution is monitored, and the modulus of the same impedance signal at 400 Hz is plotted as a function of time compared to the inline conductivity sensor. In Figures 14 and 15, the EIS signal is monitored with respect to a fixed potential, and the modulus and phase angle are generated for the bulk solution conductivity data, while the phase angle reports surface-specific changes at the same frequency. The selected frequency is intermediate, and the impedance spectrum is affected by both the conductivity effect of the bulk solution and changes in capacitance and resistance at the interface. As the elution buffer is injected towards the end of the purification cycle, the phase angle begins to increase again at the end of pH elution. For concentrated elution buffers such as those used in this example, the bulk conductivity signal of the concentrated acidic buffer eventually floods the signal. However, this problem can be bypassed by combining inline analysis (solid line) with EIS, or by tuning the buffer characteristics.For example, by interpreting impedance signal results using impedance modulus analysis, or by using inline sensors so that accurate values ​​of column binding and elution can still be obtained.

[0198] Example 3: Prevent yield loss from breakthrough by immediate identification when the chromatography support is saturated with the product. Using inline sensors makes it extremely difficult to avoid product loss due to incomplete information when the column becomes fully saturated and can no longer bind more product. Consequently, this makes it extremely difficult to truly minimize product waste using conventional chromatography. During a binding-elution chromatography cycle, there is a moment when the product breaks through. This is the point in the binding phase where the inline sensor registers that the feed solution is flowing out of the chromatography column. With a standard UV sensor, it is impossible to know when the breakthrough contains uncaptured product because the signal is contaminated with background signals from process impurities. Either some loss must be tolerated, or time-consuming offline analytical tests are performed to identify at what point in the binding cycle the column becomes saturated. Even if the yield loss due to breakthrough is small in a single chromatography step, e.g., 97%, the impact of losing this amount of sample from the material over four consecutive cycles results in an overall yield of 61%.

[0199] Figure 11 shows the binding of IgG to a support structure having a PAA polymer electrolyte coating functionalized with protein A. The EIS signal is compared to the UV absorbance signal, and the delay time of the UV signal is subtracted to superimpose the signals. In the EIS measurement, the modulus of the impedance signal at 1258 Hz is plotted as a function of time during the binding phase, and a bias potential of -0.2 V (relative to the open-circuit potential) is used. Figure 11 clearly shows how the signal reaches its maximum during sample injection (indicating saturation of the support structure by IgG). The corresponding UV signal reaches its maximum during sample injection as the sample passes through the chromatography unit. The EIS signal makes it possible to gather information that the surface of the support structure may be saturated with biomolecular products. However, in the UV signal, the peak only indicates that the sample has passed through the chromatography unit. It is not possible to know in real time whether the chromatography material is saturated or not.

[0200] Figure 11 provides a tool for real-time determination of when the column is saturated, which can be used to operate the chromatography system to prevent excessive product loss by stopping the application of new samples. Furthermore, the system can immediately proceed to the next step in the purification cycle and begin rinsing the chromatography unit. The progress of the rinsing stage can also be monitored by observing the stabilization of the EIS signal and the removal of unbound samples, as this can be measured by the low-frequency region of the impedance spectrum.

[0201] Example 3: Use information about column packing directly to minimize the time required to elute the product using elution buffer. During elution, secondary causes of yield reduction are encountered. Elution is induced by some chemical (acid, salt, surfactant, or organic molecule) that disrupts the interaction between the product and the chromatographic medium. Eluting chemicals are problematic because, if exposure is too long, they usually lead to aggregation and degradation of the product molecules. For example, in affinity chromatography using monoclonal antibodies, elution is achieved by injecting an acidic elution buffer with a pH of 3-4. The acidic environment causes some degree of aggregation of the product. The yield reduction can be as high as 60%, and for certain pH-sensitive mAbs, the production cost can become prohibitively high due to the extremely low yield. Yield optimization depends on identifying an exposure time that is long enough to ensure efficient elution, but short enough to prevent degradation without excessive yield loss. To date, there is no process analysis technique that can report the exact degree of column packing in real time, which can be used to minimize the duration of the elution buffer step.

