In-line product monitoring in integrated continuous biofabrication

Abstract

This invention provides a method for regulating and controlling process-related parameters in the integrated and continuous biological production of therapeutic protein materials and other biomolecules. [Solution] A method for controlling a biological manufacturing system includes the steps of: directing a light beam to pass through the wall of a container containing a first fluid produced by the biological manufacturing system; measuring the angle of refraction of the light beam in the first fluid, wherein the angle of refraction corresponds to the angle between the direction of propagation of the light beam in the first fluid and the normal to the interface between the wall of the container and the first fluid; determining information about the first fluid based on the measured angle of refraction; and adjusting the parameters of the biological manufacturing system based on the information about the first fluid.

Description

[Technical Field]

[0001] Cross-Reference to Related Application This application claims priority to U.S. Provisional Patent Application No. 62 / 924,551, filed on October 22, 2019, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to product monitoring systems and methods for use in integrated continuous biomanufacturing systems. [Background Art]

[0003] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce therapeutically or commercially important proteins. Integrated continuous biomanufacturing is a key aspect of reducing costs associated with such protein-based therapeutics. Monitoring systems are used in biomanufacturing to evaluate various biological products. [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] Integrated continuous biomanufacturing of therapeutic protein substances and other biomolecules holds great promise for the future production of life-saving drugs and for enhancing the widespread adoption of treatments that rely on the availability of such biomolecules. Two-column and multi-column chromatography systems of various arrangements can be used for industrial-scale biomanufacturing. In such systems, process monitoring of various eluate streams is used to adjust process-related parameters, and can control, for example, selective collection of eluate streams from a particular column and adjustment of properties of solution buffer (e.g., pH). [Means for Solving the Problem]

[0005] This disclosure features a method and system for determining solution properties, such as solute concentration and pH, using rapid in-line analysis of a solution emerging from a chromatography column or bioreactor, or a solution supplied to such a column or reactor. Angle-resolved refraction measurements, used in conjunction with calibration information, provide rapid and accurate quantitative information related to these solution parameters. The method can be implemented directly in-line without sampling the solution as it flows between containers, or on solution samples extracted from a column, reactor, or transfer line. The refraction angle measurements, combined with solution-specific calibration information, can be used to characterize the solution, and this information can be used to control various process-related biomanufacturing parameters and / or operations.

[0006] In one embodiment, the present disclosure features a method for controlling a biological manufacturing system, the method comprising: directing a light beam to pass through the wall of a container containing a first fluid produced by the biological manufacturing system; measuring the angle of refraction of the light beam in the first fluid, the angle of refraction corresponding to the angle between the direction of propagation of the light beam in the first fluid and the normal to the interface between the wall of the container and the first fluid; determining information about the first fluid based on the measured angle of refraction; and adjusting parameters of the biological manufacturing system based on the information about the first fluid.

[0007] Embodiments of this method may include one or more of the following features:

[0008] Information regarding the first fluid may include the concentration of a substance in the first fluid and / or the pH value of the first fluid and / or the ionic strength of the first fluid. The first fluid may include the fluid discharged from the system's purification unit. The purification unit may include at least one chromatography column.

[0009] The first fluid may include a supply fluid introduced into the system's reactor. The first fluid may also include a fluid discharged from the system's filtration unit (e.g., a diafiltration unit).

[0010] The parameters of the biological manufacturing system may include a fluid channel that selectively directs a continuous portion of a first fluid to one of the system's purification units. The parameters of the biological manufacturing system may include the rate at which the material is supplied to the system's reactor. The parameters of the biological manufacturing system may include the rate at which the material is supplied to the system's diafiltration unit.

[0011] The parameters of a biological manufacturing system may include the rate at which a substance is supplied to the system's purification unit. The parameters of a biological manufacturing system may also include the rate at which a continuous portion of the first fluid is supplied to the system's purification unit.

[0012] This method may include a step of determining the concentration of a substance from a calibration formula derived from measured calibration data, wherein the calibration formula expresses the concentration as a function of the angle of refraction. The calibration formula may express the concentration as a linear function of the angle of refraction. The calibration formula may also express the concentration as a nonlinear function of the angle of refraction. This method may include a step of transmitting the concentration information to a system controller, which is configured to receive the concentration information and adjust the system parameters.

[0013] The substance may include proteins and / or recombinant protein-based formulations and / or nucleic acid-based products.

[0014] The first fluid may be a process fluid produced by the system as part of a biological manufacturing process. The first fluid may include an eluate discharged from a chromatography column. The first fluid may include an eluate from a first purification unit of the system, and the information may include the concentration of a substance in the first fluid, and parameter adjustment may include directing the eluate from the first purification unit to the inlet of a second purification unit when the concentration of a substance in the first fluid exceeds a threshold. The first and second purification units may each include at least one chromatography column.

[0015] The first fluid may include an eluate from a first purification unit of the system, the information may include the concentration of a substance in the first fluid, the inlet of the first purification unit may be connected to a conduit that delivers a supply solution to the first purification unit, and the method may include the steps of disconnecting the inlet of the first purification unit from the conduit when the concentration of a substance in the first fluid exceeds a threshold, and connecting the inlet of a second purification unit of the system to the conduit to deliver a supply solution to the second purification unit.

[0016] The method comprises the steps of: directing a second light beam through the wall of a second container containing a second fluid produced by a biological manufacturing system; measuring the angle of refraction of the second light beam in the second fluid, wherein the angle of refraction corresponds to the angle between the propagation direction of the second light beam in the second fluid and the normal to the interface between the wall of the second container and the second fluid; and determining information about the second fluid based on the measured angle of refraction of the second light beam. The process may also include adjusting a second parameter of the biological manufacturing system based on information about a second fluid. The information about the second fluid may include the concentration of buffer solution components in the second fluid. The information about the second fluid may include the concentration of ionic compounds or their dissolved salts in the second fluid. The process of adjusting the second parameter may include adjusting the supply rate of the second fluid to the system's purification unit.

[0017] This method may include a step of providing concentration information to a controller as a feedback signal to adjust system parameters during a biological manufacturing process by repeating the steps of directing, measuring, determining, and transmitting.

[0018] Embodiments of this method may also include any other features or steps described herein, including any combination of features and / or steps described separately in relation to the same or different embodiments, unless otherwise expressly stated.

[0019] In another embodiment, the Disclosure features a biological manufacturing system comprising a container configured to hold or transport a first fluid produced by the system, the container characterized by a wall defining at least a portion of the container; a light source configured to direct a light beam through the wall; a detector configured to measure the angle of refraction of the light beam in the first fluid, the angle of refraction corresponding to the angle between the direction of propagation of the light beam in the first fluid and the normal to the interface between the wall and the first fluid; and a first controller connected to the detector, the first controller configured to receive information about the angle of refraction from the detector, determine information about the first fluid based on the measured angle of refraction; and adjust system parameters based on the information about the first fluid.

[0020] Embodiments of this system may include one or more of the following features:

[0021] Information regarding the first fluid may include the concentration of substances in the first fluid and / or the pH value of the first fluid.

[0022] The system may include a purification unit, where the first fluid includes the fluid discharged from the purification unit. The purification unit may include at least one chromatography column. The system may include a reactor, where the first fluid includes the supply fluid introduced into the reactor. The system may include a filtration unit (e.g., a diafiltration unit), where the first fluid includes the fluid discharged from the filtration (e.g., diafiltration) unit.

[0023] The system may include multiple purification units, and the system parameters include a fluid channel that selectively directs a continuous portion of a first fluid to one of the multiple purification units. The system may include a reactor, and the system parameters include the rate at which a substance is supplied to the reactor. The system may include a filtration (e.g., diafiltration) unit, and the system parameters include the rate at which a substance is supplied to the filtration (e.g., diafiltration) unit. The system may include a purification unit, and the system parameters include the rate at which a substance is supplied to the purification unit. The system may include a purification unit, and the system parameters include the rate at which a continuous portion of the first fluid is supplied to the purification unit.

[0024] The first controller is configured to determine the concentration of a substance from a calibration formula derived from measured calibration data, where the calibration formula expresses the concentration as a function of the angle of refraction. The calibration formula is, The concentration can be expressed as a linear function of the angle of refraction. The calibration formula may also express the concentration as a nonlinear function of the angle of refraction.

[0025] The substance may contain proteins. The substance may contain recombinant protein-based formulations. The substance may contain nucleic acid-based products.

[0026] The first fluid may be a process fluid produced by the system as part of a biological manufacturing process. The system may include a purification unit, and the first fluid may include an eluate discharged from the purification unit.

[0027] The system may include a first purification unit and a second purification unit, where the first fluid includes the eluate from the first purification unit, the information includes the concentration of the substance in the first fluid, and the parameter adjustment may include directing the eluate from the first purification unit to the inlet of the second purification unit when the concentration of the substance in the first fluid exceeds a threshold. The first and second purification units may each include at least one chromatography column.

[0028] The system may include a first purification unit and a second purification unit, the first purification unit characterized by an inlet connected to a conduit for delivering a supply solution to the first purification unit, the first fluid comprising an eluate from the first purification unit, the information comprising the concentration of a substance in the first fluid, and parameter adjustment comprising disconnecting the inlet of the first purification unit from the conduit and connecting the inlet of the second purification unit to the conduit to deliver the supply solution to the second purification unit when the concentration of a substance in the first fluid exceeds a threshold.

[0029] The system includes a second container configured to hold or transport a second fluid generated by the system, the second container characterized by a second wall defining at least a portion of the second container; a second light source configured to direct a second light beam through the second wall; and a second detector configured to measure the angle of refraction of the second light beam in the second fluid, the angle of refraction corresponding to the angle between the propagation direction of the second light beam in the second fluid and the normal to the interface between the second wall and the second fluid; and a first controller connected to the second detector and configured to receive information on the angle of refraction from the second detector, determine information on the second fluid based on the measured angle of refraction, and adjust a second parameter of the system based on the information on the second fluid.

[0030] Information regarding the second fluid may include the concentrations of buffer solution components in the second fluid. Information regarding the second fluid may also include the concentrations of ionic compounds or their dissolved salts in the second fluid.

[0031] The system may include a purification unit, and adjusting the second parameter involves adjusting the supply rate of the second fluid to the purification unit.

[0032] During the biological manufacturing process, the light source is configured to repeatedly direct a light beam through a wall, the detector is configured to repeatedly measure the refraction angle of the light beam in a first fluid, and the first controller is configured to repeatedly receive information about the refraction angle from the detector, determine information about the first fluid based on the measured refraction angle, and adjust the system parameters based on the information about the first fluid.

[0033] This system consists of multiple chromatographic columns, each having at least one chromatography column. The system may include a purification unit, a column switching mechanism, and a second controller connected to the column switching mechanism and communicating with a first controller, the first controller being configured to adjust the system parameters by sending a signal to the second controller causing the second controller to adjust the column switching mechanism to selectively direct fluid to one of a plurality of purification units. The selectively directed fluid may include an additional portion of the first fluid.

