A method for determining cell shear stress sensitivity in culture

Abstract

The present disclosure relates to a method for determining the shear stress sensitivity of eukaryotic or prokaryotic cells in a shear stress sensitive device (100) during a cultivation process, comprising: (1) providing a shear stress sensitive device (100), (2) selecting cells, a liquid medium, a cultivation mode and conditions; (3) selecting one, two or three stress parameters; (4) characterizing the shear stress sensitive device (100) for maximum shear stress by a calibration method; (5) performing the same cultivation process several times and measuring one or more performance characteristics of the cells during each run, generating a curve over time for each performance characteristic in each run; (6) performing a cultivation process as a control with a lower maximum shear stress than step (5); (7) selecting the curve from the set of curves of step (5) that is closest to the control curve, while obtaining the shear stress sensitivity or limit of the cells.

Description

Technical Field

[0001] This invention relates to a method for determining the shear stress sensitivity of eukaryotic or prokaryotic cells during culture. Background Technology

[0002] It is well known that eukaryotic or prokaryotic cells in cell culture are sensitive to external influences such as hydrodynamic and mechanical stresses, which can impair the quality and yield of cell cultures. These hydrodynamic and mechanical stresses can be generated by a variety of factors, such as the containers or reactors used for cell culture, agitators, the inflow and outflow of liquid culture media or gases, and other equipment used during the culture process. A deeper understanding of the effects of these factors, and a more accurate assessment and even quantification of their impact on cells, will help to significantly improve the stability and efficiency of cell cultures.

[0003] Several solutions to the above problems already exist in the prior art. For example, US2006 / 0223175A1 (1) A bioreactor capable of applying shear stress to cultured tissue cells is disclosed. The reactor applies shear stress via a movable shaft, on which a substrate is fixed, and tissue cells are attached to the surface of the substrate. In this technique, shear stress serves as a mechanical stimulus to enhance the efficiency of in vitro tissue cell culture.

[0004] US2008 / 0057571A1 (2) A system is described that subjectes three-dimensional cell cultures to fluid shear forces generated by fluid flowing through a flow chamber-defined channel. The purpose of this system is to apply mechanical factors to cell cultures to regulate the differentiation of human mesenchymal stem cells (MSCs), while allowing for visual observation of the cells due to the use of transparent tubing.

[0005] In addition, US2010 / 0041128A1 (3) This invention relates to a microfluidic device for applying fluid-induced stress in single-cell culture. The method is non-invasive, allows for continuous study of cell behavior, and enables spatiotemporal control of cell growth conditions. The device includes a channel with a cell culture chamber equipped with a cell attachment surface. Vacuum channels are located on both sides of the attachment surface, allowing control of shear stress by adjusting the flow rate of the culture medium.

[0006] CN102559492A (4) A cell culture apparatus is disclosed, comprising a peristaltic pump, a signal generator, an upstream reservoir, and a downstream reservoir. The signal generator is connected to a metal electrode on the lid of the cell culture apparatus, thereby subjecting the cells to uniform electrical stimulation. Shear stress and electrical stimulation are applied to adherent cells in the flow chamber by regulating fluid flow.

[0007] Additionally, CN102965333A (5) The proposed technical solution targets cell screening suitable for bioreactors or cell factories, and the large-scale culture of screened cells using such bioreactors or cell factories. This culture step involves screening cells by applying low shear stress in the appropriate culture equipment for large-scale cell culture, with a focus on the physiological characteristics of the cells; however, the technology does not quantify the shear stress.

[0008] Therefore, most existing patent applications in this field focus on using shear stress as a mechanical stimulus to improve the culture efficiency of adherent cells in vitro. Specific methods include applying shear stress through a movable axis (US2006 / 0223175A1). (1) Shear stress is generated by the fluid flowing through the chamber (US2008 / 0057571A1) (2) and CN102559492A (4) ), or apply shear stress in single-cell analysis via microfluidic devices (US2010 / 0041128A1) (3) Other technologies share similar research and development approaches, such as screening cells suitable for bioreactor (fixed bed) and cell factory culture by applying large-scale shear stress (CN102965333A). (5) However, the shear stress application devices used in these techniques differ significantly from the device of this invention. Currently, methods for quantifying the shear stress experienced by cultured cells in the prior art are still underdeveloped.

[0009] Existing literature also reports on the use of microfluidic channels to study the effects of shear stress, and the cells used in these studies are either collected from T-shaped culture flasks (Mollet et al.) (6) Alternatively, it can be pumped from the stirred bioreactor via external circulation (Godoy-Silva et al.) (7) For example, Mollet et al. (6) This paper reports a method for constructing and characterizing (simulating) a microfluidic channel through which controllable hydrodynamic stress can be applied to cells. The application of this system in studying apoptosis and necrosis of Chinese hamster ovary cells is described. Cells were collected from a T-flask culture system, not via a circulation pump. Existing technologies have disclosed techniques for placing cells in specific devices to withstand hydrodynamic shear stress, as described by Godoy-Silva et al. (7) Mollet et al. (6) and Sieck et al. (8) This type of device is referred to as a "torture chamber" or "TC". In this invention, a further improved version of this type of device is called a "shear stress sensitivity measuring device", or simply a "shear stress sensitive device".

[0010] Godoy-Silva et al. (7) The study disclosed the placement of Chinese hamster ovary cells in Mollet et al. (6) A method for withstanding hydrodynamic stress in a designed microfluidic channel (called a stress chamber (TC)). This channel is connected to a stirred bioreactor via an external circulation system. Godoy-Silva et al. (7) The microfluidic device used comprises a constricting channel, where the fluid velocity determines the maximum shear rate, and consequently, the frequency of stress application characterized by a specific energy dissipation rate (EDR). Fluid circulation is achieved by a syringe pump that continuously pumps fluid in an alternating pattern. This study enabled continuous stress application to CHO cells during culture, investigated the effects of different stress levels on cell growth, activity, and glucose and lactate metabolism, and estimated the effect of the maximum EDR in the bioreactor.

[0011] Sieck et al. (8) This study describes how to construct a scaled-down model of hydrodynamic stress in a large-scale production bioreactor to investigate the performance of CHO cells under simulated production bioreactor conditions. Different levels of hydrodynamic stress were simulated in a 2-liter bioreactor, corresponding to the stress environment in different regions of a large-scale stirred tank bioreactor. A laboratory-scale reactor was described, and its power input was characterized by measuring the current and voltage of the stirring motor. The power input conditions of the large-scale reactor were applied to the laboratory-scale reactor to simulate a large-scale hydrodynamic environment. Furthermore, by periodically adjusting the stirring rate of the laboratory-scale reactor, the process of cells repeatedly experiencing different local hydrodynamic environments in a large-scale reactor was simulated.

[0012] Neunstoecklin et al. (9) A scaled-down model is disclosed to determine the critical stress value for CHO and Sp2 / 0 cells, i.e., the value at which shear stress negatively impacts cell culture performance. The system consists of a 3-liter bioreactor and an external circulation system with a pump and nozzles. Various nozzle diameters and fluid flow rates allow for the control of different maximum shear stress levels. The maximum shear stress generated by the pump and nozzles was characterized using computational fluid dynamics (CFD) and shear-sensitive aggregate systems.

[0013] At first glance, Godoy-Silva and others (7) and Neunstoecklin et al. (9)The research approach of these studies is similar to that of this invention, both generating shear stress through external circulation. However, this is not the actual method. The technical solution of this invention is far superior to this prior art. This invention not only considers the intensity and frequency of shear stress but also incorporates the crucial parameter of stress duration. Furthermore, the shear stress generating devices used in these studies are completely different from those of this invention, employing not only different types of pumps but also nozzle structures.

[0014] Fries et al. (10) The effect of shear stress was investigated, specifically by increasing the stirring rate to increase power input. The study was conducted using CHO cells as a biological system (to measure cell death rate) and an emulsion as a non-biological system. Fries et al. (10) A magnetically levitated centrifugal pump (PuraLev® 200SU) was used in the external circulation of the bioreactor. Pressure drop and fluid flow rate were controlled via valves and reducing pipe diameters to regulate the frequency and duration of shear stress. Cells were cultured and monitored for 12 hours, and cell mortality was recorded under specific experimental parameters. The pump was characterized using computational fluid dynamics (CFD), and cell mortality was correlated with the average turbulent kinetic energy dissipation rate, which is positively correlated with the average shear stress. In short, this study aimed to determine the appropriate operating range of the pump for its biological system, using cell mortality as an evaluation metric.