[0202] Figure 12 shows EIS and UV measurements of IgG elution from a PAA polymer electrolyte coating coated with protein A. EIS spectra were measured in the range of 100 Hz to 1e6 Hz, with a frequency value of 1258 Hz shown in Figure 12. EIS scans were generated at a bias potential of -0.2 V to ensure passive, non-invasive monitoring of the elution process, and a solution maintaining pH 3 was injected to induce elution of bound IgG. The EIS signal and its derivatives are plotted as crosses (x) and circles (o), respectively, with the UV absorbance signal shown as a solid line (-). The column-sensor delay time is subtracted from the UV absorbance signal to superimpose the signals. Figure 12 shows how the EIS signal provides direct, real-time continuous information of how the column empties from 100% full to 0% and to what extent, while the UV absorbance signal generates a delayed peak. EIS monitoring allows for reduction of the amount of elution buffer used and the duration for which the product needs to be exposed.

[0203] Example 4: Direct in-line quantification (concentration) of the product during the purification process. Current purification workflows using standard chromatography (see scheme in Figure 6) lack true real-time information regarding the concentration of the product flowing through the system. This is because current analytical techniques are primarily used for process control, making it difficult, and simply too difficult, to be certain that any of the signals specifically detect the product. Therefore, product quantification is performed using offline techniques. However, inline quantification would be highly desirable.

[0204] The system for monitoring the purification process described herein (see Figure 3) allows for real-time detection of the binding curve using the EIS signal, similar to SPR experiments. In fact, Figures 5 and 15 illustrate an example highlighting how column packing can be measured at any given point in the chromatogram, ranging from completely empty (0%) to completely full (100%).

[0205] While the relative packing of a column is important in bioproduction processes, it is even more useful to be able to output the actual concentration of the product at any given time after it has been bound to the chromatography column. Using the system described herein, the concentration of the product directly bound to the column surface during the purification process can be calculated in real time by utilizing knowledge of a combination of (A) device surface area and (B) polymer coating binding capacity.

[0206] (A) Determination of the electroactive surface area. The surface area of ​​an electrode can be determined using redox pairs such as ferricyanides. This provides information about the available internal surface area of ​​the system. The effective surface area of ​​the working electrode was calculated by calculating the peak area of ​​the cathode CV peak using potential cyclic voltammetry in the presence of redox pairs and applying the following relationship. A = Q / (482 μC cm⁻²)

[0207] Another alternative is to use EIS to find the double-layer capacitance of the electrode and compare it to the capacitance using a known area.

[0208] (B) Determination of polymer coating bonding capacity The polymer coating bonding capacity can be determined in several ways: for example, by knowing the bonding capacity of the coating on the analytical sensor QCM-D or SPR, the coating capacity on a scaled-up microporous electrode can be determined by knowing the effective surface area of ​​electrode (A).

[0209] Another option is to directly calibrate the device using different samples of bioproducts at known concentrations, as shown in Figure 1. The corresponding EIS signals and measured eluted samples can then be used to benchmark the binding capacity.

[0210] In affinity chromatography, there is a high degree of certainty that the bound readout is the specific product of the target. Therefore, the best place to measure the overall concentration of the product in the process is during the rinsing and washing steps after product binding.

[0211] Example 5: Direct determination of the potency equilibrium binding constant of products from a chromatographic purification process using EIS signal readout. Current chromatography workflows rely entirely on offline analysis of collected fractions to assess product potency. Common techniques for performing equilibrium binding constant determination include SPR and ELISA. Different concentrations of the target molecule bind to a surface, and the binding constant is extracted from the readout. Using the chromatography systems described herein, the binding constant can be determined directly in line by measuring the binding curves of the product to the chromatographic support for different concentrations using EIS.