[0034] The system may include a reservoir containing a second fluid and a second controller that communicates with a first controller, the first controller being configured to adjust system parameters by transmitting control signals to a second processor based on information about the first fluid, and the second controller being configured to introduce a portion of the second fluid from the reservoir into a continuous portion of the first fluid in response to the control signals. The second fluid may include a buffer solution. The second fluid may include a solution of an ionic compound or its dissolved salt.

[0035] Embodiments of this system may also include any other features or processes described herein, including any combination of features and / or processes described separately in relation to the same or different embodiments, unless otherwise expressly stated.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials described herein may be used in the practice or testing of the subject matter herein, but preferred methods and materials are described below. All publications, patent applications, patents and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0037] Details of one or more embodiments are shown in the accompanying drawings and the following specification. Other configurations and advantages will be apparent from the specification, drawings and claims. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 is a schematic diagram showing a light beam that crosses the interface between two materials and is refracted at the interface. [Figure 2] Figure 2 is a schematic diagram showing a light beam that crosses the interface between different materials and is refracted at the interface. [Figure 3] Figure 3 is a schematic diagram showing a system for performing angle-resolved refraction measurements of a light beam. [Figure 4] Figure 4 is a schematic plot showing the solute concentration as a function of the refraction angle of the solution. [Figure 5] Figure 5 is a schematic diagram showing an in-line system for performing angle-resolved refraction measurements of a light beam. [Figure 6] Figure 6 is a schematic diagram showing a system for performing angle-resolved refraction measurements of a light beam in reflection mode. [Figure 7] Figure 7 is a schematic diagram of a continuous biological manufacturing system. [Figure 8] Figure 8 is a schematic diagram illustrating the steps involved in switching chromatography columns in a continuous biological manufacturing system. [Modes for carrying out the invention]

[0039] Similar symbols in the drawing indicate similar elements.

[0040] Detailed explanation Introduction Industrial-scale biological manufacturing, involving the purification of intermediates and / or products, can be carried out in two-column and multi-column chromatography systems of various configurations. In these complex systems, product yield, purity, and waste rate are functions of numerous process-related parameters and steps. During the production of therapeutic proteins and other commercially valuable biomolecules, the product outcome is strongly influenced by these parameters and steps. Therefore, appropriate control over such parameters and steps is a crucial aspect of large-scale manufacturing. Configurations and configurations of biological manufacturing systems are disclosed, for example, in International Publication 2014 / 137903 of the PCT Patent Application Publication, the entire contents of which are incorporated herein by reference.

[0041] The proper control of bioproduction parameters, including automated control, is facilitated by in-process, in-line monitoring of intermediate solution flows, specifically the concentrations of intermediates and products in such flows, as well as other characteristics of such flows (e.g., pH). Conventional methods of solution monitoring include techniques such as UV absorbance measurement. Unfortunately, however, such methods suffer from drift during the measurement period of several days due to factors such as temperature, humidity, ambient light intensity, and local sample heterogeneity.

[0042] Disclosed herein are methods and systems for using angle-resolved refractometry to determine properties of a process solution including concentrations of products and intermediate components and buffer concentration / pH levels. Such refractive measurements are rapid, highly reproducible, and when combined with appropriate calibration information, can be used to determine accurate quantitative information about the solution. This quantitative information can then be used as feedback to an automatic or semi-automatic process control system that adjusts process conditions and starts or stops specific process steps.

[0043] In-line angle-resolved refractometry Figure 1 is a schematic diagram showing a light ray passing from a gaseous environment (e.g., air) to a liquid environment (e.g., an aqueous solution). In Figure 1, an interface 106 separates the gaseous environment 102 from the liquid environment 104. An incident light beam 110 propagates through the gaseous environment 102, passes through the interface 106, and then propagates through the liquid environment 104 as a refracted beam 112. The incident light beam 110 is incident on the interface 106 at an angle θ relative to the normal 108 of the interface 106 I incident on the interface 106. The refracted light beam 112 forms an angle θ with the normal 108 r .

[0044] The refraction angle θ r depends on the incident angle θ I and the respective refractive indices n of the gaseous environment and the liquid environment gas and n liquid . The relationship between these variables is given by Snell's law: n gas sin(θ i )=n liquid sin(θ r ) [1] Therefore, rearranging Equation (1) gives θ r calculated as follows: θ r =sin -1 (n gas sinθ I / n liquid ) [2]

[0045] θ r is a function of n liquidIf n is known, it can be calculated from equation (2). liquid The value of n has been measured for various pure liquids. For solutions consisting of solute dissolved in a solvent, the refractive index n solution ≒n solvent +Δn solute And here Δn solute This represents the additional contribution to the refractive index of the solution caused by the presence of the solute. Generally, a dissolved solute makes the solution optically denser than a pure liquid solvent, and this is because, in most cases, n solution >n solvent This means that it will be as follows.

[0046] Typically, as the concentration of a solute in a solution increases, the optical density and refractive index of the solution also increase. There is no general analytical formula to explain the relationship between the concentration of a particular solute in a given solvent and the refractive index of the resulting solution. Instead, only the following general relationship holds: n solution =f(c) [3] Here, c is the concentration of a specific solute in a specific solvent that forms a solution, and f(c) is an unknown functional form.

[0047] Assuming that f(c) is a linear function of the form f(c) = uc + v, and that u and v are unknown constants, then the following occurs: n solution =uc+v [4]

[0048] Substituting this into equation (1), we get the following: n gas sinθ i =(uv+c)sinθ r [5]

[0049] Rearranging equation (5) for solute concentration c, we obtain the following equation:

number

[0050] Equation (6) gives the concentration c as c = m·csc(θ) r The form is ) + b, and the slope m = (ngas / u)·sin(θ i ), the intercept b = -(v / u) csc(θ r It is expressed as a linear function of θ. r For a certain value, csc(θ r )≒θ r If the following condition is met, then the result will be as follows: c = m·θ r +b [7] Here, the values ​​of m and b are as described above. In other words, for example, if the values ​​of the constants m and b are known from the calibration information of a specific solute dissolved in a specific solvent, then for a solution in which the concentration of the solute dissolved in the solvent is unknown, the solute concentration c can be given by equation (7) and the refraction angle θ. r It can be determined directly from the measured values.

[0051] The situation becomes slightly more complex when the light ray being investigated passes through the walls of a container or conduit in which a solution is held or flowing. Figure 2 is a schematic diagram showing a light beam 210 propagating through a gas 202. The beam is directed through the solid, translucent, or transparent walls 203 of a tube or container containing the liquid 204. Interface 206 separates the gas 202 from the solid wall 203, and a second interface 207 separates the solid wall 203 from the liquid 204. The gas 202 has a refractive index n gas The solid wall 203 has a refractive index n solid Formed from a material having a refractive index n, liquid 204 has a refractive index n liquid It holds.

[0052] The light beam 210 is incident on interface 206 at an angle θ1 with respect to the normal 208 of interface 206, and is refracted within the solid wall 203 at an angle θ2 with respect to the normal 208, forming a refracted beam 212. Assuming that interfaces 206 and 207 are parallel, the refracted beam 212 is incident on interface 207 at an angle θ2 with respect to the normal 209, and is refracted within the liquid 204 at an angle θ3 with respect to the normal 209, forming a refracted beam 214.

[0053] At interface 206, Snell's Law explains the following relationship between the refractive index and the angle of incidence and angle of refraction: ngas sinθ1=n solid sinθ²[8] Similarly, at interface 207, Snell's Law gives the following: n solid sinθ²=n liquid sinθ3[9]

[0054] Combining equations (8) and (9) eliminates the dependence on the solid wall 203: n gas sinθ1=n liquid sinθ3

[10]

[0055] Equation (10) is in the same form as equation (1), θ i =θ1, θ r =θ3. Therefore, for a specific solute that dissolves in a specific solvent to form liquid 204, the concentration c of the dissolved solute can be expressed as follows, assuming the same corresponding assumptions as above. c = m·θ³ + b

[11] Here, the slope m = (n gas The equation is ( / u)·sin(θ1), and the intercept b = -(v / u). In other words, if the values ​​of the constants m and b are known, for example from calibration data, the concentration c can be directly determined from the measured angle of refraction θ3 and equation (11).

[0056] Figure 3 is a schematic diagram showing one embodiment of a measurement system 300 for measuring the refraction angle of a light beam in a liquid. The system 300 includes a light source 308 and an angle-resolved photodetector 310 connected to a controller 312. A liquid 304, an intermediate or product solution containing a certain amount of a biological manufacturing product, such as a protein dissolved in a solvent, is contained between the walls 302 and 306 of a container or conduit. The liquid 304 can be stationary or moving, i.e., flowing, within the container or conduit. The walls 302 and 306 are formed from a solid, translucent or transparent material, such as glass or a polymer material, with a refractive index n wall It has. Liquid 304 has a refractive index n solution It holds.

[0057] During operation, the controller 312 directs the light source to generate an incident light beam 314 that is incident on the interface 322 between the wall 302 and the liquid 304 at an angle θ1 with respect to the normal 318 of the interface 322. The refracted beam 316 is refracted at an angle θ2 with respect to the normal 318.

[0058] The refracted beam 316 is incident on a detector 310 located at the interface 324 at an angle θ2 with respect to the interface normal 320. The detector 310 is configured to measure the refraction angle θ2 of the beam 316 and transmit information about θ2 to the controller 312. The geometric arrangement in Figure 3 is similar to the arrangement in Figure 1, and θ i =θ1, θ r =θ². Therefore, Snell's Law gives the following: n wall sinθ1=n solution sinθ²

[12]

[0059] Therefore, for a specific solute that dissolves in a particular solvent to form liquid 304, the concentration c of the dissolved solute can be expressed as follows, assuming the same corresponding assumptions as above. c = m·θ² + b

[13] Here, the slope m = (n wall The equation is ( / u)·sin(θ1), and the intercept b = -(v / u). In other words, if the values ​​of the constants m and b are known, for example from calibration data, the concentration c can be directly determined from the measured angle of refraction θ2 and equation (13).

[0060] To obtain a quantitative concentration c of a specific solute in a specific solvent, calibration data is measured first. Typically, for a series of solutions, each with a known concentration of the solute of interest dissolved in the solvent of interest, each solution is measured using System 300 to generate a series of calibration data including the solute concentration c and the corresponding refraction angle θ2. Figure 4 is a schematic plot showing a series of refraction angles θ2 measured for solutions with known concentrations c of a specific solute.

[0061] After the data is measured, it is sent to the controller 312 for analysis. The controller 312 is configured to determine the values ​​of the constants m and b in equation (13). In some embodiments, for example, the controller 312 performs a linear regression analysis to determine the best-fitted line 402 through a set of (c,θ2) calibration data points. The regression analysis determines the values ​​of the constants m and b from equation (13). Once the values ​​of m and b are determined, the system 300 is calibrated and ready for inline measurement of process solutions in a biomanufacturing system.