[0015] Villiger et al. (11) This paper describes a calibration method using a shear stress-dependent physical quantity, specifically the aggregate particle size of polymethyl methacrylate nanoparticles. The calibration device used in this study, as described in the embodiment of this invention, is a converging nozzle device with various nozzle diameters. Related designs and illustrations of such devices can be found in Soos et al. (14) The research, as described in embodiments of the present invention, by Villiger et al. (11) Characterization experiments were also conducted (see the second paragraph in the right column on page 1741 of the document), using the relationship between the maximum stable aggregate particle size measured in the nozzle device and the maximum effective hydrodynamic shear stress applied to convert the aggregate particle size measured in the stirred tank into the corresponding shear stress value.

[0016] However, unlike this invention, Villiger et al. (11) The previous study only focused on simple stirred tanks, and the shear stress generating device used was completely different from that of this invention. This study failed to identify the key factors generating maximum shear stress in the device (in this invention, the pump speed within the shear stress sensing device is crucial for generating maximum shear stress), therefore the practical application value of the measured shear stress values ​​is questionable. Villiger et al. (11)In this study, the stirred tank contained only water or a solution containing surfactants. No actual cell culture experiments were conducted, therefore the results cannot be directly applied to real-world cell culture processes. Furthermore, the study lacked a control experiment, making it impossible to verify the practical significance of the measured shear stress values. It also did not determine the shear stress sensitivity or critical shear stress value during cell culture, i.e., the maximum shear stress level that cells can tolerate.

[0017] In addition, Villiger et al. (11) By measuring the maximum hydrodynamic shear stress, the study investigated the jet stress generated by bubble detachment at the aerator, the stress generated during bubble rise and collapse, and the stress generated by turbulence formed by the rotation of the agitator. However, this study found that in conventional bioreactors, the shear stress generated by aeration or agitation has negligible impact on the total hydrodynamic shear stress, and regardless of the culture conditions, the level of this type of shear stress does not exceed 10 Pa. The shear stress generated by aeration and agitation is only background shear stress and has almost no effect on the total shear stress (shear stress intensity) generated during cell culture. In the culture system, only shear stress parameters (maximum shear stress, shear stress duration, and shear stress frequency) play a decisive role in the total shear stress. Therefore, Villiger et al. (11) The experimental results are not relevant to this invention.

[0018] For complete illustration, this invention also references US2007 / 034014A1 (26) and US2023 / 103671A1 (27) US2007 / 034014A1 (26) A wall shear stress sensor is disclosed, which can measure the shear stress on the test surface. However, this sensor is only a very small component of a complex system such as a bioreactor and cannot reflect the actual culture conditions inside the entire bioreactor.

[0019] Finally, US2023 / 103671A1 (27) This paper describes a method for quantifying the effect of shear stress on cells, comprising the following steps: (a) subjecting immobilized cells to a force that generates shear stress; and (b) performing nanoindentation testing on the cells in step (a) to determine their mechanical properties under different stress levels. In this study, the cells were first shaken in a flask or stirred in a bioreactor, and then the shear stress was measured using a nanoindenter. Nanoindentation is a materials testing method used to determine the hardness of materials at the microscale, and is particularly suitable for hardness testing of thin-layer materials. The nanoindenter is an optical probe-type indentation device that can achieve in-situ measurement. However, the type of device can significantly affect the test results and may lead to distortion. Furthermore, US2023 / 103671A1 (27)The previous study required sampling and measurement, which cannot reflect the actual cultivation conditions in dynamic systems such as bioreactors. This invention, however, can directly measure shear stress during the cultivation process. Furthermore, the study measured mechanical shear stress using a nanoindenter, which is unrelated to the hydrodynamic shear stress studied in this invention. The described method lacks practical application value.

[0020] The purpose of this invention is to overcome the deficiencies of existing technologies and determine the maximum shear stress that a biological or cell line can withstand, i.e., the shear stress level that is the critical value before key performance indicators of cell culture (such as biomass / viable cell density, production efficiency, and product quality indicators) undergo significant changes. Furthermore, this invention also aims to investigate the contribution of equipment and / or process parameters used during the culture process to shear stress. Summary of the Invention

[0021] The objective of this invention is achieved by a method for determining the shear stress sensitivity of cells during a culture process containing liquid culture medium in a shear stress-sensitive device, the method comprising the following steps: (1) A shear stress sensitive device is provided, comprising -Bioreactor - A closed-loop system connected to and located outside the bioreactor; - A pump placed in the loop system, which is a power pump, and - A device for controlling fluid resistance in a control loop system; (2) Select the cells, liquid culture medium, culture mode and culture conditions to be used in the culture process to be carried out; (3) Select one, two, or three shear stress parameters to be studied from the following groups: - Maximum shear stress expressed as pump speed; - Duration of shear stress exposure, expressed as the residence time of cell cultures within the pump; and / or - Shear stress frequency, expressed as the number of times the cell culture (including culture medium and cells) passes through the loop system per unit time; (4) The shear stress values ​​appearing under different maximum shear stresses are determined by calibration methods, thereby characterizing the shear stress sensitive equipment; (5) In a shear stress sensitive device, for the same culture process in step (2), one, two, three or more culture runs are performed within the same selected time period. In each subsequent run, one, two or three shear stress parameters are increased or decreased in turn, and one or more performance characteristics of the cells are measured in each culture run, generating curves of the change of each performance characteristic over time in each run. (6) In the shear stress sensitive device, for each performance characteristic, a control culture operation is performed using the same culture process as in step (5), wherein a lower maximum shear stress than in step (5) is used, and a control curve of each performance characteristic changing over time is generated. (7) Select the performance characteristic curve that is closest to the control curve of the performance characteristic in step (6) from the curve set in step (5). This curve represents the shear stress sensitivity of the cell and constitutes the shear stress limit of the cell. This ensures that the maximum shear stress in the closed-loop system is higher than the maximum shear stress in the bioreactor.

[0022] The core concept of this invention lies in the stress exerted on cells, particularly by a pump connected to the bioreactor via an external loop. This allows the bioreactor to operate in various culture modes (e.g., batch, fed-batch, continuous, perfusion, or other process control strategies) by continuously applying shear stress in a defined manner by circulating a cell culture containing liquid culture medium and cells within the loop. This external loop allows for the study of the single effect of shear stress on the culture without altering the internal conditions of the bioreactor. Not only can the maximum shear stress (adjusted by pump speed, as detailed later) be controlled and studied, but also the frequency and duration of shear stress exposure can be controlled and studied.

[0023] Shear stress-sensitive devices can provide in-depth analysis of the relationship between cell behavior and hydrodynamic shear stress, thereby accurately inferring the level of applied shear stress and providing a basis for prediction of other culture processes of the same, larger or smaller scale using similar or different hardware components.

[0024] Because cell shear stress was quantified through mechanical model calibration, the results are independent of the specific shear stress-sensitive equipment used (such as individual pumps), but reflect the conditions of the corresponding complete culture system.

[0025] The present invention also relates to a cell culture process in liquid cell culture, wherein the shear stress sensitivity measured according to the method of the present invention is applied to the same culture process, but on a different scale than that used when measuring the shear stress sensitivity.

[0026] The present invention also relates to a process for culturing eukaryotic or prokaryotic cells in a liquid cell culture in a bioreactor, and performing a method according to the invention for determining the shear stress sensitivity of cells in a shear stress sensitivity device during the culture process.

[0027] This invention also relates to a process for producing a recombinant protein, the process comprising the following steps: Step I) Culture eukaryotic or prokaryotic cells expressing the recombinant protein in a cell culture environment in a bioreactor; Step II) Harvest the recombinant protein; Step III) Purify the recombinant protein; In step I), the method according to the invention for determining the shear stress sensitivity of cells in a shear stress sensitivity device during culture is performed.

[0028] The terms "method" and "process" both refer to procedures related to this invention, are synonyms, and are used interchangeably. These two terms are used for ease of understanding and distinction between different procedures: "method" refers to the method according to the invention, while "process" refers to other procedures used in conjunction with the method of the invention, or procedures applying the method of the invention.