[0212] Figure 17 shows what a network of smaller chromatography columns connected in a flow scheme looks like, where eluents holding products of known concentrations can be used with background buffer and mixing valves to obtain binding data at different concentrations. Association and dissociation are measured in the analytical column to generate inline potency and Kd values ​​that can be used to verify critical quality attributes and make direct decisions regarding final product confirmation / clarification.

[0213] Since the analyzed sample can be returned to the entire process flow, the system described in Figure 17 is even more wasteful. Compared to SPR and ELISA, which utilize micrograms of sample, it is possible to significantly scale the amount of sample tested, but chromatography-based Kd measurement can validate a much larger proportion of product supply with fewer resources and a wider range of automation.

[0214] Example 6: Use of EIS signals to monitor the pH and conductivity of bulk solutions The frequency of the EIS signal measures charge transport through the polymer coating between the electrolyte and the electrode chromatography support at different timescales. There is a frequency dependence that can be used to understand bulk solution properties and distinguish them from surface-specific properties, such as product binding within the polymer coating. In Figure 13, conductivity is measured at high frequencies, while binding to the surface is measured at lower frequencies. Using EIS measurements performed on the chromatography support structure, properties such as the conductivity and pH of the bulk electrolyte environment, as well as the buffer solution flowing through the chromatography unit, can be monitored in situ. Another example is shown in Figure 14, where inline conductivity is plotted against the measured conductivity signal, demonstrating a very close similarity between the two signals. In effect, certain inline sensors can be replaced with conductive chromatography materials that passively monitor the bulk and surface properties of the chromatography unit.

[0215] Example 7: Real-time monitoring using a porous support structure including stacking of separate layers The support structure, having a layered structure, has a thickness of 1 cm to 100 cm and achieves resolution along the axial direction of the support structure and along the flow direction from the solution inlet to the solution outlet.

[0216] For preparative purification applications, it may be necessary to increase the length of the support structure and working electrode to accommodate the required binding capacity. With longer electrodes, determining the real-time binding capacity at the column inlet versus outlet can be difficult. To achieve resolution along the column flow direction, it may be advantageous to divide the support structure and working electrode into multiple physically separated layers and connect each working electrode layer to a potentiostat unit that simultaneously monitors the AC signals of all working electrode layers in real time using a parallel multiplexing scheme.

[0217] An example of what such a design might look like is shown in Figures 18a–18c. The fluid first flows through the counter electrode 102, which acts as a flow distributor, after which the liquid flows into the first individual layer 101a of the working electrode, and then flows through each individual layer of the electrode until it reaches the final individual layer 101n of the working electrode stack before exiting the chromatography column.

[0218] In Figure 18b, the working electrode stack is mounted in the center of the device, and the counter electrode 102 is wrapped around the working electrode stack 101. In this way, the counter electrode is efficiently and equally interfaced with all the working electrodes in the system.

[0219] As the fluid flows through the chromatography column device, the AC signal is used to track in real time the concentration of biological material bound to the electrode, as well as the flow of impurities through the bulk and through all individual layers of the working electrode. By collecting all surface and bulk solution information across the entire chromatography column, users have the potential to adjust binding and elution temporally and spatially (see Figures 19a–19c).

[0220] Efficient elution of biological substances is achieved by sequentially performing elution in real time using ground truth data feedback, as shown in the flowchart of Figure 20. Figure 20 shows a graph illustrating saturated working electrode discrete layers (black bars) and fully eluted discrete layers (white bars), as well as the location of eluted biological substances measured using AC voltage signals from EIS, providing real-time information on both the degree of product binding to the working electrode surface and the presence of eluted biological substances from upstream electrode discrete layers.