[0062] Figure 5 is a schematic diagram showing part of a biomanufacturing system 500, including a measurement system 300. In Figure 5, a process solution 508 containing a product or intermediate such as a protein, polypeptide, antibody, or other biomolecule (i.e., solute) dissolved in a solvent flows from a first vessel 502 (e.g., a chromatography column, bioreactor, or other reaction or purification vessel, or tank) through a conduit 506 to a second vessel 504 (e.g., a chromatography column, bioreactor, or other reaction or purification vessel, or tank). Integrated into the conduit 506 are a light source 308 and an angle-resolved detector 310 connected to a controller 312. As shown in Figure 5, the controller 312 also includes connections 510 to another control / logic unit that modulates various operational parameters and / or process steps related to the biomanufacturing process.

[0063] During operation, as described above, the controller 312 includes calibration information (i.e., values ​​of m and b) determined for the product or intermediate species dissolved in the solute. In some embodiments, the controller 312 determines this information before performing an in-line measurement of the solution 508. Alternatively, in certain embodiments, the calibration information is retrieved by the controller 312 from a storage unit 370 (e.g., a memory unit or other persistent storage medium such as a magnetic or optical storage medium). As another alternative, in some embodiments, the calibration information is encoded within the controller 312, for example, in the controller's firmware. As a further example, in certain embodiments, the calibration information is stored in a network-accessible database, and the controller 312 is configured to retrieve the calibration information from the database via a wired or wireless network interface (e.g., a local area network, a wide area network, or an interface of any other type of distributed network architecture connecting two or more computing devices).

[0064] To measure solution 508, light source 308 generates a light beam that refracts upon entering solution 508, and detector 310 measures the refraction angle θ2. Controller 312 receives information on the refraction angle θ2 from detector 310 and then uses the angle information and calibration information of equation (13) to determine the concentration c of the product or intermediate species in solution 508. Optionally, controller 312 can communicate the concentration information of solution 508 to another control / logic unit via connection 510.

[0065] Generally, the conduit 506 is formed from a material that is transparent or translucent to the light beam generated by the light source 308. In some embodiments, for example, the conduit 506 is formed from a flexible polymer material such as polyethylene, polypropylene, or polybutylene, but is not limited to these. In certain embodiments, the conduit 506 is formed from one or more glass materials.

[0066] A wide variety of devices can be used to implement the light source 308. In some embodiments, for example, the light source 308 features one or more light-emitting diodes (LEDs). Alternatively or additionally, in certain embodiments, the light source 308 includes one or more laser diodes. In some embodiments, the light source 308 includes one or more lasers (e.g., gas lasers, solid-state lasers, organic dye-based lasers). This includes lasers. In certain embodiments, the light source 308 includes one or more fluorescent and / or incandescent light sources, such as a flash lamp-based light source.

[0067] The light source 308 can be configured to generate a light beam having central wavelengths in various spectral regions. In some embodiments, for example, the light beam has central wavelengths from about 400 nm to about 800 nm in the visible region of the spectrum. In certain embodiments, the light beam has central wavelengths greater than 800 nm in the near-infrared or infrared region of the spectrum. In some embodiments, the light beam has central wavelengths less than 400 nm in the ultraviolet region of the spectrum. Wavelengths in the visible region of the spectrum may be advantageous in that they are easily observable for the adjustment and calibration of the measurement system 300. Wavelengths in the near-infrared and infrared regions of the spectrum may be advantageous in that they can at least partially penetrate materials that are otherwise relatively opaque to wavelengths in the visible region of the electromagnetic spectrum.

[0068] In some embodiments, the light source 308 is a relatively broadband light source that generates a light beam having a full width at half maximum (FWHM) spectral bandwidth of 5 nm or more (e.g., 7 nm or more, 10 nm or more, 12 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 50 nm or more). In certain embodiments, the light source 308 is a relatively narrowband light source that generates a light beam having an FWHM spectral bandwidth of 3 nm or less (e.g., 2 nm or less, 1 nm or less, 0.5 nm or less).

[0069] In general, any sensor capable of performing angle-resolved measurements of light passing through solution 508 can be used in detector 310. That is, a wide variety of sensors capable of measuring the refraction angle θ2 for a light beam passing through solution 508 can be used. As an example, in certain embodiments, detector 310 may include an InVue® CR288 concentration monitor (available from Entegris, Inc., Billerica, MA).

[0070] In Figure 5, the light source 308 is in contact with the surface of the conduit 506, and the light beam generated by the light source 308 is directly connected to the wall of the conduit 506. However, more generally, the light source 308 does not need to be in direct contact with the conduit 506 and is positioned at a distance from the conduit 506. If the light source 308 is positioned at a distance from the conduit 506 so that the light beam propagates through a gas (e.g., air) before entering the wall of the conduit, then the same relationships as in equations (12) and (13) still hold, and n wall n instead gas The formula used is θ1, where θ1 refers to the angle of incidence of the light beam onto the wall of the conduit.

[0071] In Figures 3 and 5, the detector 310 is integrated into the wall 306 of the container or conduit containing the solution to be analyzed, at a position approximately opposite (laterally offset) the light source 308. The measurement system 300 thus performs transmission mode measurements of the light beam refracted through the solution 508.

[0072] Depending on the circumstances, either or both of the light source 308 and the detector 310 can be integrated into the wall of the container or conduit containing the solution to be analyzed. Various different implementations can be used to integrate these components into the container wall. For example, in some embodiments, the container wall includes an opening sized to accommodate the light source 308 or the detector 310. The light source 308 or detector 310 is placed within the opening and secured in place using an appropriate adhesive. In certain embodiments, the light source 308 or detector 310 is positioned within the opening such that the surface of the light source 308 or detector 310 protruding through the container wall is flush with the inner surface of the container wall, so that the solution flowing through the container is not obstructed by the light source or detector.

[0073] In some embodiments, the measurement system 300 can be configured to perform reflection mode measurements of a light beam refracted through the solution 508. Figure 6 is a schematic diagram showing a measurement system 600 for performing reflection mode measurements. The system 600 includes a light source 308, a detector 310, and a controller 312. The light source 308 is positioned to produce an incident light beam 314 that refracts at the interface 322 and enters the interface 324 at an angle θ2 with respect to the normal 320 of the interface, as previously described in relation to Figure 3.

[0074] In the measurement system 600, the mirror 606 is positioned to block the refracted light beam 316 at a location where, otherwise, the refracted light beam 316 would intersect the interface 324. The mirror 606 reflects the refracted beam 316, forming a reflected beam 602 reflected at an angle θ2 with respect to the normal 320. The reflected beam 602 is then incident on the interface 322 at an angle θ2 with respect to the interface normal 604. At the interface 322, the reflected beam 602 is blocked by the detector 310, which measures the angle θ2. Due to the symmetry in the measurement system 600, the incident angle θ2 of the reflected beam 602 with respect to the normal 604 is the same as the refraction angle of the refracted beam 316 with respect to the normal 318. Thus, equations (12) and (13) remain valid for the reflect mode measurement performed by the system 600, and calibration and quantitative concentration determination can be performed in the manner described above.

[0075] Solvent / solute-specific calibration Generally, the calibration data measured is the refractive index n of the solution. solution The complex and non-analytical relationship between solute concentration c and the target solute and solvent is specific to that particular solute and solvent. Therefore, generally, for each new combination of solute and solvent in the measurement systems 300 and 600, a new set of calibration data specific to that solute and solvent is measured or acquired. In some embodiments, the measurement systems 300 and 600 can each store multiple sets of calibration data, each set corresponding to a different combination of solute and / or solvent. When the controller 312 receives an appropriate control signal from the control / logic unit of the biomanufacturing system indicating that a particular combination of solute and solvent is to be measured, the controller 312 retrieves the appropriate set of calibration data from the storage unit 314 or measures a new set of calibration data specific to that solute and solvent combination.

[0076] It should be noted that the stored calibration data can take on a number of forms. In some embodiments, the stored calibration data includes the measured data point (c, θ²). In certain embodiments, the stored calibration data includes both the measured data point and the values ​​of the constants m and b determined for equation (13). Conversely, in some embodiments, the stored calibration data includes only the values ​​of m and b; the raw data point is not stored.

[0077] In general, the correlation between concentration c and refractive angle θ2 in equation (13) (an example of which is plotted in Figure 4) is nearly linear at relatively high solute concentrations and more nonlinear at relatively low solute concentrations. The exact nature of the correlation between c and θ2 is a complex function, and the change in solute concentration c is related to the refractive index n. solution This depends on the mode of change that occurs, which in turn depends on the inherent nature of the interaction between the solute and the solvent, and the pure solvent n solvent It depends on the refractive index.

[0078] In equation (13), it is assumed that the solute concentration c depends linearly on the refraction angle θ2. However, as shown in Figure 4, this linear relationship does not necessarily hold for all concentrations c. Therefore, while assuming a linear relationship between c and θ2 is convenient for calibration and concentration calculation purposes, more generally, in some embodiments, it is also possible to assume that c is a nonlinear function of θ2. Furthermore, in order to express c as a function of θ2, various Various nonlinear function forms can be used. For example, in certain embodiments, c can be expressed as one or more of the following: a polynomial function of θ², a hyperbolic function of θ², an exponential function of θ², a logarithmic function of θ², and a trigonometric function of θ².

[0079] If these or any other arbitrary nonlinear functional forms are assumed for the relationship between c and θ2, the controller 312 can determine appropriate values ​​for the tunable parameters of the assumed functional form using various methods, including, for example, nonlinear regression analysis. One advantage of storing the measured data points (c, θ2) in the calibration information is that the controller 312 can easily change to different assumptions about the functional form governing the relationship between c and θ2. If the measured data points have been acquired and the parameter values ​​for the new functional form do not yet exist in the calibration information, the controller 312 can determine the parameter values, for example, by performing regression analysis on the raw measured data points. In some cases, the controller 312 can then update the calibration information by storing the parameter values.

[0080] For example, if the concentration c is csc(θ) r Consider equation (6), which is a linear function of ). For small angles, csc(θ r ) ≈ 1 / θ r Therefore, a small θ r So, it would be as follows: c ≈ m / θ r +b

[14]

[0081] Therefore, the angle of refraction θ r If it is small, c is 1 / θ r It can be expressed as a linear function of θ, where the constants m and b have the values ​​described above for equation (6). That is, c is θ r It can be expressed as a hyperbolic function of (or θ²), where c vs 1 / θ r The plot of this can be an almost straight line with slope m and y-intercept b.

[0082] The above discussion focuses on determining the concentration c of a solute as a function of the measured refraction angle θ², but more generally, other parameters can also be measured as a function of the refraction angle θ². For example, in some embodiments, the pH of a solution can be determined as a general function g of the refraction angle. pH=g(θ2)

[15]

[0083] In general, pH can be a linear or nonlinear function of the angle of refraction and may have any of the functional forms described herein in relation to the concentration c. Furthermore, equation (15) can also be applied to other solution parameters that can be measured by the systems described herein, including but not limited to pOH, viscosity, tension, osmotic pressure, and light scattering (e.g., light scattering intensity).