[0029] The present invention also relates to the use of a device comprising... -Bioreactor - A closed-loop system connected to and located outside the bioreactor; - A pump placed in the loop system, which is a power pump, and - A device for controlling fluid resistance in a control loop system; As a shear stress sensitivity measuring device or shear stress sensitivity instrument This device has no nozzles. Used to culture cells in liquid culture medium and to set a specific shear stress during the culture process. Brief description of the attached diagram Embodiments of the prior art and the present invention will be described by way of example with reference to the accompanying drawings, which are schematic diagrams and not intended to be drawn to scale, and therefore cannot make assumptions about precise geometric values ​​regarding the original dimensions. The accompanying drawings, which are incorporated in and form part of this specification, also illustrate embodiments of the invention, but are not limited to the specific embodiments described. The drawings, together with the abstract and detailed description, serve to explain the principles of this disclosure. In all the drawings, the same reference numerals are used for the same features. In the drawings: Figure 1A A schematic diagram of a shear stress-sensitive device setup for fed-batch or batch culture according to an embodiment of the present invention is shown. Figure 1B A schematic diagram of a shear stress-sensitive device setup for continuous culture, such as perfusion culture, according to an embodiment of the present invention is shown. Figure 2 The results of shear stress measurements were shown in a simple 3L bioreactor without an external loop, under different aeration and stirring rates. Figure 3A and 3B The flow rate [mL / min] of the pump at different speeds [rpm] is shown in different configurations of a 2L water storage tank with an external circuit. Figure 4A The pressure drop (dP) [bar] measured at different pipe inner diameters (ID) in a 2L water storage tank with an external circuit is shown as a function of flow rate [mL / min]. Figure 4B The pressure drop (dP) [bar] measured with flow rate [mL / min] is shown in a 2L water storage tank with an external loop, with hollow fiber filter modules of different lengths. Figure 5 The relationship between the aggregate size of polymethyl methacrylate (PMMA) nanoparticles (expressed as radius of gyration Rg [µm]) and pump speed [rpm] is shown for tubes with different inner diameters (ID) and two different hollow fiber filter modules; Figure 6 A schematic simplified cross-sectional view of the main components of a magnetic levitation centrifugal pump is shown. Figure 7 A visualization of the radial velocity distribution in a cylindrical laminar flow tube is shown; Figure 8A A schematic diagram of a known calibration method in the prior art is shown; Figure 8B This illustrates an embodiment of the invention, employing... Figure 8A A schematic diagram illustrating the calibration method for characterizing shear stress-sensitive devices; Figure 9A The graph shows the relationship between the aggregate size (radius of gyration Rg [µm]) of polymethyl methacrylate (PMMA) nanoparticles and the rotational speed [rpm] in a shear stress-sensitive device using a power pump (i.e., a magnetically levitated centrifugal pump); Figure 9B The diagram illustrates a calibration curve generated from a prior art replication device using a known calibration method according to an embodiment of the present invention, used to correlate the measured aggregate size (radius of gyration [µm]) with a known shear stress value [Pa]. Figure 9C This illustrates an embodiment of the invention, in which... Figure 9A The aggregate size (radius of gyration [µm]) of polymethyl methacrylate (PMMA) nanoparticles, measured in a shear stress-sensitive device, was determined by... Figure 9B The graph showing the relationship between shear stress value [Pa] and pump speed [rpm] after the calibration curve is converted to shear stress value [Pa]; Figures 10A to 10E An overview of different operating modes of the shear stress sensitive device according to an embodiment of the present invention is shown, corresponding to cases 1 to 4; Figure 11A and 11BAn embodiment of the invention is shown, without characterizing the shear stress sensitive device. Example curves of culture processes using CHO cell lines are shown in a production-scale bioreactor (curve P, selected culture process) and a small-scale bioreactor (curve S, selected control culture process), with two performance characteristics measured: changes in viable cell density (VCD) [10^6 cells / mL] and titer [g / L] over time [h]. Figure 12A and 12B Showing Figure 11A and 11B The curves and additional control culture runs performed in a shear stress-sensitive device, which varied the shear stress intensity by rotational speed, shear stress frequency, and shear stress exposure duration, according to embodiments of the invention; Figures 13A to 13F The invention illustrates the results of performance characteristics of fed-batch CHO cell cultures over time based on measured offline data, according to an embodiment of the invention, wherein the shear stress parameter studied is the maximum shear stress expressed as a function of pump speed. Figure 14 A and 14B show Figures 13A to 13F The measured online data; Figures 15A to 15D The invention illustrates the results of performance characteristics of CHO cell perfusion cultures over time based on measured offline data, according to an embodiment of the invention, wherein the shear stress parameter studied is the maximum shear stress expressed as a function of pump speed. Figure 16 A and 16B show Figures 15A to 15D The measured online data; Figures 17A to 17D The invention illustrates the results of performance characteristics of CHO cell perfusion cultures over time based on measured offline data according to an embodiment of the invention, wherein two shear stress parameters, namely shear stress frequency and shear stress exposure duration, and the variation of bypass flow rate were studied. Figure 18 A and 18B show Figures 17A to 17D The measured online data; Figures 19A to 19D The results show the performance characteristics of other CHO cell perfusion cultures over time based on measured offline data according to an embodiment of the present invention, wherein three shear stress parameters, namely pump speed, shear stress frequency and shear stress exposure duration, and the bypass flow rate variation were studied. Figure 20 A and 20B show Figures 19A to 19D The measured online data.

[0031] Legends for the diagrams are provided at the end of the instruction manual. Invention Details Terminology Definition Terms not specifically defined herein shall have the meanings that a person skilled in the art would assign to them based on the content of this disclosure and the context.

[0033] A bioreactor (also referred to herein as a fermenter) is a container, vessel, apparatus, device, or equipment in which living organisms, particularly specific microorganisms, cells, or small plants, are cultured or fermented under optimal conditions. A bioreactor may consist of or contain a biocompatible container in which chemical or biochemical methods involving organisms and / or bioactive substances derived from those organisms are carried out. Bioreactors utilize additional equipment such as agitators, baffles, one or more atomizers, and / or ports, which specifically allow for the culture and propagation of cells. Typically, bioreactors are cylindrical tubes with two ends forming the top and bottom of the bioreactor. Bioreactors range in size from liters to cubic meters, are typically made of stainless steel, and are designed for multiple uses. Cells or cell components or metabolites are cultured in bioreactors for the purpose of obtaining them. These products are used, for example, as active pharmaceutical ingredients in the pharmaceutical industry, such as antibiotics, antibodies, or insulin; or as basic chemicals in the chemical industry, such as in wastewater treatment, the food industry, pest control, or in the biodegradation of waste or pollutants, such as in oil spills. The bioreactor according to this disclosure can be used from laboratory scale to large-scale production and can be used as a component of a shear stress-sensitive device for measuring shear stress during the culture process.

[0034] There are no particular restrictions on the cells cultured in the bioreactor. They are preferably prokaryotic or eukaryotic cells, especially eukaryotic cells. These eukaryotic cells are particularly animal cells, such as mammalian cells, insect cells, plant cells, bacterial cells, yeast, fungi, etc. Eukaryotic cells, such as Chinese hamster ovary (CHO) cells or yeast cells, are used, for example, for the production of antibodies such as monoclonal antibodies and / or recombinant proteins such as therapeutic recombinant proteins. Alternatively, the cells can produce, for example, peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites or any other useful substances. The term "eukaryotic cell" as used herein specifically refers to a cell having a nucleus surrounded by a nuclear membrane, including animal cells, human cells, plant cells, and yeast cells. In this invention, "eukaryotic cell" specifically encompasses mammalian cells, such as Chinese hamster ovary (CHO) cells or HEK293 cell-derived cells, and yeast cells.

[0035] The term "cell culture" encompasses cell culture and fermentation methods at all scales (e.g., from sub-mL to >10,000 L), all different methodologies (e.g., batch, fed-batch, continuous culture (e.g., perfusion)), all method control protocols (e.g., uncontrolled, fully automated, and controlled systems, such as pH, temperature, and oxygen control), and all types of fermentation systems (e.g., disposable systems, stainless steel systems, glassware systems). In exemplary embodiments, cell culture refers to cell cultures with volumes ≥10 mL, ≥15 mL, ≥250 mL, ≥500 mL, ≥1 L, ≥2 L, ≥10 L, ≥1000 L, ≥5000 L, or ≥10000 L.

[0036] As used herein, the term "liquid culture medium" or "cell culture medium" refers to a culture medium used for culturing cells (e.g., mammalian cells) containing essential nutrients and components such as vitamins, trace elements, salts, macrosalts, amino acids, lipids, and carbohydrates, preferably in a buffered medium. Typical mammalian cell culture media have a pH of approximately neutral, for example, from about 6.5 to about 7.5, preferably from about 6.8 to about 7.3, and more preferably about 7. Non-limiting examples of such cell culture media include commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco modified Eagle medium (DMEM; Sigma), Limit Essentials medium (MEM; Sigma), Iscove modified Dulbecco medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-Invitrogen), serum-free CHO medium (Sigma), protein-free CHO medium (Sigma), and proprietary media from various sources. Cell culture media can be basal cell media. Cell culture medium can also be a basic cell culture medium supplemented with feed medium and / or additives. Cell culture medium can also be called fermentation broth.