[0221] Elution is achieved by changing the bias potential, such as by electrochemical release of bound material, or by injecting a chemical that induces elution. When electrochemistry is used, each segment of the working electrode is eluted using feedback so that the concentration of the eluate is maximized and the minimum amount of electrochemistry is applied. When the eluted biological material is registered in the bulk solution of an adjacent electrode discrete layer, an algorithm controlling the potentiostat automatically responds by eluting from that electrode discrete layer. In this way, elution can be manipulated to maximize the output concentration of the biological material. Even as chemicals are consumed, the process can be optimized using real-time monitoring and rapidly fed back to the upstream pump to ensure that the working electrode structure is completely emptied of the biological material using the minimum amount of eluting chemical and solvent.

[0222] Figure 21 shows continuous tracking of the concentration of the binding material to the electrode surface. In-situ real-time monitoring along the direction of the chromatography support structure stack enables highly efficient use of the supernatant solution, even for working electrodes with large stack heights where sample loss due to oversupply is minimized. In Figure 21, black bars correspond to fully saturated working electrode individual layers, gray indicates partially blackened, and white indicates empty working electrode individual layers. By dividing into n segments and monitoring the binding, it is possible to track the degree of binding to thick electrode stacks from 0 to 100% / n, where n is the number of electrode individual layers. All working electrodes are connected in parallel to a potentiostat device, which measures an AC voltage that produces an EIS spectrum in which the separation between bulk solution properties and surface properties can be decoupled, for example. In this way, information regarding the location of impurities rinsed from the working electrode can be monitored with high precision through the chromatography column along the working electrode stack, allowing algorithms operating the purification process to limit the amount of excess solution supplied through the system.

[0223] Example 8: In-situ real-time monitoring of chromatography column devices Figure 18c shows a chromatography column in which the support structure / working electrode 101 consists of three individual layers 101a, 101b, and 101c. Each electrode contains a different polymer electrolyte coating. The first coating is a capture step in which the coating is functionalized with affinity ligands; the second coating is for polishing, in which this electrode segment contains a polymer electrolyte coating with ion exchange properties; and the third segment is also a polishing step in which the biological substance of interest is separated from remaining impurities by hydrophobic interactions. Each individual layer of the working electrode is connected in parallel to a potentiostat device so that all steps, all different purification mechanisms, are monitored sequentially in real time. All steps are controlled by setting the bias potential to a value that results in the elution of each segment of interest. When the AC signal detects a saturated bond, combined with the absence of impurities in the bulk solution of a single individual layer, the algorithm generates elution from that individual layer, which triggers a downstream cascade of decisions regarding the monitoring of adjacent individual layers.

[0224] Purification using the column shown in Figure 18c may be the only chromatographic step required in the purification process, as it can cover all the mechanisms by which biological substances are purified today. Avoiding multiple separate unit operations (passing the product through multiple different columns) is time-consuming and results in yield loss at each step; even small yield losses result in large combined yield losses when there are multiple steps. Naturally, eliminating combined yield losses is of interest. However, creating a chromatographic device that incorporates multiple separation mechanisms is difficult because the chromatographic column is a black box, controlled upstream and analyzed downstream, inline, and offline. Furthermore, most chromatographic materials use elution buffers that are incompatible with each other. For example, strong acids or imidazoles are elution buffers used in affinity and IMAC chromatography, and these may not be compatible with resin and membrane materials for ion exchange and hydrophobic interactions. In summary, the underlying purification devices and technologies enable the use of multiple (>2) different purification mechanisms into a single chromatography unit step by utilizing non-invasive EIS monitoring for in-situ tracking of products and impurities, and invasive electrochemical elution as a universal in-situ local elution mechanism.

[0225] Consideration The inventors have demonstrated a chromatography system that uses EIS as a process analysis technique. In contrast to using sensors as inline or in-situ tools for monitoring the process, the inventors use a whole chromatography support to directly measure the binding of the entire product sample in real time. The scale of measurement is preparative rather than analytical, highlighting the potential for this technique to be used in industrial-scale manufacturing and its industrial applicability. The scope of using this technique is to reduce losses, directly measure critical quality attributes in the purification process, increase yields, and create more efficient manufacturing workflows. This should reinforce the shift towards digitalization and automation that is so desperately needed in bioproduction processes. As a result of this technological shift, product costs will be reduced because product losses will be lower and production times will be faster.