[0084] Information reporting As described above, the measurement systems disclosed herein can be used to measure the solute concentration of a wide variety of solute-solvent solution pairs. Generally, the solutions on which the measurement is performed include, but are not limited to, product solutions coming from chromatography columns, bioreactors, holding tanks, and other containers in a bioproduction system. Alternatively or additionally, the solutions may be intermediate solutions, effluents or eluents, or other liquid process flows from chromatography columns, bioreactors, holding tanks, and other containers in a bioproduction system.

[0085] Certain solutes for which concentration information can be quantitatively determined include proteins, polypeptides, amino acids, antibodies, and various other biological molecules. In certain embodiments, the solute of interest Recombinant therapeutic proteins are recombinant therapeutic proteins. Examples of such proteins include, but are not limited to, immunoglobulins (including light-chain and heavy-chain immunoglobulins, antibodies or antibody fragments (e.g., any of the antibody fragments described herein)), enzymes (e.g., galactosidases (e.g., alpha-galactosidase), myozymes or cerezymes), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or interferon alpha or beta), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in vaccines). Recombinant therapeutic proteins can be engineered antigen-binding polypeptides containing at least one polyfunctional recombinant protein scaffold (see, for example, Gebauer et al., Current Opin. Chem. Biol. Vol. 13: pp. 245-255, 2009; and recombinant antigen-binding proteins described in U.S. Patent Application Publication No. 2012 / 0164066 (both of which are incorporated herein by reference in their entirety)).Examples of recombinant therapeutic proteins that are antibodies include, but are not limited to, panitumumab, omalizumab, avagovomab, absiximab, actoxumab, adalimumab, adecatumumab, aferimomab, aftuzumab, aracizumab, alemtuzumab, alirocumab, altumomab, amatsuximab, anatumomab, anlukinzumab, apolizumab, alsitumomab, atinumab, tocilizumab, basiliximab, vectumomab, belimumab, bevacizumab, besilezumab, besilesomab, bezlotoxumab, bisilomab, canakinumab, certolizumab, cetuximab, cizutumumab, daclizumab, and denos Examples include mab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, epratuzumab, erzumakisomab, etalasizumab, figtumumab, golimumab, ibritumomab tiuxetan, igobomab, imagatuzumab, infliximab, inorimomab, inotuzumab, lavetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, olegobomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotzumab, trastuzumab, beltuzumab, zaltumumab, and zatuximab. Additional examples of recombinant therapeutic proteins that can be measured include alglucosidase alpha, laronidase, abatacept, galsulfase, alteplase alpha, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alpha, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, imiglucerase, dorunase alpha, epoetin alpha, insulin or insulin analogs, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin-11, idursulfase, galsulfase, α-1-proteinase inhibitors, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alpha (e.g., Thyrogen®), and alteplase.Further examples of recombinant proteins that can be measured include acid α-glucosidase, alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase (e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, and N-acetylglucosamine-1-phosphotransferase. Examples include ceramidase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase, α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

[0086] Analysis of calibration data between solute concentration (or another parameter of interest) and the refractive index of the solution. Since it is used to establish a relationship, the solute concentration value can be determined in real time or near real time using the methods and systems disclosed herein, which can be important for feedback and control to the biomanufacturing process. In particular, in some embodiments, the elapsed time between the start of refractive index measurement and the time when the solute concentration is determined may be less than one minute (e.g., less than 30 seconds, less than 15 seconds, less than 10 seconds, less than 5 seconds, less than 3 seconds, less than 2 seconds, less than 1 second).

[0087] Concentration, pH, and any other aforementioned types of information can be transmitted by controller 312 to another control / logic unit within the biomanufacturing system for the purpose of monitoring and feedback control of various process parameters and processes. As will be detailed in the following sections, the transmitted information can be used for a variety of purposes, including increasing the yield of valuable products, reducing the generation of undesirable by-products and waste streams, and controlling the rate of reactions that occur as part of the manufacturing process.

[0088] In particular, when implemented as part of an in-line measurement system for continuous biofabrication, the methods and systems disclosed herein can offer significant advantages. For example, certain conventional measurement techniques, such as UV absorbance and conductivity measurements, may be affected by signal degradation / drift over time, requiring relatively frequent recalibration to ensure accuracy. However, in the case of continuous biofabrication operations, near-continuous process monitoring may be required, making frequent recalibration extremely inconvenient or even impossible. In contrast, reflection-based measurements and measurement systems are typically unaffected by signal degradation / drift and are therefore ideally suited for continuous manufacturing applications.

[0089] Integration with bio-manufacturing systems The measurement systems disclosed herein can be integrated with biomanufacturing systems to provide feedback control to various components and processes in the synthesis process. As shown in Figure 5, the measurement systems are typically implemented inline between components of the manufacturing system, allowing for real-time analysis without sampling or bypassing flowing or stationary solutions. The measurement systems can also be used conventionally with samples extracted from reaction vessels, holding tanks, or chromatography columns before performing refractometer measurements.

[0090] In addition to those described below, aspects of bio-manufacturing systems are described in U.S. Patents No. 9,650,412, No. 10,071,364, and No. 10,087,214, and U.S. Patent Publications No. 2018 / 0051054 and No. 2020 / 0063082, the entire contents of each of these are incorporated herein by reference.

[0091] (a) Purification of the product and intermediates An integrated, fully continuous process for producing therapeutic protein drugs and other substances may include, for example, providing a liquid culture medium containing a substantially cell-free recombinant therapeutic protein, and then feeding the liquid culture medium into a first multi-column chromatography system (MCCS1). The next step may include using MCCS1 to capture the recombinant therapeutic protein in the liquid culture medium, and then continuously feeding the eluate from MCCS1 containing the recombinant therapeutic protein into a second multi-column chromatography system (MCCS2), and using MCCS2 to purify and polish the protein. The eluate obtained from MCCS2 is considered to be the therapeutic protein drug substance. The process can be integrated and operated continuously from the liquid culture medium to the eluate from MCCS2 which is the therapeutic protein drug substance.

[0092] Biological manufacturing systems are typically used to carry out the processes described above. For example, Such a system may include an MCCS1 including an inlet and an MCCS2 including an outlet. In these systems, the first and second MCCSs are in fluid communication with each other. The system is also configured such that fluid enters the manufacturing system through the first and second MCCSs at the inlets and exits through the outlets.

[0093] Such a system can provide continuous and time-efficient generation of therapeutic drug substances from liquid culture media. For example, the elapsed time between supplying a fluid containing the therapeutic protein (e.g., liquid culture medium) to the first MCCS and eluting the therapeutic protein drug substance (containing the therapeutic protein) from the outlet of the second MCCS is, for example, about 4 hours to about 48 hours.

[0094] Figure 7 is a schematic diagram illustrating an example of a biological manufacturing system. System 1 includes a first MCCS, i.e., a four-column periodic countercurrent chromatography system (PCCS) 2, where three of the four columns in the four-column PCCS2, columns 3, 4, and 5, perform a unit operation to capture recombinant therapeutic proteins from a fluid containing recombinant therapeutic proteins (e.g., a liquid culture medium substantially free of mammalian cells), and one of the columns 6 in the PCCS2 performs a unit operation to inactivate viruses present in the elutes from columns 3, 4, and 5 in the PCCS2 containing recombinant therapeutic proteins. Columns 3, 4, and 5 may contain resins that utilize a protein A binding and capture mechanism. Column 6 can hold the fluid at a pH of approximately 3.75 for approximately 1 hour. PCCS1 also has an inlet 7, which is, for example, an opening that allows fluid to flow into PCCS1.

[0095] System 1 also includes a second MCCS which is PCCS8 containing three chromatography columns 9, 10, and 11, and one chromatography membrane 12. Columns 9, 10, and 11 in PCCS8 may contain cation exchange resins. Chromatography membrane 12 in PCCS8 may contain cation exchange resins. PCCS8 also has a fluid conduit 13 positioned between columns 9, 10, and 11 and the chromatography membrane 12 in PCCS8. PCCS8 also has an in-line buffer adjustment reservoir 14 which is in fluid communication with the fluid conduit 13, and is configured so that a buffer contained in the in-line buffer adjustment reservoir 14 is introduced into the fluid present in the fluid conduit 13. PCCS8 also includes an outlet 15, which is, for example, an opening that allows fluid to exit PCCS8.

[0096] System 1 may further include a fluid conduit 16 positioned between PCCS2 and PCCS8. System 1 may also include an inline buffer adjustment reservoir 17 that is in fluid communication with the fluid conduit 16 and configured to introduce a buffer contained within the reservoir 17 into the fluid present in the fluid conduit 16. System 1 may include a filter 18 positioned in the fluid conduit 16 for filtering the fluid present in the fluid conduit 16. System 1 may also include a break tank 19 positioned in the fluid conduit 16 and configured to hold any fluid in the fluid conduit 16 that cannot be easily supplied to PCCS8.

[0097] System 1 may further include a pump system 20 that is in fluid communication with the inlet 7. The pump system 20 may include a pump 21 for pushing fluid into the inlet 7. System 1 may also include a fluid conduit 22 positioned between the pump 21 and the inlet 7. System 1 may also include a filter 23 positioned in the fluid conduit 22 for filtering out fluid present in the fluid conduit 22 (e.g., liquid culture medium). System 1 may also include a break tank 24 positioned in the fluid conduit 22, which is in fluid communication with the fluid conduit 22 and is configured to store any fluid present in the fluid conduit 22 that cannot enter the inlet 7.

[0098] System 1 may also include a bioreactor 25, as well as a fluid conduit 26 positioned between the bioreactor 25 and the pump 21. A filtration system 27 may be positioned in the fluid conduit 26 to filter the liquid culture medium present in the fluid conduit 26 (for example, to remove cells from it).

[0099] The first MCCS (PCCS2) includes an inlet through which a fluid (e.g., a liquid culture medium substantially free of cells) can pass. The inlet is any structure known in the art for such purposes. It may include, for example, threading, rib fittings or seals that allow the fluid conduit to be inserted such that, after insertion of the fluid conduit into the inlet, the fluid enters the first MCCS through the inlet without significant leakage of fluid from the inlet.

[0100] The first MCCS comprises at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane, as well as an inlet. For example, the first MCCS may comprise, in total, four chromatography columns, or three chromatography columns and one chromatography membrane, or any of the other exemplary MCCS described herein, or may have one or more of the exemplary configurations of the MCCS described herein (in any combination).