[0037] As used herein, "basal medium" or "basal cell medium" is a liquid culture medium or cell culture medium used for culturing mammalian cells. It refers to the medium used to culture cells from the beginning of a cell culture run and is generally not used as an additive to another medium, although various components can be added to the basal medium. The basal medium serves as a foundation, to which further additives (or supplements) and / or feed media can be optionally added during the culture run, ultimately forming the cell culture medium. The basal cell medium is provided from the start of the cell culture process. Typically, the basal cell medium provides nutrients such as carbon sources, amino acids, vitamins, macrosalts (e.g., sodium chloride or potassium chloride), various trace elements (e.g., manganese sulfate), pH buffers, lipids, and glucose. Macrosalts are usually only present in the basal medium, and the final osmotic pressure in cell culture should not exceed approximately 280–350 mOsm / L to allow the cell cultures to grow and proliferate under reasonable osmotic stress.

[0038] As used herein, the term "feed" or "feed medium" refers to a nutrient concentrate / concentrated nutrient composition, particularly used as a feed in mammalian cell culture. Therefore, it is provided as a concentrate and added to cell cultures. It is provided as a "concentrated feed medium" to minimize dilution of cell cultures, typically provided at 10-50 ml / L / day, preferably 15-45 ml / L / day, more preferably 20-40 ml / L / day, and even more preferably 30 ml / L / day, based on the culture starting volume (CSV, i.e., the starting volume on day 0) in the container. This corresponds to adding approximately 1-5%, preferably approximately 1.5-4.5%, more preferably approximately 2-4%, and even more preferably approximately 3% of the culture starting volume daily. For cultures using high-density or ultra-high-density seeding, higher feed rates may be beneficial, such as 10-50 ml / L / day, 15-45 ml / L / day, or 25-45 ml / L / day. This corresponds to adding approximately 1-5%, 1.5-4.5%, or 2.5-4.5% of the culture starting volume daily. The feeding rate should be understood as the average feeding rate during the feeding period. The feed medium typically has a higher concentration of most (but not all) of the components of the basal cell culture medium. Generally, the feed medium replaces nutrients consumed during cell culture, such as amino acids and carbohydrates, while salts and buffers, of lower importance, are usually provided along with the basal medium. The feed medium is typically added to the (basal) cell culture medium / fermentation broth in a feed-batch manner. The feed medium added to the basal medium (repeatedly or continuously) forms the cell culture medium. Feeding can be done in different modes, such as continuous or push-feed, or via perfusion-related techniques (chemostat or mixed perfusion system). Preferably, the feed medium is added daily, but it can also be added more frequently, such as twice daily, or less frequently, such as once every two days. More preferably, the feed medium is added continuously. Nutrient addition is typically carried out during the culture period (i.e., after day 0). Compared to basal media, fed-batch media typically consist of a high concentration of nutrient solutions (e.g., >6 times), providing all the components similar to basal media, except for "highly osmotically active compounds," such as large amounts of salts (e.g., NaCl, KCl, NaHCO3, MgSO4, Ca(NO3)2). Typical basal media, concentrated 6 times or higher (containing little or no large amounts of salts), maintain good compound solubility and sufficiently low osmolarity (e.g., 270-1500 mosmol / kg, preferably 310-800 mosmol / kg) to maintain an osmolarity of approximately 270-550 mosmol / kg, preferably approximately 280-450 mosmol / kg, and more preferably approximately 280-350 mosmol / kg in cell culture. Feed-batch media can be added as a complete feed-batch solution or may contain one or more feed supplements for individual addition to cell culture.Due to varying feeding protocols, such as periodic feeding and on-demand feeding (typically for glucose addition), one or more feeding supplements may be required and are therefore typically provided at least as separate supplements. The use of one or more feeding supplements may also be necessary due to the low solubility of certain compounds, differences in solubility of certain compounds at different pH levels, and / or interactions of compounds at high concentrations in the feeding medium. The feeding medium preferably has a well-defined chemical composition (optionally containing recombinant proteins such as insulin or IGF). It is cell-free, not in contact with cells in culture, or free of cell-derived metabolic waste products. Therefore, the term "feeding medium" as used herein excludes pretreatment media derived from cell culture or media in the presence of cells (also referred to herein as liquid media or cell media).

[0039] As used herein, the term "feed supplement" refers to a nutrient concentrate that can be added to the feed medium prior to use, or can be added separately from the feed medium to the basal medium and / or cell culture medium. Therefore, compounds can be provided either with the feed medium or the feed supplement, or both. For example, in a dual-feed strategy for cysteine, it can be added via both the feed medium and the feed supplement. Like the feed medium, the "feed supplement" is provided as a concentrate to avoid diluting the cell culture.

[0040] Liquid culture media or cell culture media, including basal media and supplemental media, are preferably serum-free and have a defined chemical composition. The basal media and / or supplemental media may further be protein-free. As used herein, "serum-free culture medium" refers to cell culture media used for in vitro cell culture that do not contain animal-derived serum. This is preferred because serum may contain contaminants from the animal, such as viruses, and because the composition of serum is undefined and varies significantly from batch to batch. The basal media and supplemental media according to the invention are serum-free.

[0041] As used herein, "chemically defined culture medium" refers to a cell culture medium suitable for in vitro cell culture in which all components are known. More specifically, it contains no supplements, such as animal serum or plant, yeast, or animal hydrolysates. Therefore, a chemically defined culture medium is also serum-free. The basal and supplemental culture media according to the invention are preferably chemically defined. In one embodiment, the basal and / or supplemental culture media are serum-free and chemically defined, and optionally contain recombinant growth factors, such as insulin or insulin-like growth factor (IGF). The basal and / or supplemental culture media referred to herein contain no other proteins except those produced by the mammalian cells to be cultured after the cell culture medium is provided in cell culture.

[0042] As used herein, "protein-free culture medium" refers to a liquid culture medium or cell culture medium used for in vitro cell culture that does not contain proteins (except for proteins produced by the cells being cultured during cell culture). Proteins refer to polypeptides of any length, but do not include single amino acids, dipeptides, or tripeptides. Specifically, growth factors such as insulin and insulin-like growth factor (IGF) are absent from the culture medium. Preferably, the basal and feed media according to the invention are chemically defined and protein-free.

[0043] As used herein, the term "viability" refers to the percentage of living cells in a cell culture as determined by methods known in the art, such as trypan blue rejection assays using a Cedex device (Roche Diagnostics; Rotkreuz) based on automated microscopy cell counting. However, many other methods exist for determining viability, such as fluorescence methods (e.g., based on propidium iodide), calorimetry, or enzymatic methods, which reflect the energy metabolism of living cells, such as methods using LDH (lactate dehydrogenase) or certain tetrazolium salts like Alamar blue, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), or TTC (tetrazolium chloride).

[0044] The term "batch culture" refers to a culture method in which cells are inoculated with a specific volume of culture medium. The cells grow in the medium and experience constantly changing conditions as nutrients are consumed and waste products accumulate. This system can be considered a closed system, with no additions or removals during culture, but oxygen is periodically added to the system through aeration.

[0045] The term "feed-batch" as used herein refers to a cell culture method in which cells are fed continuously or periodically with a feed-batch medium containing nutrients. Feeding can begin shortly after the start of cell culture (day 0), or more typically one, two, or three days after the start of culture. Feeding can follow a pre-set schedule, such as daily, every two days, every three days, etc. Alternatively, cell growth, nutrients, or toxic byproducts of the culture can be monitored, and feeding adjusted accordingly. Typically, the following performance characteristics are measured daily: viable cell concentration, product concentration (titer), pH, osmolarity, and several metabolites, such as glucose, lactate (a salt content indicator), and ammonium (a growth inhibitor that negatively affects growth rate and reduces viable biomass). Higher product titers can be achieved in fed-batch culture compared to unfeeded culture. Typically, fed-batch culture is stopped at a certain time point to harvest cells and / or culture medium, and to isolate and / or purify the target product, such as a heterologous protein or recombinant virus. The fed-batch process typically lasts about 2-3 weeks, for example, about 10-24 days, about 12 to 21 days, about 12 to 18 days, preferably about 12 to 16 days. In particular, the fed-batch process for producing heterologous proteins typically lasts about 2-3 weeks, for example, about 10-24 days, about 12 to 21 days, about 12 to 18 days, preferably about 12 to 16 days.