Claims

1. A chromatography unit (200) for use in a chromatography system for real-time monitoring of the purification of biological substances, A chromatography column (100) comprising a housing (110) provided with a solution inlet (107) and a solution outlet (108), A support structure (101, 101a to 101n) is disposed in the space between the solution inlet (107) and the solution outlet (108) within the housing (110), wherein an electrolyte (F) is arranged to flow from the solution inlet to the solution outlet and is in contact with at least a portion of the support structure (101, 101a to 101n), and at least a portion of the surface of the support structure is conductive. a) A counter electrode (102) arranged to be electrically connected to the support structure (101, 101a to 101n) via the electrolyte, which is disposed within the housing (110) and is arranged to flow from the solution inlet (107) to the solution outlet (108), or b) A counter electrode (102) and a reference electrode (103) arranged within the housing and electrically connected to each other and to the support structures (101, 101a to 101n) via the electrolyte which is arranged to flow from the solution inlet (107) to the solution outlet (108), A polymer electrolyte coating (111) is provided on at least a portion of the surface of the support structure (101), and the polymer electrolyte coating (111) is arranged to be switchable between a first state in which the biological substance to be purified is trapped on the support structure and a second state in which the trapped biological substance is released from the support structure, and a counter electrode (102), or a counter electrode (102) and a reference electrode (103), Connected to the aforementioned support structures (101, 101a to 101n), a) Arranged to be connected to the counter electrode (102) and to provide an AC voltage signal with a bias between the support structure (101, 101a to 101n) and the counter electrode (102), or b) Arranged to be connected to the counter electrode (102) and the reference electrode (103), and arranged to provide an AC voltage signal with a bias between the support structure (101, 101a to 101n) and the reference electrode (103). Voltage generator (300), A recording device (300) is arranged to record current signals and / or potential signals generated between the support structures (101, 101a to 101n) and the counter electrode (102), or between the support structures (101, 101a to 101n) and the reference electrode (103). A chromatography unit (200) is provided with the following:

2. Chromatography unit (200) according to claim 1, wherein the support structure (101) is a laminated structure comprising at least two separate layers (101a to 101n) that are physically separated from each other, and an electrolyte (F) arranged to flow from the solution inlet (107) to the solution outlet (108) is arranged to be in contact with at least a portion of each of the separate layers (101a to 101n), at least one of the separate layers is provided with a polymer electrolyte coating, the voltage generator (300) is arranged to be connected to each or at least a large portion of the separate layers (101a to 101n) of the support structure (101), and the recorder (300) is arranged to record current signals and / or potential signals generated between the support structure (101) and each of the separate layers (101a to 101n) of the counter electrode (102) or the reference electrode (103), or between a large portion of the separate layers (101a to 101n).

3. The chromatography unit (200) according to claim 2, wherein the support structure (101) comprises at least first and second individual layers (101a, 101b), and the polymer electrolyte coating disposed on the first individual layer (101a) has a different chemical composition from the polymer electrolyte coating disposed on the second individual layer (101b).