[0101] The chromatography columns and / or chromatography membranes present in the first MCCS may contain one or more different types of resins. For example, the resins present in one or more of the chromatography columns and / or chromatography membranes present in the first MCCS may be resins that utilize capture mechanisms (e.g., protein A binding capture mechanisms, protein G binding capture mechanisms, antibody or antibody fragment binding capture mechanisms, substrate binding capture mechanisms, cofactor binding capture mechanisms, aptamer binding capture mechanisms, and / or tag binding capture mechanisms). The resins present in one or more of the chromatography columns and / or chromatography membranes of the first MCCS may be cation exchange resins, anion exchange resins, molecular sieve resins, or hydrophobic interaction resins, or any combination thereof. Additional examples of resins that can be used to purify recombinant therapeutic proteins are known in the art and may be present in one or more of the chromatography columns and / or chromatography membranes present in the first MCCS. The chromatography columns and / or chromatography membranes present in the first MCCS may contain the same and / or different resins (e.g., any of the resins described herein or any of the resins known in the art for use in recombinant protein purification).

[0102] Two or more chromatography columns and / or chromatography resins present in the first MCCS can perform one or more unit operations (e.g., capturing recombinant therapeutic proteins, purifying recombinant therapeutic proteins, polishing recombinant therapeutic proteins, inactivating viruses, adjusting the ion concentration and / or pH of a fluid containing recombinant therapeutic proteins, or filtering a fluid containing recombinant therapeutic proteins). In non-limiting examples, the first MCCS can perform unit operations such as capturing recombinant therapeutic proteins from a fluid (e.g., liquid culture medium) and inactivating viruses present in a fluid containing recombinant therapeutic proteins. The first MCCS can perform any combination of two or more unit operations described herein or known in the art.

[0103] Chromatographic columns and / or chromatographic membranes present in the first MCCS can be connected to or moved relative to one another by a switching mechanism (e.g., a column switching mechanism). The first MCCS may also include one or more (e.g., two, three, four, or five) pumps (e.g., automatic pumps, e.g., automatic peristaltic pumps). Column switching events can be triggered by detecting the level of recombinant therapeutic protein in the fluid passing through the first MCCS (e.g., input to and / or eluate from one or more chromatographic columns and / or chromatographic membranes in the first MCCS), a specific volume of liquid (e.g., buffer), or a specific elapsed time. Column switching generally means a mechanism that allows at least two different chromatographic columns and / or chromatographic membranes in the MCCS (e.g., two or more different chromatographic columns and / or chromatographic membranes present in the MCCS (e.g., the first or second MCCS)) to pass through substantially simultaneously different processes (e.g., equilibration, loading, elution, or washing) for at least part of the process.

[0104] The first MCCS, PCCS2, can include four chromatography columns. The first three columns perform unit operations to capture recombinant therapeutic proteins from a fluid (e.g., liquid culture medium), while the fourth column of the PCCS performs unit operations to inactivate viruses in a fluid containing recombinant therapeutic proteins. The first MCCS, PCCS, can utilize a column switching mechanism. The PCC system can utilize a modified AKTA system (GE Healthcare, Piscataway, NJ) that can operate up to, for example, four, five, six, seven, or eight columns, or more.

[0105] As described above, column switching events are triggered by the detection of the concentration of a particular protein or other substance in the fluid eluting from one of the columns of PCCS2 or PCCS8, the flow through the filter in the MCCS contained in the MCCS break tank, or the flow through the conduit in the MCCS (e.g., between MCCS1 and MCCS2). The concentration of such proteins can be measured using the angle-resolved refractometer system disclosed herein, and the concentration information can be transmitted to a controller in System 1 that initiates events such as column switching, filtration, and fluid transport in System 1.

[0106] The first MCCS may comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) angle-resolved refractometer systems (e.g., Systems 300, 600), one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. The first MCCS may also comprise a controller running an operating system that utilizes software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to determine when column switching should occur (e.g., based on concentration information derived from refractometer measurements, liquid volume, or elapsed time) and to influence (induce) column switching events. The angle-resolved refraction measurement system may be installed, depending on the circumstances, at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) inlets of the chromatography columns and / or chromatography membranes in the first MCCS, and / or at one or more outlets of the chromatography columns and / or chromatography membranes in the first MCCS.

[0107] The first MCCS is one or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, The system may further include 23 or 24 inline buffer adjustment reservoirs and / or buffer reservoirs. In other examples, the first MCCS may include one or more (e.g., 2, 3, 4, 5, or 6) break tanks that can hold fluids that cannot easily pass through one or more chromatography columns and / or chromatography membranes in the first MCCS. The systems described herein may include one or more break tanks (e.g., break tanks described herein) in the first and / or second MCCS. Other examples of the systems described herein do not include break tanks in the first or second MCCS, or do not include break tanks in the system as a whole. Other examples of the systems include up to 1, 2, 3, 4, or 5 break tanks in the system as a whole.

[0108] The second MCCS comprises at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane, as well as an outlet. For example, the second MCCS may comprise, in total, four chromatography columns, three chromatography columns and one chromatography membrane, or any of the other exemplary MCCS described herein, or may have one or more of the exemplary configurations of the MCCS (in any combination) described herein. The chromatography columns and / or chromatography membranes present in the second MCCS may have one or more of the shapes, sizes, volumes (bed volumes), and / or unit operations described herein. The resin contained in one or more of the chromatography columns and / or chromatography membranes present in the second MCCS may be a resin that utilizes a capture mechanism (e.g., a protein A binding capture mechanism, a protein G binding capture mechanism, an antibody or antibody fragment binding capture mechanism, a substrate binding capture mechanism, a cofactor binding capture mechanism, a tag binding capture mechanism, and / or an aptamer binding capture mechanism). Examples of useful resins include cation exchange resins, anion exchange resins, molecular sieve resins, and hydrophobic interaction resins. The chromatography columns and / or chromatography membranes present in the second MCCS may contain the same and / or different resins (e.g., any of the resins described herein or any of the resins known in the art for use in recombinant protein purification).

[0109] Chromatographic columns and / or chromatographic membranes present in the second MCCS can perform one or more unit operations (e.g., any of the unit operations described herein, or any combination thereof). In an unspecified example, the second MCCS can perform the unit operations of purifying recombinant therapeutic proteins from a fluid and polishing recombinant therapeutic proteins present in a fluid containing recombinant therapeutic proteins. In another unspecified example, the second MCCS can perform the unit operations of purifying recombinant therapeutic proteins present in a fluid, polishing recombinant therapeutic proteins present in a fluid, and filtering a fluid containing recombinant therapeutic proteins. In yet another example, the second MCCS can perform the unit operations of purifying recombinant therapeutic proteins present in a fluid, polishing recombinant therapeutic proteins present in a fluid, filtering a fluid containing recombinant therapeutic proteins, and adjusting the ion concentration and / or pH of a fluid containing recombinant therapeutic proteins. The second MCCS can perform any combination of two or more unit operations described herein or known in the art.

[0110] The second MCCS may also include one or more (e.g., two, three, four, or five) pumps (e.g., automatic pumps, e.g., automatic peristaltic pumps).

[0111] Chromatographic columns and / or chromatographic membranes present in the second MCCS can be connected to or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). Column switching events can be triggered, as discussed above, by detecting the level of recombinant therapeutic protein or other substances by angle-resolved refraction measurements, a specific volume of liquid (e.g., buffer solution), or a specific elapsed time, in order to determine the level of recombinant therapeutic protein in the fluid passing through the second MCCS (e.g., input to one or more of the chromatographic columns and / or chromatographic membranes in the second MCCS and / or eluate therefrom).

[0112] The PCCS8 forming the second MCCS may include three columns for performing unit operations to purify recombinant therapeutic proteins from a fluid, and a chromatographic membrane for performing unit operations to polish recombinant therapeutic proteins present in the fluid. For example, the three columns for performing unit operations to purify recombinant therapeutic proteins from the fluid may contain, for example, a cation exchange resin, and the chromatographic membrane for performing the polishing unit operation may also contain a cation exchange resin. The PCCS, which is the second MCCS, can utilize a column switching mechanism. For example, the PCCS can utilize a modified AKTA system (GE Healthcare, Piscataway, NJ) that can operate up to, for example, four, five, six, seven, or eight columns, or more.

[0113] Similar to the first MCCS, the second MCCS may also comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) angle-resolved refractometers (e.g., systems 300 and / or 600), one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. One or more angle-resolved refractometers transmit concentration information about proteins or other substances in the fluid being measured to a controller that uses the concentration information to determine whether to trigger a column switching event. The second MCCS may include a controller that runs an operating system, which is performed by a controller that receives concentration information, for determining when a column switching event should occur (e.g., based on angle-resolved refractometry, liquid volume, or elapsed time) and for initiating the column switching event, utilizing software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ). In an example where the second MCCS includes one or more angle-resolved refractometry systems, the refractometry systems may be installed at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) inlets of the chromatography columns and / or chromatography membranes in the second MCCS, and / or at one or more outlets of the chromatography columns and / or chromatography membranes in the second MCCS.

[0114] The second MCCS may further include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) inline buffer adjustment reservoirs and / or buffer reservoirs. In other examples, the second MCCS may include one or more (e.g., 2, 3, 4, 5, or 6) break tanks (e.g., any of the break tanks described herein) that can hold fluids that cannot easily pass through one or more chromatographic columns and / or chromatographic membranes in the second MCCS.

[0115] The second MCCS includes an outlet from which the therapeutic protein drug substance can exit the system. The outlet may include, for example, threading, rib fitting or seal, or a vial designed to contain or store the therapeutic protein drug substance, which allows for the insertion of a fluid conduit. The outlet may also include a surface that can be used to seal a sterile vial or other such storage container, in order to allow the recombinant protein formulation to flow directly into a sterile vial or storage container.

[0116] One or more angle-resolved refraction measurement systems, such as those disclosed herein, may also be configured to measure the concentration of a protein-based drug substance (or another substance) flowing out of the outlet. This information is transmitted to an MCCS controller, which can then determine the purity of the substance based on the information.

[0117] The system described herein may also include a fluid conduit positioned between a first MCCS and a second MCCS. One or more angle-resolved refraction measurement systems can be positioned along the fluid conduit to determine information (e.g., concentration information) about the fluid held within the conduit (e.g., flowing through the conduit). This information is communicated to the MCCS controller, which can then decide whether to initiate a column switching event based on the information, as described above.

[0118] Any of the fluid conduits described herein are, for example, tubes made of polyethylene, polycarbonate, or plastic. The fluid conduits located between the first MCCS and the second MCCS may, in any combination, further include one or more of the following: one or more inline buffer conditioning reservoirs that are in fluid communication with the fluid conduit and are positioned so that buffer stored in the inline buffer conditioning reservoir is added to the fluid present in the fluid conduit; a break tank (for example, any of the break tanks described herein) that is in fluid communication with the fluid conduit and is positioned to hold any excess fluid present in the fluid conduit that cannot be easily supplied to the second MCCS; and one or more filters positioned in the fluid conduit so as to filter the fluid present in the fluid conduit (for example, to remove bacteria). Any of the inline buffer conditioning reservoirs may contain, for example, a volume of buffer (for example, at 25°C or below, 15°C, or 10°C) in volumes of about 0.5 L to 50 L.