[0046] "Perfusion" or "perfusion culture" is understood herein as a continuous process. It is a specific cell culture method in which nutrients, provided in the form of fresh culture medium, are added to the cell culture while cellular waste products and nutrient-depleted medium are continuously removed from the cell culture. According to this disclosure, a cell-pass filter (hollow fiber filter module) retains these substances. Typically, fresh culture medium is provided to the cells at the same rate as the depleted medium is removed, thereby maintaining a constant bioreactor volume.

[0047] The term "laboratory scale" refers to experimental setups conducted on a small scale. For example, these are protocols using volumes ranging from a few milliliters to a few liters.

[0048] The terms "industrial scale" and "large scale" are used interchangeably and are synonymous, referring to products obtained in large quantities, typically with cost advantages, where unit output costs decrease as scale increases. All other things being equal, large-scale production units are expected to have lower unit output costs than smaller units. In the context of cell culture, industrial scale can be understood as the use of bioreactors with a volume equal to or greater than approximately 100 L. According to another embodiment, the volume of bioreactors used in industrial scale can be equal to or greater than 600, 800, 1000, 1200, 1500 L, or even larger.

[0049] For background information on cell culture, cell types, culture media used, and related methods, see WO 2021 / 165302 A1, the contents of which are incorporated herein by reference in their entirety. In particular, see paragraphs

[0054] through

[0088] .

[0050] The term "loop" or "loop system" refers to the external circulation piping system of a bioreactor, which is part of shear stress-sensitive equipment. The terms "loop," "external loop," and "loop system" are synonymous and used interchangeably.

[0051] The term "closed-loop system" means that the bioreactor is connected to a loop on both sides, and the loop has no other outlet. The bioreactor is connected in such a way that the culture medium and cell flow enter the bioreactor from one side and exit from the other side. A closed-loop system is schematically shown in [illustrative image]. Figure 1A and 1B A closed-loop system is used for the overall circulation of cell cultures containing cells and liquid culture medium in shear stress-sensitive equipment. Here, "circulation" is understood as the process of circulating the cell culture, containing cells and liquid culture medium, as a whole from the bioreactor to the closed-loop system, back to the bioreactor, through the closed-loop system again, and so on. Circulation is performed by pumps located within the closed-loop system, which continuously pump to circulate the cell culture containing cells and liquid culture medium within the closed-loop system. The pumps used are power pumps.

[0052] The term "shear stress" refers to force divided by the area of ​​the surface on which it acts. In engineering mechanics, shear stress is the load produced on an object by antiparallel forces acting on a parallel surface. This means that opposite lateral forces are acting. These forces subsequently cause what is called shear load. Just as the stress generated between two adjacent layers of the Earth during an earthquake is called shear stress. Typically, the unit of shear stress T (tau) is Pascal [Pa]. The shear stress experienced by cells is due to external influences caused by the type and process of cell culture.

[0053] A shear stress sensitivity measurement device, also referred to herein as a "shear stress sensitive device," is a device used to measure the shear stress acting on cells during culture. A shear stress sensitive device includes a bioreactor, a circuit located outside the bioreactor, a power pump, and a device for controlling the fluid dynamics shear stress in the circuit (such as a variable tube), as well as an optional cell retention device (such as a hollow fiber filter module). In a shear stress sensitive device, liquid culture medium for culturing cells and cell cultures are continuously circulated by the pump to measure the shear stress under corresponding conditions by changing specific parameters.

[0054] "Fluorodynamic shear stress" refers to the shear stress generated by fluid dynamic effects during the culture process, particularly caused by any form of motion, such as liquid media, agitation, and gas supply in the cell culture. "Shear stress" and "fluid dynamic shear stress" are considered synonyms here and can be used interchangeably.

[0055] Like living organisms, individual living cells, such as bacteria and yeast, especially those without cell walls, such as living cells derived (in principle) from multicellular organisms (like animals), are sensitive to shear stresses that affect their morphological integrity, overall physical stability, and general biological functions. Therefore, a cell's "shear stress sensitivity" measures the extent to which a cell can withstand shear stresses, particularly its resistance to shear stress intensity, which is directly reflected in its properties, especially product-related properties referred to herein as "performance characteristics."

[0056] The "shear stress limit" of a cell is considered here as the shear stress or shear stress intensity that a cell can withstand without significantly adversely affecting the cell culture process. This affects performance characteristics in cell culture, such as viable cell density, cell productivity (e.g., production of proteins or other molecules of interest (qualitative and / or quantitative)), etc. The term "limit" or "threshold" refers to the upper limit at which adverse degradation occurs in cell culture. The shear stress limit allows for a quantitative statement of a cell's shear stress sensitivity.

[0057] "Shear stress intensity" refers to the strength or magnitude of the total shear stress acting on a cell, such as in a shear stress-sensitive device. Shear stress intensity represents the combination or sum of all shear stress parameters. Shear stress parameters are the maximum shear stress, shear stress frequency, and shear stress exposure duration. Parameters are quantifiable variables or quantities whose values ​​depend on the selected conditions; therefore, parameters are specified using values, which is common knowledge to those skilled in the art. Thus, shear stress parameters are quantifiable variables or quantities whose values ​​depend on the selected conditions; that is, shear stress parameters are given in the form of their quantified values. Both "shear stress parameter" and "shear stress value" are technical terms whose meanings are within the knowledge of those skilled in the art. Furthermore, the mathematical equations describing the various shear stress parameters are given in the section "Relationships between Shear Stress Parameters and the Influence of Bypass Flow."

[0058] "Maximum shear stress" is a shear stress parameter determined and expressed by the rotational speed of the pump used (i.e., the power pump in a shear stress-sensitive device). The value given for the shear stress parameter "maximum shear stress" Tmax (tau) also has the unit Pascal [Pa].

[0059] "Shear stress exposure duration" is a shear stress parameter that is determined and expressed by the residence time of cell cultures containing liquid culture medium and cells in the pump used (i.e., the power pump in a shear stress sensitive device).

[0060] "Shear stress frequency" is a shear stress parameter determined and represented by the number of times a cell culture containing liquid culture medium and cells passes through a shear stress sensitive device loop system per unit time.

[0061] The term "bypass flow" refers to the flow of culture medium along with cells within a loop system of a shear stress-sensitive device. A loop system represents the external loop or external circulation of a bioreactor. Flow rate can be measured, for example, using a flow meter.

[0062] The term "characteristic" refers to any measurable variable that changes over time during cell culture. "Performance characteristic" refers to any measurable variable that changes over time during cell culture, which is a characteristic parameter of a particular culture process for the cultured cells and can characterize them. As understood in this invention, performance characteristics of cell culture are particularly so-called product-related quality parameters. Examples of performance characteristics are the growth and productivity of cell cultures, particularly relative viable cell density, viability, glucose concentration, lactate concentration, LDH concentration, relative titer (product concentration), glycosylation of the product, etc. The term "performance characteristic" is known to those skilled in the art (e.g., in the references of Kompala et al.). (24) As mentioned in [the article], many performance characteristics of cell culture affected by shear stress are also known to those skilled in the art (Sieck et al.). (8) Neunstoecklin et al. (9) and Gaugler et al. (25) ).

[0063] There are numerous "parameters" and "conditions" that can be set and changed in a shear stress-sensitive apparatus. These are all the variable parameters and conditions that can be used for cell culture. These are known to those skilled in the art in the prior art. For example, these include aeration settings, stirring in the bioreactor, temperature, feed rate, dissolved oxygen tension, pH, etc. When studying shear stress levels in a shear stress-sensitive apparatus, the parameters and conditions in the apparatus are selected and set once, and then kept as constant as possible throughout the study to avoid interfering with the research.

[0064] The expressions “including,” “contains,” and “comprising” should also cover the more specific term “composes of,” unless otherwise stated or obvious from the context.

[0065] Furthermore, it should be noted that the singular and plural forms are not used in a restrictive manner in this disclosure. The singular forms "a," "an," "the," and "the" used herein therefore refer to both the singular and the plural, unless otherwise stated or obvious from the context.

[0066] The expression "about" or "approximately" means within 10%, particularly within 5%, and even more particularly within 1% or 0.1% of the specified or indicated upper or lower limits.

[0067] Embodiments of the present invention The present invention will now be explained with reference to various exemplary embodiments.

[0068] According to this embodiment of the invention, the method comprises or consists of steps (1), (2), (3), (4), (5), (6), and (7). According to one embodiment, these steps are performed in the indicated order. According to another embodiment, no intermediate steps are performed between these steps.