4. A chromatography system (400) comprising a chromatography unit (200) according to any one of claims 1 to 3.

5. A method for real-time monitoring of chromatographic purification of biological substances, i) A step of obtaining an electrolyte volume containing biological substances and contaminants to be purified, ii) The step of providing the chromatography system (400) according to claim 4, iiia) Applying an AC potential having a bias potential difference between the support structure (101, 101a to 101n) and the counter electrode (102), and optionally measuring the open-circuit potential between the support structure (101, 101a to 101n) and the counter electrode (102), or iiib) A step of applying an AC potential having a bias potential difference between the support structure (101, 101a to 101n) and the reference electrode (103), iv) A step of supplying electrolytic running buffer to the housing (110) at the solution inlet (107) of the chromatography column (100), and equilibrating the polymer electrolyte coating (111) of the support structure (101, 101a to 101n) in the electrolytic running buffer, v) A step of setting the polymer electrolyte coating (111) placed on the support structure (101, 101a to 101n) to its first state, vi) A step in which the biological substance is captured in the polymer electrolyte coating on the support structure (101, 101a to 101n) by supplying the volume of electrolyte containing the biological substance to be purified and contaminants to the housing (110) at the solution inlet (107) of the chromatography column (100), vii) The step of setting the polymer electrolyte coating (111) placed on the support structure to its second state so that the captured biological material is released from the support structure (101, 101a to 101n), viiii) A step of recording the current signal and / or potential signal generated between the support structure (101, 101a-101n) and the counter electrode (102), or between the support structure (101, 101a-101n) and the reference electrode (103) during steps iv) to vii). Methods that include...

6. If the support structure (101) has a laminated structure comprising at least two individual layers (101a to 101n), in step iiia), an AC potential having a bias potential difference is applied between each or at least a large portion of the individual layers (101a to 101n) and the counter electrode (102), and optionally, an open-circuit potential is measured between each or at least a large portion of the individual layers (101a to 101n) and the counter electrode (102), or in step iiib), having a bias potential difference The method according to claim 5, wherein an AC potential is applied between each or at least a large portion of the individual layers (101a to 101n) and the reference electrode (103), and in step viiii), current signals and / or potential signals generated between each or at least a large portion of the individual layers (101a to 101n) and the counter electrode (102), or between each or at least a large portion of the individual layers (101a to 101n) and the reference electrode (103) during steps iv) to vii), are recorded.

7. The method according to claim 6, wherein step v) the step of setting the polymer electrolyte coating (111) disposed on the support structure to its first state includes changing / switching the bias potential difference of the applied AC potential.

8. The method according to any one of claims 57, wherein step vii) the step of setting the polymer electrolyte coating (111) disposed on the support structure to its second state includes changing / switching the bias potential difference of the applied AC potential.

9. The method according to any one of claims 5 to 8, wherein in step v), the step of setting the polymer electrolyte coating (111) disposed on the support structure to its first state is performed in step iv) via the electrolytic running buffer used.

10. The method according to any one of claims 5 to 9, wherein step vii) the step of setting the polymer electrolyte coating (111) disposed on the support structure to its second state includes adding an elution buffer to the chromatography unit.

11. The method according to any one of claims 5 to 10, further comprising a rinsing step after step vi).

12. The method according to any one of claims 5 to 11, further comprising the step of collecting biological material released from the support structure (101, 101a to 101n) and exiting through the solution outlet (108).

13. The method according to any one of claims 5 to 12, wherein the applied bias AC potential has a bias potential of + / - 1.5V.

14. The method according to any one of claims 5 to 13, wherein the applied bias AC potential has a frequency of 1 μHz to 1 MHz.

15. The method according to any one of claims 5 to 14, wherein the amplitude of the AC potential is 1 μV to 100 mV.

16. The method according to any one of claims 5 to 15, further comprising the step of providing real-time information relating to bulk electrolyte properties, the degree of binding / saturation of the biological substance captured by the polymer electrolyte coating, the degree of column packed with the biological substance, the potency of the biological substance, the concentration of the biological substance captured by the polymer electrolyte coating, the pH of the electrolyte, and / or the conductivity of the electrolyte, based on the recorded current signal and / or potential signal from step viiii).

17. The method according to claim 16, wherein the provided real-time information is used in a feedback loop to control step vi) and / or step vii).

18. The method according to any one of claims 5 to 17, wherein the volume of the electrolyte, which includes the electrolytic running buffer and the biological substance to be purified, is supplied to the housing (110) at a flow rate of 0.1 to 10 ml / min through the solution inlet (107).

19. The method according to claim 18, wherein the flow rate is a continuous flow rate having a maximum change in flow rate of 0.5 ml / min.

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