[0119] The systems described herein may optionally include a fluid conduit positioned between the final chromatography column or chromatography membrane and the outlet in the second MCCS. The systems described herein may further include one or more filters in fluid communication with the fluid conduit positioned between the final chromatography column or chromatography membrane and the outlet in the second MCCS, so that the filters can remove, for example, precipitates, particulate matter, or bacteria from the fluid present in the fluid conduit positioned between the final chromatography column or chromatography membrane and the outlet in the second MCCS.

[0120] Some examples of the systems provided herein include a bioreactor that is in fluid communication with the inlet of the first MCCS. Any of the exemplary bioreactors described herein or known in the art may be used in this system.

[0121] Some examples of systems provided herein include pump systems. A pump system may include one or more of the following: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pumps (e.g., as described herein) A pump (or any pump known in the art), one or more (e.g., two, three, four, or five) filters (e.g., any of the filters described herein or known in the art), one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) UV detectors, and one or more (e.g., two, three, four, or five) break tanks (e.g., any of the break tanks described herein). Some examples of systems provided herein further include a fluid conduit (e.g., any of the exemplary fluid conduits described herein or known in the art) positioned between the pump and the inlet of the first MCCS. In some examples, this particular fluid conduit may include one or more (e.g., two, three, or four) pumps (e.g., any of the pumps described herein or known in the art) and / or one or more (e.g., two, three, or four) break tanks (e.g., any of the exemplary break tanks described herein), and these pumps and / or break tanks are in fluid communication with the fluid present in the fluid conduit.

[0122] Some examples of the systems described herein further include a further fluid conduit connected to the fluid conduit between the pump and the inlet, where one end of the further fluid conduit is fluidically connected to the bioreactor and the other end is fluidically connected to the fluid conduit between the pump and the inlet. This further fluid conduit may include a filter (e.g., an ATF cell retention system) that can remove cells from the liquid culture medium removed from the bioreactor.

[0123] The aforementioned biological manufacturing systems enable the continuous production of therapeutic protein drug substances. For example, the systems provided herein enable a percentage of recombinant therapeutic protein yield (from starting material, e.g., starting liquid culture medium) of about 70%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98%. The systems described herein can also yield a percentage of recombinant therapeutic protein yield (from starting material, e.g., starting liquid culture medium) of about 80% to about 90%, about 82% to about 90%, about 84% to about 90%, about 84% to about 88%, about 84% to about 94%, about 82% to about 92%, or about 85% to about 95%.

[0124] The systems described herein can also result in the production of therapeutic protein drug substances containing recombinant therapeutic protein concentrations higher than approximately 1.0 mg / mL, approximately 1.5 mg / mL, approximately 2.0 mg / mL, approximately 2.5 mg / mL, approximately 3.0 mg / mL, approximately 3.5 mg / mL, approximately 4.0 mg / mL, approximately 4.5 mg / mL, approximately 5.0 mg / mL, approximately 5.5 mg / mL, approximately 6.0 mg / mL, approximately 6.5 mg / mL, approximately 7.0 mg / mL, approximately 7.5 mg / mL, approximately 8.0 mg / mL, approximately 8.5 mg / mL, approximately 9.0 mg / mL, approximately 10.0 mg / mL, approximately 12.5 mg / mL, or approximately 15.0 mg / mL.

[0125] As discussed above, in some embodiments, the first and / or second MCCS is a periodic countercurrent chromatography system (PCCS). A PCCS may include two or more switchable chromatography columns (e.g., three or four columns) to allow for the continuous elution of recombinant therapeutic proteins from two or more chromatography columns. A PCCS may include two or more chromatography columns, two or more chromatography membranes, or at least one chromatography column and at least one chromatography membrane. Column operations generally involve loading, washing, It consists of elution and regeneration steps. In PCCS, multiple columns are used to operate the same steps individually and sequentially in a cyclical manner. Since the columns are operated in series, the flow-through and washing from one column are captured by another column. This unique configuration of PCCS allows for resin loading that is close to its static binding capacity rather than its dynamic binding capacity, as is typical during batch mode chromatography.

[0126] Figure 8 shows an example of a three-column switching technique used in a PCCS containing three columns. A cycle is defined as three complete column operations that yield eluate pools from each of the three columns used in the column switching technique. Once all steps of a cycle are complete, the cycle is restarted. As a result of continuous cycling and elution, the fluids entering the PCCS are processed continuously, but the elution of recombinant therapeutic proteins from each column is individual and periodic.

[0127] A column switching strategy is used to move from one step to another in a PCCS cycle, such as the exemplary cycle shown in Figure 8. The column switching method uses two automated switching operations per column in the three columns of the exemplary PCCS system shown in Figure 8: the first is related to the appearance of the first product, and the second coincides with column saturation. The decision of when to perform the column switching operation is based on information about the concentration of recombinant therapeutic protein in the eluate from each chromatographic column in the PCCS.

[0128] As described above, the angle-resolved refraction measurement system disclosed herein can be used to determine the concentration of recombinant therapeutic protein in the eluate from a PCCS column. The concentration information serves as feedback control for the biological manufacturing system and is transmitted to the MCCS controller, which initiates column switching after determining that switching is justified.

[0129] For example, during column loading (Step 1; Figure 8), the PCC control system can determine the baseline concentration (typically zero) of the therapeutic protein material eluting from the column using the angle-resolved refraction measurement system discussed above. During active elution, as the protein material appears (Step 2; Figure 8), the measured protein concentration increases (e.g., above the baseline concentration). The system continues to monitor the increasing protein concentration, and once the concentration reaches a predetermined threshold, the flow-through from column 1 is directed to column 2 instead of being discarded. Nominally, this occurs at time t1.

[0130] As the supply to column 1 continues, column 1 will eventually become nearly saturated with protein products. At this point, the measured concentration of protein in the eluate has reached another predetermined value that occurs at time t2. At this point, the MCCS controller switches the inlet supply to column 2.

[0131] The column switching strategy described above allows for uniform loading of the column regardless of the concentration and volume of the feed product. Similar column switching can be implemented based on the level of recombinant protein detected in the eluate from each column. Column switching can also be based on elapsed time or the amount of fluid (e.g., buffer) that has passed through one or more chromatographic columns and / or chromatographic membranes in the first or second MCCS.

[0132] In addition to providing feedback information for control column switching events, the angle-resolved refraction measurement system disclosed herein can also provide feedback information for the adjustment of various other biological manufacturing processes and operational parameters. One example is the controlled regulation of buffer concentrations at various stages of a biological manufacturing process.

[0133] In general, one or more different types of buffers (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) can be used between the use of two or more MCCS in any of the processes described herein. As is known in the art, one or more types of buffers used in two or more MCCS used in the processes described herein depend on the resins, recombinant therapeutic proteins, and specific chromatographic operations performed by the chromatographic columns and / or chromatographic membranes of the two or more MCCS (e.g., the first and second MCCS). The volume and type of buffers used between the use of two or more MCCS in any of the processes described herein can also be determined by those skilled in the art (e.g., discussed in more detail below). For example, the volume and type of buffer used between the use of two or more MCCS in any of the processes described herein can be selected to optimize one or more of the following in the recombinant protein formulation: the total yield of the recombinant therapeutic protein, the activity of the recombinant therapeutic protein, the level of purity of the recombinant therapeutic protein, and the removal of biological contaminants from the fluid containing the recombinant therapeutic protein (e.g., the absence of active viruses, mycobacteria, yeast, bacteria, or mammalian cells).

[0134] Unit operations to adjust the ion concentration and / or pH of a fluid containing recombinant therapeutic proteins can be performed using an MCCS (e.g., the first and / or second MCCS) that includes and uses a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) to add new or additional buffer solution to the fluid containing recombinant therapeutic proteins (e.g., the first and / or second MCCS). The inline buffer adjustment reservoir can be of any size (e.g., greater than 100 mL) and may contain any buffered solution (e.g., a buffer having one or more of the following: increased or decreased pH compared to a fluid containing recombinant therapeutic protein, increased or decreased ion (e.g., salt) concentration compared to a fluid containing recombinant therapeutic protein, and / or increased or decreased concentration of a drug that competes with recombinant therapeutic protein for binding to a resin present in at least one chromatographic column or at least one chromatographic membrane of an MCCS (e.g., a first or second MSCCS)).

[0135] (b) Upstream identification, verification, and quantitative measurement Angle-resolved refraction measurements can also be used in upstream (i.e., before obtaining purified products) continuous biofabrication operations. For example, in some embodiments, the solute of interest includes components of a buffer solution used to maintain a specific pH range within the chromatography column and bioreactor. The concentrations of these buffer solution components can also be determined quantitatively. Furthermore, once the buffer solution components are quantified, the controller 312 can also calculate other information related to the solution being measured, including the pH of the solution (i.e., based on the buffer composition).

[0136] In some embodiments, the MCCS controller determines the amount of buffer solution to be added to the process fluid, which is obtained from angle-resolved refraction measurements performed as described above. The measurement is based on concentration information regarding the components of the body. For example, the solute for the purpose of such measurement is a component of the buffer solution, or a component of the process fluid whose concentration relates to the buffer composition of the fluid, the pH of the process fluid, and / or the ionic strength of the process fluid. The measurement of concentration information about the components is provided to the MCCS controller as feedback information, which is used to determine the timing and amount of one or more buffer solutions to be discharged into the process fluid. Angle-resolved refraction measurement systems can generally be located anywhere in the biological manufacturing system (Figure 7) for the purpose of measuring the process fluid and providing feedback information related to the buffer to the MCCS controller.

[0137] In certain embodiments, angle-resolved refraction measurements can be used in inline dilution, mixing, and / or conditioning operations involving one or more buffer solutions. For example, in operations involving quality control verification of buffer solutions, such measurements can be used to verify the identity of buffer solution components before the components are mixed to form the buffer solution. Similarly, such measurements can be used to verify the identity of one or more prepared buffer solutions before the solution is introduced into a feed stream, before it is mixed with other solutions, before it is injected into a bioreactor, or before it is otherwise introduced into a continuous bioproduction system.

[0138] Identification of buffer solutions can also be performed using angle-resolved refraction measurements before introducing the buffer solution into a chromatography column for the separation and / or purification of analytes, intermediates, and waste.

[0139] The identity of components introduced into such systems can also be verified by angle-resolved refraction measurements of components other than buffers. Such components include, but are not limited to, antifoaming agents, various types of reactor nutrients, and poloxamers (e.g., Pluronic, Symperonic, and / or Coryphore).

[0140] To verify the identity of buffer components, buffer solutions, and other components as described above, angle-resolved refraction measurements are typically compared to stored information (e.g., library or database records) about various substances. Typically, for example, library records contain a set of calibration information including refraction angles and corresponding substance concentrations. Verification of a substance's identity can be performed by confirming that the refraction angle measured at one or more measurement wavelengths corresponds to an angle (or range of angles) associated with the substance in the library record. Such verification can be performed automatically by the system described herein, and if the identity of a particular substance cannot be verified (e.g., the verification process fails), a warning message may be generated (or other control functions may be performed).