[0069] The method according to the invention for determining cell shear stress sensitivity during culture is performed in a shear stress sensitivity apparatus. This apparatus enables better assessment of the shear stress effects acting on cells during culture, and even allows for the measurement of shear stress sensitivity in different operating modes of the apparatus. The focus here is on the specific effects on cells, where variations in the parameters of the shear stress sensitivity apparatus and changes in culture conditions lead to different effects and results. In this context, the intensity of shear stress that cells can withstand / be exposed to is important, as it is crucial for cell performance. The intensity of shear stress directly affects so-called product-related quality parameters of cell culture, referred to herein as "performance characteristics," such as cell growth.

[0070] General setup of shear stress sensitive devices The general setup commonly used in cell culture equipment forms the basis of the "shear stress-sensitive device" of this invention. In this device, shear stress is measured, particularly the shear stress sensitivity or limit that cells can withstand.

[0071] The shear stress sensitive device according to the present invention comprises: -Bioreactor - A closed-loop system for the overall circulation of cell cultures containing cells and liquid culture medium, wherein both ends of the closed-loop system are connected to the bioreactor and are located outside the bioreactor; - A pump placed in the loop system, particularly downstream of the bioreactor, continuously pumps to circulate cell cultures containing cells and liquid culture medium within the loop system. The pump used is a power pump. - A device for controlling fluid resistance, placed in a loop system, particularly downstream of the pump.

[0072] According to step (1) of the method of the present invention, a shear stress sensitive device is provided.

[0073] To better illustrate, refer to Figure 1A and 1B The illustrated schematic embodiment of the invention describes in detail the shear stress sensitive device of the present invention. The shear stress sensitive device can be adapted according to the culture operation mode to be studied (i.e., batch, fed-batch, or continuous (e.g., perfusion)). Therefore, Figure 1A and 1B Two different shear stress-sensitive device settings are described in the paper. Figure 1A The settings for fed-batch culture are displayed. These settings also apply to batch processes. Figure 1B The setup for continuous culture, particularly perfusion culture, is shown.

[0074] First, one embodiment of the shear stress-sensitive device for fed-batch or batch culture according to the present invention will be explained, such as... Figure 1A As shown. In Figure 1A In this embodiment, the shear stress-sensitive device 100 has a bioreactor 110 connected to a loop system. For circulating a cell culture containing cells and culture medium 115, a closed loop system is provided connected to the bioreactor 110, and this loop system is arranged outside the bioreactor 110. In this embodiment, the outlet at the bottom of the bioreactor is connected to the inlet at the top of the bioreactor. Other configurations in which the loop system is connected to the bioreactor are also possible. Figure 1A In this embodiment, bioreactor 110 contains liquid culture medium 115 and cells to be cultured (not shown). In the illustrated embodiment, the outlet 114 at the bottom of bioreactor 110 is connected to the inlet 116 at the top of bioreactor 110 via pipes 125a, 125b, 125c, 125d1, and 125d2. The type of pipe is not further limited and includes any type of fluid conduit known to those skilled in the art, such as tubing, hoses, pipes, etc. The pipes may be selected from polymers, such as rubber, or metal.

[0075] A loop system, located outside the bioreactor 110, is used to circulate the entire cell culture containing liquid culture medium 115 and cells. In this embodiment, the flow direction of the cell culture containing cells and liquid culture medium 115 is as shown by arrows A and B. An additional stirrer 118 may be provided in the bioreactor 110. For other embodiments of the loop system and other locations where the loop system is connected to the bioreactor, [further details are needed]. Figure 1A Compared to the example shown, it is also possible according to the present invention. For example, cell cultures can be pumped from the bioreactor into the loop system using an infiltration tube that extends into the bioreactor from above.

[0076] A pump exists in the loop system that continuously circulates cell cultures containing liquid culture medium and cells. Figure 1A In the process, bioreactor 110 is connected to pipe 125a. Pipe 125a is then connected to the suction side of pump 130. Pump 130 is a powered pump, particularly a centrifugal pump, especially a magnetically levitated centrifugal pump. This type of pump has proven particularly advantageous for shear stress testing. On the discharge side, pump 130 is again connected to pipe 125b. Pipe 125b is then connected to devices 125c1, 125c, and 125c2 for controlling fluid resistance. "Device for controlling fluid resistance" refers to one, two, or more devices, auxiliary devices, or tools used to generate a pressure drop in the loop system, particularly a higher or lower pressure drop. This comes into play when an increase in one shear stress parameter causes another parameter, which should remain constant, to increase undesirably. This will be explained in more detail later.

[0077] exist Figure 1A In the implementation scheme, the device for controlling fluid resistance is a pipe 125c with a variable length and / or variable diameter, also referred to herein as a "variable pipe". Therefore, the length and / or diameter can be adjusted according to operating conditions. This can be achieved, for example, by replacing pipe 125c with another pipe having a different length and / or a different diameter. To change the diameter of the variable pipe 125c, a reducing fitting 125c1 can be provided at one end of pipe 125c, and a reducing fitting 125c2 at the other end. For example, increasing the length of the variable pipe 125c or decreasing its diameter will increase the pressure drop. Other devices for controlling fluid resistance are also possible. Valves, such as pinch valves, are exemplarily mentioned. Other practical implementations of this device are also possible, such as variable winding of the variable pipe 125c to, for example, increase the pressure drop.

[0078] exist Figure 1A In the illustration, the exemplarily shown devices 125c1, 125c, and 125c2 for controlling fluid resistance are connected to pipe 125d1, which in turn is connected to bioreactor 110 via pipe 125d2. A flow meter 140 may be installed between bioreactor 110 and the devices 125c1, 125c, and 125c2 for controlling fluid resistance. Furthermore, if desired, a pressure gauge or pressure sensor (not shown) may be used to determine the pressure drop across the devices 125c1, 125c, and 125c2 for controlling fluid resistance. Figure 1A In this configuration, devices 125c1, 125c, and 125c2 for controlling fluid resistance are located downstream of pump 130. Other configurations are also conceivable.

[0079] Cell cultures are cultured in a liquid within a bioreactor and continuously pumped out of the bioreactor by a powered pump (such as a magnetically driven centrifugal pump). As observed in the experiments, the highest shear stress is applied by the pump, which is controlled by its rotational speed [rpm]. Several parameters are used to set the shear stress level in a shear stress-sensitive device: in addition to the maximum shear stress expressed by the pump speed, there are the residence time of the cell culture within the pump (referred to here as the shear stress exposure duration) and the number of times the cell culture, containing both liquid culture medium and cells, passes through the loop system per unit time (referred to here as the shear stress frequency). These parameters will be explained in more detail later.

[0080] In continuous mode, for example Figure 1B Under the perfusion pattern shown, Figure 1A The setup incorporates cell retention devices, such as hollow fiber filter modules. Figure 1B The image shows the setup of a shear stress-sensitive device 100 for continuous mode, particularly perfusion mode, including a hollow fiber filter module 150 with an permeate flow (arrow C). Cells are retained by the hollow fiber filter 150. Figure 1B In the middle, pipe 125c is connected to hollow fiber filter module 150 through pipe 125d1, such as Figure 1B As shown. Also in Figure 1B In this embodiment, a flow meter 140 may be installed between the bioreactor 110 and the hollow fiber filter module 150. A pressure gauge or sensor (not shown) may be used to determine the pressure drop across the hollow fiber filter module 150. According to the invention, there are no further limitations on the hollow fiber filter module. Any hollow fiber filter module suitable for culturing cells in continuous mode (e.g., perfusion) and any cell retention method / apparatus may be used.

[0081] In step (2) of the method according to the invention, the cells, liquid culture medium, culture mode, and culture conditions are selected. Within the scope of the invention, these selections are not further limited, and therefore any culture method having any cells, any liquid culture medium, any culture mode, and any culture conditions can be used in the method of the invention. The only prerequisite is that the culture must be carried out in a liquid medium so that it can be circulated in a shear-sensitive device. The object of the invention is that various culture methods exist (e.g., large-scale methods for producing new biological entities (NBEs)) in which increased shear stress causes or may cause performance loss. The method of the invention enables the simulation of culture methods in a shear stress-sensitive device, thereby characterizing / examining shear stress under appropriate conditions.

[0082] Based on these selection steps in step (2), it can be determined which of the two settings of the shear stress-sensitive device to use, i.e., according to... Figure 1A Settings or based on Figure 1BThe setup also includes a cell retention device, which can be positioned between the fluid resistance control device (such as a variable tube) and the bioreactor if the shear stress-sensitive device is to operate in a continuous mode (such as perfusion). Other configurations are possible.