[0141] In addition to verifying the identity of buffer components, buffers, and other constituents, angle-resolved refraction measurements can provide concentration information for these analytes, as described above. This concentration information can be used in various in-line dilution, mixing, and conditioning processes that introduce buffers, drugs, and other substances into bioproduction systems at various points. For example, in some bioproduction systems, a flow meter with one or more dosing pumps and / or flow control valves is used to dynamically mix buffer components to form a buffer solution, which is then introduced into a bioreactor in the feed stream. In addition to verifying the identity of the mixed components as described above, angle-resolved refraction measurements provide a feedback signal to the controller, which adjusts the dosing pump to combine the appropriate amounts of each component to create a mixed buffer solution configured as desired. Before performing the mixing operation, measurements can be performed on stock solutions of the buffer components to determine their respective concentrations, for example.

[0142] In certain embodiments, angle-resolved refraction measurements can also be used to obtain concentration information of constituents during mixing or dilution operations involving growth media and other substances, including but not limited to nutrients such as iron. As described above, the identity and concentration of these substances in the mixed and / or diluted solutions can be verified before the solutions are introduced into the bioreactor's feed stream.

[0143] As an example, in a perfusion bioreactor, the culture medium supply is continuous throughout the manufacturing process. Angle-resolved refraction measurements can be used as part of a feedback loop to adjust the amount of growth medium introduced into the feed stream over time. A stock solution of growth medium can be metered by a controller (e.g., via a dosing pump or flow control valve) and combined with water and / or other components to generate a feed stream for delivery to the bioreactor. The metering of the growth medium solution can be performed by a controller connected to a pump or control valve, and the controller receives information on the concentration of growth medium in the feed stream obtained from angle-resolved refraction measurements.

[0144] In addition to confirming the concentration of growth medium in the supply stream, such measurements can be used to dynamically adjust the amount of growth medium delivered to the bioreactor over time. For example, as the cell population in the reactor increases, angle-resolved refraction measurements can be used to increase the amount of growth medium delivered to the reactor to support a larger cell population.

[0145] In some embodiments, angle-resolved refraction measurements can be used to provide feedback signals for filter selection during product filtration operations. For example, a particular reaction product is subjected to a diafiltration and / or ultrafiltration process (e.g., a continuous diafiltration process), and one or more molecular-size-selective filters are used to separate reaction intermediates or products in the product stream from the bioreactor from reaction by-products and other substances. Angle-resolved refraction measurements performed as described above can be used to identify the reaction products or intermediates and determine the concentrations of these substances on one or both sides of the filter. For example, such measurements can be used to confirm that target concentrations of buffer, salt, and / or product have been achieved. Alternatively or additionally, one or more filters can be adjusted (e.g., inserted into or removed from the solution flow path) based on the measured concentrations of substances on one or both sides of the filter to facilitate the achievement of target concentrations of product, buffer, and salt components.

[0146] As described above, angle-resolved refraction measurements can be used to measure the concentrations of the stock solution, components, and other agents before mixing these substances, forming a feed stream solution that is delivered to the bioreactor and / or one or more purification units downstream of the bioreactor. Because there is variability in the concentration of the stock solution (which is relatively common), using the concentration values ​​measured during the mixing operation involving the stock solution and other substances for feedback control of the dosing pump and / or flow control valve achieves more precise control over the component concentrations in the final solution.

[0147] Angle-resolved refractometers are particularly well-suited for controlling such operations because they can perform such measurements relatively quickly and do not require routine calibration. Such methods are especially useful in continuous bioproduction operations where calibration-related downtime is undesirable. In contrast, alternative measurement methods such as pH detection and conductivity monitoring typically exhibit calibration drift (e.g., pH detection systems may require daily recalibration) and / or may not be sensitive enough for the purpose of determining the identity and concentration of certain substances. Furthermore, certain pH and conductivity sensors are not effectively "visualized" by some salines. It has been observed to be "concealed," and therefore, it is difficult to use under certain bio-manufacturing conditions.

[0148] Furthermore, certain pH sensors contain an electrolyte solution, and if such sensors are used for inline monitoring, the electrolyte solution may leak into the process flow where the sensor is located, contaminating the flow and leading to inaccurate measurements. Additionally, some pH sensors have specific storage requirements (such as immersion in solution), which may necessitate intervention by the system operator when the sensor is not in use or when activating it for measurement. Angular-resolved measurements performed according to the systems and methods described above do not involve electrolyte solution leakage or intervention steps to activate or deactivate the sensor.

[0149] Conductivity-based measurements can be used to determine the concentrations of buffer components. However, such measurements can be highly temperature-dependent, and it has been observed that for certain buffers, even a 1°C temperature change can lead to a change in conductivity of more than an order of magnitude. To compensate for such changes, some conductivity-based measurement techniques use temperature compensation values ​​to adjust the conductivity measurements. However, such values ​​typically only apply to a single component of the solution. Therefore, for multi-component solutions, a set of such values ​​is commonly used, and this set of values ​​varies depending on the temperature deviation from the ideal measurement temperature or the baseline measurement temperature. Angle-resolved measurements performed according to the systems and methods described above are unaffected by such temperature changes, and as a result, there is no need to use complex temperature compensation calibration values ​​to adjust the measurements. Instead, such measurements can be used with a set of temperature-independent calibration data to calculate the concentration values ​​of multiple different analytes.

[0150] Angle-resolved refraction measurements can also be used to improve the efficiency and reduce costs of specific operational steps in bioproduction systems. For example, as described above, following product generation, a product stream carrying one or more products can be directed from a bioreactor to one or more chromatography columns for purification and characterization. The columns used in this process are loaded with the product stream (or eluate stream from another column), and the components of the stream are separated and eluted sequentially from the column. The productivity curve of a column typically increases first when the column is first loaded and then eluted, and then decreases over time.

[0151] During such operations, the column is typically loaded to about 80% of its capacity to prevent one or more products in the flow from being discarded from the downstream (i.e., elution) end of the column. However, this reduces the overall efficiency of the process because about 20% of the column's loaded capacity is not used. Furthermore, any buffer used in the elution process will be consumed in additional amounts compared to the amount that would have been used if the column had been loaded to near its full capacity.

[0152] Dynamic angle-resolved refraction measurements can be used to monitor the concentration of the product at the downstream end of the column, allowing for increased column load and thereby improving the efficiency of the purification process. The concentration measurements can be used to provide feedback signals to pumps or flow control valves that adjust the product flow rate, allowing for the cessation of flow if product is detected at the downstream end of the column. Once product is detected at the downstream end, the column is considered to be loaded to its effective capacity, and the maximum percentage of the column's nominal load capacity is used.

[0153] Furthermore, by utilizing the detection of the column's temporary maximum load endpoint, the column's evolution can be triggered by flowing buffer solution and other substances through the column as soon as it is loaded (or equilibrated in some applications). In this way, the waiting time for purification is reduced. This allows for more efficient purification of the product obtained from the product stream.

[0154] Hardware and software implementation The controller 312 may include one or more processors, one or more memory units, and one or more interfaces for interconnecting with other components of the system described herein. The interfaces may include wired and / or wireless interfaces for receiving information and transmitting control instructions to the components of the system and other components and devices described herein. The controller 312 is connected to the storage unit 370 as shown in Figure 5.

[0155] The processor may process instructions for execution within the controller, including instructions stored in one or more memory units and / or storage units 370. For example, instructions may instruct the processor to perform any of the analysis and control steps disclosed herein.

[0156] The memory unit may store executable instructions from the processor, information about system parameters, and measurement information such as measured refraction and / or reflection angle information and measured refraction and / or reflected light intensity. The storage unit 370 may be a computer-readable medium such as a floppy disk device, hard disk device, optical disk device, or tape device, flash memory or other similar solid-state memory device, or an array of devices, including devices in a storage area network or other configuration. The storage unit 370 may store any of the executable instructions from the processor and other information stored by one or more memory units.

[0157] In some embodiments, the controller 312 may include a graphics processing unit for displaying graphical information on an external input / output device (e.g., using a GUI or text interface). The graphical information may be displayed by a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying any of the information disclosed herein. The user may provide input to the controller 312 using an input device (e.g., a keyboard, pointing device, touchscreen, or speech recognition device).

[0158] Users of the system described herein may provide the controller 312 with various different types of instructions and information via an input device. These instructions and information may include, for example, calibration information, information about one or more parameters measured by the system, and information about a model used to correlate the measured angles with the parameters. The controller 312 may use any of these different types of information to perform the methods and functions described herein. It should also be noted that any of these types of information can be stored (for example, in the storage unit 370) and retrieved by the controller 312 when needed.

[0159] The methods, processes, and functions described herein can be implemented by the controller 312 by executing instructions in one or more computer programs that are executable and / or interpretable by the controller 312. These computer programs (also called programs, software, software applications, or code) contain machine instructions for a programmable processor and can be implemented in high-level procedural and / or object-oriented programming languages ​​and / or assembly / machine languages. For example, the computer program may include one or more memory units, storage units 370, and / or tangible objects. This may include instructions stored on a computer-readable medium and executed by one or more processors of the controller 312 as described above. As used herein, the term “computer-readable medium” means any computer program product, apparatus, and / or device (e.g., magnetic disks, optical disks, memory, programmable logic devices (PLDs), ASICs, and electronic circuits) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions.

[0160] Alternatively or additionally, the methods, processes, and functions described herein can be implemented in whole or in part in hardware by electronic circuits and circuit elements specifically configured to perform the methods, processes, and functions. The circuits and circuit elements may be programmable or non-programmable. In certain embodiments, for example, the circuit may be an application-specific integrated circuit (ASIC).

[0161] Other Embodiments Numerous embodiments have been described. However, it will be understood by those skilled in the art that other embodiments are also included in this disclosure and the following claims. [Explanation of Symbols]

[0162] 1 System 2. 4-column periodic countercurrent chromatography system (PCCS) 3 columns 4 columns 5 columns 6 columns 7 Entrance 8 PCCS 9 Chromatography Columns 10 chromatography columns 11. Latcolinear column 12. Chromatographic membranes 13 Fluid conduit 14. In-line buffer adjustment reservoir 15 Exit 16 Fluid conduit 17. In-line buffer adjustment reservoir 18 filters 19 Break Tank 20 Pump System 21 pumps 22 Fluid conduit 23 filters 24 Break Tank 25 Bioreactors 26 Fluid conduit 27 Filtration System 102 Gaseous Environment 104 Liquid environment 106 Interface 108 Normal 110 Incident light beam 112 Refracted light beam 202 Gas 203 Wall 204 Liquid 206 Interface 207 Second Interface 208 Normal 209 Normal 210 Light beams 212 Refraction beam 214 Refraction beam 300 Measurement Systems 302 Wall 304 Liquid 306 Wall 308 Light source 310 Angle-resolved photodetector 312 Controller 314 Incident light beam 316 Refraction beam 318 Normal 320 Normal to the interface 322 Interface 324 Interface 370 storage units 402 Best-fitting line 500 Bio-manufacturing Systems 502 First container 504 Second container 506 Conduit 508 Process Solution 510 connection 600 Measurement Systems 602 Reflected beam 604 Normal to the interface 606 Mirror

Claims

1. A method for controlling a biological manufacturing system, A step of directing a light beam to pass through the walls of a container containing a first fluid produced by a biological manufacturing system; A step of measuring the refraction angle of a light beam in a first fluid, wherein the refraction angle corresponds to the angle between the propagation direction of the light beam in the first fluid and the normal to the interface between the container wall and the first fluid; A step of determining information about a first fluid based on the measured refraction angle, and A step of adjusting the parameters of a biological manufacturing system based on information about a first fluid. The method, including the method described above.

2. The method according to claim 1, wherein the information relating to the first fluid includes the concentration of a substance in the first fluid.

3. The method according to claim 1, wherein the information relating to the first fluid includes the pH value of the first fluid.

4. The method according to claim 1, wherein the first fluid includes a fluid discharged from the purification unit of the system.

5. The method according to claim 4, wherein the purification unit includes at least one chromatography column.

6. The method according to claim 1, wherein the first fluid includes a supply fluid introduced into the reactor of the system.

7. The method according to claim 1, wherein the first fluid includes a fluid discharged from the system's diafiltration unit.

8. The method according to claim 1, wherein the parameters of the biological manufacturing system include a fluid channel that selectively directs a continuous portion of a first fluid to one of a plurality of purification units of the system.

9. The method according to claim 1, wherein the parameters of the biological manufacturing system include the rate of supply of material to the reactor of the system.

10. The method according to claim 1, wherein the parameters of the biological manufacturing system include the rate of supply of material to the system's diafiltration unit.

11. The method according to claim 1, wherein the parameters of the biological manufacturing system include the rate of supply of a substance to the purification unit of the system.

12. The method according to claim 1, wherein the parameters of the biological manufacturing system include the supply rate of a continuous portion of the first fluid to the purification unit of the system.

13. The method according to claim 2, further comprising the step of determining the concentration of a substance from a calibration formula derived from measured calibration data, wherein the calibration formula expresses the concentration as a function of the angle of refraction.

14. The method according to claim 13, wherein the calibration formula expresses the concentration as a linear function of the angle of refraction.

15. The method according to claim 13, wherein the calibration formula expresses the concentration as a nonlinear function of the angle of refraction.

16. The method according to claim 13, further comprising the step of transmitting concentration information to a system controller, the controller being configured to receive the concentration information and adjust system parameters.

17. The method according to claim 2, wherein the substance contains a protein.

18. The method according to claim 2, wherein the substance comprises a recombinant protein-based formulation.

19. The method according to claim 2, wherein the substance comprises a nucleic acid-based product.

20. The method according to claim 1, wherein the first fluid is a process fluid produced by the system as part of a biological manufacturing process.

21. The method according to claim 1, wherein the first fluid includes an eluate discharged from a chromatography column.

22. The method according to claim 1, wherein the first fluid comprises an eluate from a first purification unit of the system, the information comprises the concentration of a substance in the first fluid, and the parameter adjustment comprises directing the eluate from the first purification unit towards the inlet of a second purification unit when the concentration of a substance in the first fluid exceeds a threshold.

23. The method according to claim 22, wherein the first and second purification units each include at least one chromatography column.

24. The first fluid includes an eluate from a first purification unit of the system, the information includes the concentration of a substance in the first fluid, the inlet of the first purification unit is connected to a conduit that delivers a supply solution to the first purification unit, and the method is used when the concentration of a substance in the first fluid exceeds a threshold. The step of disconnecting the inlet of the first purification unit from the conduit; and The process involves connecting the inlet of the second purification unit of the system to a conduit and delivering the supply solution to the second purification unit. The method according to claim 1, further comprising:

25. A step of directing a second light beam so as to pass through the wall of a second container containing a second fluid produced by a biological manufacturing system; A step of measuring the refraction angle of a second light beam in a second fluid, wherein the refraction angle corresponds to the angle between the propagation direction of the second light beam in the second fluid and the normal to the interface between the wall of the second container and the second fluid; A step of determining information about a second fluid based on the measured refraction angle of a second light beam; and A step of adjusting a second parameter of a biological manufacturing system based on information about a second fluid. The method according to claim 1, further comprising:

26. The method according to claim 25, wherein the information relating to the second fluid includes the concentration of the buffer solution component in the second fluid.

27. The method according to claim 25, wherein the information relating to the second fluid includes the concentration of an ionic compound or its dissolved salt in the second fluid.

28. The method according to claim 25, wherein the step of adjusting the second parameter includes adjusting the supply rate of the second fluid to the purification unit of the system.

29. The method according to claim 16, further comprising the step of providing concentration information to a controller as a feedback signal to adjust system parameters during a biological manufacturing process, by repeating the steps of directing, measuring, determining, and transmitting.

30. A biological manufacturing system, A container configured to hold or transport a first fluid generated by a system, the container comprising a wall defining at least a portion of the container; A light source configured to direct a light beam so that it passes through a wall; A detector configured to measure the refraction angle of a light beam in a first fluid, wherein the refraction angle corresponds to the angle between the propagation direction of the light beam in the first fluid and the normal to the interface between the wall and the first fluid; and A first controller connected to the detector, Information regarding the refraction angle is received from the detector, and information regarding the first fluid is determined based on the measured refraction angle; Adjust the system parameters based on information about the first fluid. A first controller configured as follows The biological manufacturing system, including the above.

31. The system according to claim 30, wherein the information relating to the first fluid includes the concentration of a substance in the first fluid.

32. The system according to claim 30, wherein the information relating to the first fluid includes the pH value of the first fluid.

33. The system according to claim 30, further comprising a purification unit, wherein the first fluid includes a fluid discharged from the purification unit.

34. The system according to claim 33, wherein the purification unit includes at least one chromatography column.

35. The system according to claim 30, further comprising a reactor, wherein the first fluid includes a supply fluid introduced into the reactor.

36. The system according to claim 30, further comprising a diafiltration unit, wherein the first fluid comprises a fluid discharged from the diafiltration unit.

37. The system according to claim 30, further comprising a plurality of purification units, wherein the system parameters include a fluid channel that selectively directs a continuous portion of the first fluid to one of the plurality of purification units.

38. The system according to claim 30, further comprising a reactor, wherein the parameters of the system include the rate of supply of material to the reactor.

39. The system parameters also include a diafiltration unit, and the dia The system according to claim 30, including the rate of supplying material to the filtration unit.

40. The system according to claim 30, further comprising a purification unit, wherein the system parameters include the rate of supply of a substance to the purification unit.

41. The system according to claim 30, further comprising a purification unit, wherein the parameters of the system include the supply rate of a continuous portion of the first fluid to the purification unit.

42. The system according to claim 31, wherein the first controller is configured to determine the concentration of a substance from a calibration formula derived from measured calibration data, the calibration formula expressing the concentration as a function of the angle of refraction.

43. The system according to claim 42, wherein the calibration formula expresses the concentration as a linear function of the angle of refraction.

44. The system according to claim 42, wherein the calibration formula expresses the concentration as a nonlinear function of the angle of refraction.

45. The system according to claim 31, wherein the substance contains a protein.

46. The system according to claim 31, wherein the substance comprises a recombinant protein-based formulation.

47. The system according to claim 31, wherein the substance comprises a nucleic acid-based product.

48. The system according to claim 30, wherein the first fluid is a process fluid produced by the system as part of a biological manufacturing process.

49. The system according to claim 30, further comprising a purification unit, wherein the first fluid includes an eluate discharged from the purification unit.

50. Further comprising a first purification unit and a second purification unit, The first fluid comprises the eluate from the first purification unit; The information includes the concentration of the substance in the first fluid; The system according to claim 30, wherein parameter adjustment includes directing the eluate from the first purification unit to the inlet of the second purification unit when the concentration of a substance in the first fluid exceeds a threshold.

51. The system according to claim 50, wherein the first and second purification units each include at least one chromatography column.

52. The system further includes a first purification unit and a second purification unit, the first purification unit including an inlet connected to a conduit for delivering a supply solution to the first purification unit. The first fluid comprises the eluate from the first purification unit; The information includes the concentration of the substance in the first fluid; The system according to claim 30, wherein parameter adjustment includes disconnecting the inlet of the first purification unit from the conduit and connecting the inlet of the second purification unit to the conduit when the concentration of a substance in the first fluid exceeds a threshold, thereby delivering the supply solution to the second purification unit.

53. A second container configured to hold or transport a second fluid generated by a system, the second container comprising a second wall defining at least a portion of the second container; A second light source configured to direct a second light beam so as to pass through a second wall; and A second detector configured to measure the refraction angle of a second light beam in a second fluid, wherein the refraction angle corresponds to the angle between the propagation direction of the second light beam in the second fluid and the normal to the interface between the second wall and the second fluid. It further includes, The first controller is connected to the second detector. Information regarding the refraction angle is received from the second detector, and information regarding the second fluid is determined based on the measured refraction angle; Adjust the second parameter of the system based on information about the second fluid. The system according to claim 30, configured as described above.

54. The system according to claim 53, wherein the information relating to the second fluid includes the concentration of the buffer solution component in the second fluid.

55. The system according to claim 53, wherein the information relating to the second fluid includes the concentration of an ionic compound or its dissolved salt in the second fluid.

56. The system according to claim 53, further comprising a purification unit, wherein adjusting a second parameter includes adjusting the supply rate of a second fluid to the purification unit.

57. During the biological manufacturing process: The light source is configured to repeatedly direct a beam of light through the wall; The detector is configured to repeatedly measure the angle of refraction of a light beam in a first fluid; The first controller repeats: The detector receives information about the refraction angle; Based on the measured refraction angle, information about the first fluid is determined; Adjust the system parameters based on information about the first fluid. The system according to claim 30, configured as described above.

58. Multiple purification units, each containing at least one chromatography column; Column switching mechanism; and A second controller connected to a column switching mechanism, which communicates with the first controller. It further includes, The system according to claim 30, wherein the first controller is configured to adjust the parameters of the system by transmitting a signal to the second controller, causing the second controller to adjust a column switching mechanism for selectively directing fluid to one of a plurality of purification units.

59. The system according to claim 58, wherein the fluid to which the fluid is selectively directed includes an additional portion of the first fluid.

60. A reservoir containing a second fluid; and A second controller that communicates with the first controller. It further includes, The first controller is configured to adjust the system parameters by transmitting control signals to the second processor based on information about the first fluid; The system according to claim 30, wherein the second controller is configured to introduce a portion of the second fluid from the reservoir into a continuous portion of the first fluid in response to a control signal. Hmm.

61. The system according to claim 60, wherein the second fluid comprises a buffer solution.

62. The system according to claim 60, wherein the second fluid comprises a solution of an ionic compound or a dissolved salt thereof.

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