[0083] There are no further restrictions on the cells used. Any type of cell can be used, particularly eukaryotic or prokaryotic cells. Eukaryotic cells are especially animal cells, such as mammalian cells, insect cells, plant cells, bacterial cells, yeast, fungi, etc. Eukaryotic cells specifically include mammalian cells, such as Chinese hamster ovary (CHO) cells or HEK293 cells, as well as yeast cells.

[0084] There are no particular restrictions on liquid culture media; any type of liquid culture medium used for cell culture may be used. Liquid culture media include, for example, basal media, media with well-defined chemical compositions, and protein-free media, as detailed in the terminology definition section.

[0085] Culture conditions include all options that can be selected for cell culture. These include, for example, cell seeding density, oxygen concentration, selected culture scale, type of feed medium, feed rate, pH range used, temperature set during culture, glucose supply, type of hollow fiber module selected during perfusion, perfusion rate, aeration rate, unit power input, etc.

[0086] The cultivation modes for the cultivation process to be carried out include batch, fed-batch, and continuous (e.g., irrigation) processes.

[0087] Shear stress characterization The following section provides a general explanation of the shear stress characterization that forms the basis for the development of the aforementioned shear stress-sensitive device, as well as the results found therein. Here, all parts of the previously described device have been characterized in relation to shear stress. Furthermore, the source of the maximum shear stress has been identified, which plays a central role in this invention.

[0088] First, experiments were conducted to determine whether the shear stress triggered by aeration or agitation in a typical bioreactor was indeed lower than the shear stress in the loop used in a shear stress-sensitive device. Only by clarifying this could meaningful results regarding shear stress be obtained. To this end, shear stress in a 3L bioreactor was measured in a separate experiment. The shear stress values ​​were calculated according to the method of the present invention. This is explained in detail in the section "Method for Determining Maximum Hydrodynamic Shear Stress" and subsequent sections. This is achieved by using a shear stress-dependent quantity that is explicitly correlated with the maximum shear stress value that occurs. In the present experiment, the shear stress-dependent quantity is the aggregate size, more precisely the radius of gyration of aggregates of polymethyl methacrylate (PMMA) nanoparticles subjected to shear stress in the device (here, the 3L bioreactor). The shear stress here is aeration and agitation. It is known that PMMA nanoparticle aggregates are sensitive to shear stress, that the aggregates decompose in a defined manner under shear stress, and that the resulting aggregate size is adjusted as a result of the shear stress that occurs.

[0089] Based on existing technologies, the correlation between aggregate size and shear stress value is known (e.g., Villiger et al.). (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) As a calibration curve, the obtained aggregate sizes are converted to the maximum shear stress values ​​that must exist in the device (here: 3L bioreactor) to obtain these aggregate sizes. Examples of how to perform this operation in detail will be explained in the section "Methods for Determining Maximum Hydrodynamic Shear Stress", particularly under "Exemplary Calibration Methods" and "Exemplary Characterization Methods".

[0090] Figure 2 Shear stress measurements were performed at different aeration and stirring rates in a simple 3L bioreactor without an external loop. Shear stress Tmax [Pa] was plotted against impeller stirring speed [rpm]. The shear stress values ​​of Tmax [Pa] were determined using polymethyl methacrylate nanoparticle aggregates, which will be explained later. Gas flow rates were set to constant values ​​for each case, as shown below. Figure 2 As shown, it is given in [vvm]. The unit "vvm" refers to the air volume per minute per volume of culture medium.

[0091] In the experiment, 1 g / L of surfactant (Pluronic F-68) was added to prevent bubble-aggregate adhesion between bubbles generated by the atomizer used for ventilation and the polymethyl methacrylate nanoparticle aggregates. Cell adhesion to the bubbles was prevented in the same way.

[0092] Figure 2 The results show that in a 3L bioreactor, shear stress increases with increasing impeller stirring rate. However, the effects of aeration rate and stirring speed on hydrodynamic shear stress are negligible. In particular, shear stress never reaches levels >10 Pa, regardless of the conditions applied. Therefore, the actual shear stress factor exists in other parts of the culture system. Thus, stirring and aeration exist only as background shear stress and have almost no effect according to the present invention.

[0093] To verify the intensity of shear stress experienced by cells during culture, further experiments were conducted to characterize the additional shear stress occurring within the external circuit. In this context, the maximum hydrodynamic shear stress that occurs is of particular interest, and is important according to the present invention.

[0094] Maximum hydrodynamic shear stress The shear stress sensitive device used is characterized by the maximum shear stress value that occurs therein. This is done using calibration methods, particularly those known in the prior art. In this regard, it must be ensured that only the maximum shear stress source is used for characterization. The "characterization" of the shear stress sensitive device is a calibration method used to determine the maximum shear stress value under different conditions. This calibration method of the shear stress sensitive device is referred to as "characterization" here to clearly distinguish it from the calibration methods known in the prior art. The maximum shear stress is used so that it can be measured using calibration methods known in the prior art. This will be explained in detail later. It has been found that the maximum shear stress source in the shear stress sensitive device of the present invention is the pump speed, as will be demonstrated in detail below: Using known calibration methods Figure 1B The shear stress-sensitive device was characterized experimentally. In this experiment, a known calibration method was selected based on determining a shear stress-dependent quantity, in this case, the radius of gyration of a particle aggregate of polymethyl methacrylate nanoparticles. This is known from existing techniques (e.g., Villiger et al.) (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) The breakage of these particle aggregates is a function of the maximum hydrodynamic shear stress, thus it is possible to infer the maximum shear stress present in the device from the size of the aggregates. These abiotic aggregates, such as PMMA aggregates, have irregular dimensions, expressed as the (average) radius of gyration (Rg). The radius of gyration (Rg) is a known quantity characterizing the spatial extent or size of irregularly shaped aggregates and can be compared to the average particle size. This quantity is known to those skilled in the art.

[0095] Furthermore, the effects of flow rate and pressure in shear stress-sensitive devices were investigated, and the results were combined with... Figure 3A , 3B 4A and 4B will be explained below.

[0096] A simplified experimental setup was used, replacing the previously used 3L bioreactor with a 2L water tank that was only slightly agitated to prevent high stress. To determine whether the highest stress occurred in the variable-length tube or in different types of hollow fiber filter modules, the experimental setup was adjusted accordingly. Either tubes with a specific inner diameter (ID) or hollow fiber filter modules with a specific length were used in the experiment. In other words, a setup similar to... Figure 1A and 1B A simplified setup, but using a 2L water tank instead of a bioreactor.

[0097] exist Figure 3A and 3B The figure shows the variation of pump flow rate [mL / min] with various rotational speeds [rpm] under different settings. The pump used was a magnetically levitated centrifugal pump. For the characterization settings, the tube length was fixed at 245 mm.

[0098] Figure 3A The effect of the inner diameter (ID) on the flow rate (mL / min) at different pump speeds (rpm) is shown in three studies. The flow rate (mL / min) increases with increasing speed (rpm). For a tube with an inner diameter (ID) of 6.4 mm, a flow rate >6500 mL / min is achieved at approximately 13000 rpm, while a tube with a minimum inner diameter (ID) of 1.6 mm produces a flow rate <1000 mL / min at the same speed (rpm). Similarly, compared to the short hollow fiber filter module (HF short), the long hollow fiber filter module (HF long) reduces the flow rate at the same speed (see [link to relevant documentation]). Figure 3B ).

[0099] also, Figure 4A The pressure drop (dP) [bar] measured at different tube inner diameters (ID) is shown as a function of flow rate [mL / min]. Figure 4B The pressure drop (dP) [bar] measured for hollow fiber filter modules of different lengths is shown as a function of flow rate [mL / min]. As already discussed and... Figure 3A and 3B As shown, a smaller inner diameter (ID) and a longer hollow fiber filter module result in a lower flow rate at the same rotational speed [rpm], due to an increased pressure drop, such as... Figure 4A and 4B As shown.

[0100] Based on the aforementioned flow rate and pressure studies, it has been demonstrated that the shear stress-sensitive device can operate at different flow rates while maintaining a constant pump speed [rpm] by varying the pressure drop through different inner diameters (ID) or hollow fiber filter modules of varying lengths. Alternatively, the pump speed [rpm] can be increased while maintaining a constant flow rate due to the increased pressure drop.

[0101] exist Figure 5 In this study, the average size of the polymethyl methacrylate nanoparticle aggregates was measured and plotted against the radius of gyration (Rg) [µm] relative to the corresponding set rotational speed of the pump [rpm]. The pump used in this case was a power pump, i.e., a magnetically levitated centrifugal pump.

[0102] In detail, Figure 5 The relationship between the aggregation radius of gyration (Rg) [µm] and the centrifugal pump speed [rpm] is shown, wherein the device for controlling fluid resistance is selected from variable tubes with different inner diameters (ID), and the cell retention device is selected from two different hollow fiber filter modules: a long hollow fiber filter module (HF long) and a short hollow fiber filter module (HF short). Figure 5 The two black dashed lines in the figure represent the 95% prediction interval of the fitted graph. It is noteworthy that the aggregate size (Rg) decreases in the same manner as the pump speed increases, regardless of the piping or hollow fiber filter module used. This trend follows a power-law function, R... 2 It is 0.92, such as Figure 5 As shown. Figure 5 The fitted curve in the function y = a * x follows the function y = a * x b Where a = 5.91e+04, b = -1.34, R 2 = 0.92, adjusted R 2 = 0.92.

[0103] Since the aggregate size (Rg) can be attributed to the maximum hydrodynamic shear stress in the system (Villiger et al.) (11) The experiment showed that the highest shear stress in the system did not originate from the piping or the hollow fiber filter module (used in this setup), but rather depended largely on the pump speed [rpm].

[0104] Furthermore, the pump speed can be considered as the maximum shear stress occurring in the shear stress-sensitive device. This means that the characterization of the shear stress-sensitive device is based on the maximum shear stress generated by the pump speed. Moreover, the maximum shear stress serves as a guide to ensure that the maximum shear stress in the loop system of the shear stress-sensitive device is higher than the maximum shear stress in the bioreactor. In this case, the calibration methods of the prior art are guaranteed to produce correct results.

[0105] Shear stress parameters According to the present invention, the shear stress acting on cells is studied based on three parameters: maximum shear stress, shear stress frequency, and shear stress exposure duration. These shear stress parameters are also referred to herein as stress parameters.

[0106] Accordingly, in step (3) of the method according to the invention, at least one stress parameter is selected, and the effect of shear stress on the cells is studied based on this parameter. The effect of the shear stress parameter on the cells is determined by changing the selected shear stress parameter in multiple culture runs of a culture process that is always carried out in the same manner, so that, in particular, the change of the shear stress parameter affects the data available from the cell culture and thus provides knowledge about the shear stress load on the cells.

[0107] The variable shear stress parameters are selected from the following group, which consists of the following: - Maximum shear stress expressed as pump speed; - Duration of shear stress exposure, expressed as the residence time of cell cultures containing liquid culture medium and cells within the pump; and / or - The shear stress frequency, expressed as the number of times a cell culture containing liquid culture medium and cells passes through the loop system per unit time.

[0108] In the shear stress sensitive device of this invention, each shear stress parameter is achieved by changing specific physical parameters: Shear stress intensity represents the strength or magnitude of the total shear stress acting on a cell. Since—as already shown—shear stress intensity represents the combination or sum of all shear stress parameters.

[0109] According to the present invention, the maximum shear stress is controlled by using a pump, thereby setting different maximum shear stress values ​​acting on the cells by changing the pump's rotational speed. The unit of pump rotational speed is [rpm]. The higher the pump rotational speed is set, the higher the maximum shear stress acting on the cells. Therefore, the pump can operate at different rotational speeds [rpm], in which case the pump induces actual shear stress. The pump used is a power pump, such as a centrifugal pump, especially a magnetically levitated centrifugal pump. Other power pumps may also be used.

[0110] Therefore, the shear stress in the pump is determined by the rotational speed, which may correspond to a specific flow rate in the loop system. If only one selected stress parameter is changed in a shear stress-sensitive device, all other parameters should be kept constant as much as possible to determine the effect of only that one shear stress parameter. For example, if the pump rotational speed [rpm] is changed, a constant bypass flow rate is set in the loop system in each case. This is obtained through the pressure drop in the loop system in the form of fluid resistance, i.e., the device controlling the fluid resistance. This is, for example, a variable tube with variable length and / or diameter, the presence of one or more valves (e.g., pinch valves), and / or providing variable winding of the variable tube. It is convenient to measure the pressure drop by providing one or more pressure gauges in the shear stress-sensitive device.

[0111] Our experiments show that the pump's rotational speed or frequency corresponds to the maximum shear stress occurring in shear stress-sensitive equipment. Therefore, the maximum shear stress is an important shear stress parameter. Furthermore, it should be ensured that the maximum hydrodynamic shear stress is induced by the pump, and that it occurs in a closed loop rather than in the bioreactor, to obtain meaningful results.

[0112] Shear stress exposure duration is determined by the residence time of the cell culture within the pump. A longer residence time of the cell culture within the pump means a longer shear stress exposure duration. A shorter residence time of the cell culture within the pump means a shorter shear stress exposure duration. For example, a longer residence time of the cell culture within the pump can be achieved by simultaneously connecting multiple pumps in series in the circuit of a shear stress-sensitive device. This increases the residence time of the cells in the pump. In other words, exposure duration means how long the cells remain in the pump. In other words, the dead volume in the pump head actually corresponds to a specific residence time of the cells in the pump.

[0113] To better illustrate the duration of shear stress exposure or the residence time of cell cultures within the pump, refer to Figure 6 The figure shows a schematic simplified cross-sectional view of the main parts of one embodiment of a magnetically levitated centrifugal pump. The magnetically levitated centrifugal pump 130 shown consists of a single motor / bearing unit that provides both drive and magnetic levitation bearing functions. Essentially, the pump 130 comprises three parts: an impeller 133 containing rotating magnets (such as an overmolded magnetic ring 134), a bottom shell, and a top shell, which form the entire pump housing 131. The pump housing 131 is the outer shell of the entire pump head 132. The impeller 133 is suspended non-contactly within the pump housing 131 and is driven by the magnetic field of the motor (motor / bearing winding 135). The rotational speed of the impeller 130 can be precisely controlled electronically. The pump head 132 can be easily removed from the motor / bearing stator 136 and cleaned without difficulty. In some applications, the pump head 132 is a disposable unit.

[0114] Figure 6 The two arrows illustrate the path of the cell culture liquid containing liquid culture medium and cells through pump 130, with the inlet at arrow D entering pump 130 and the outlet at arrow E exiting pump 130. Thus, the cell culture containing liquid culture medium and cells is drawn in from the center by the created suction and discharged from the side. If cells are present in pump 130 along with the liquid culture medium, they are subjected to shear stress as long as they remain in pump 130. According to the invention, this is referred to as the residence time within the pump, i.e., the duration of shear stress exposure. For example, if two pumps are connected in series in a shear stress-sensitive device, the residence time of cells in the pump is longer, possibly doubling; if three pumps are connected, the residence time is even longer, possibly tripling.

[0115] According to the invention, a power pump, such as a magnetically levitated centrifugal pump, is used. However, any type of power pump can be used. It is particularly advantageous that the pump head is a disposable pump head, meaning the pump head can be easily replaced. For example, the pump head can be used only for one application (e.g., multiple runs of the same culture process) and then the pump head can be replaced. The advantage of doing so is that the measurement results have particularly good reproducibility.

[0116] Shear stress frequency is the frequency at which a cell is exposed to shear stress. According to the present invention, this is equivalent to... Figure 1A Or 1B, the number of times it passes through the external loop in a shear stress-sensitive device. Figure 1A In the diagram, the external loop of bioreactor 110 is represented by pipes 125a + 125b + 125c + 125d1 + 125d2. Figure 1B In this circuit, the loop is represented by tubes 125a + 125b + 125c + 125d1 + 125d2 + 125d3. Cell cultures containing liquid culture medium with cells are continuously recirculated within the loop, flowing from the bioreactor through a tube in the external loop, back into the bioreactor, then from the bioreactor again through a tube in the external loop, and so on. The shear stress frequency can be increased, for example, by providing additional loops through which cell cultures containing liquid culture medium and cells pass. Another possibility for adjusting the frequency is to adjust the bioreactor's packing volume (see Equations 1-4, explained in the section "Relationships between Shear Stress Parameters and the Effect of Bypass Flow").

[0117] In this context, it should be noted that the shear stress frequency is actually more like a distribution. For simplicity, ideal conditions for the shear stress frequency are assumed here, but this is acceptable for the method according to the invention.

[0118] According to the present invention, not only can a single stress parameter be changed and its effect tested in a shear stress-sensitive device, but multiple stress parameters can also be changed simultaneously and their effects studied. This will be explained in detail later.

[0119] The relationship between shear stress parameters and the influence of bypass flow rate In devices for controlling fluid resistance, besides increasing pipe length or decreasing diameter to increase pressure drop, valves (e.g., pinch valves) or other methods (e.g., coiled pipes) can also be used to increase pressure drop, provided that the maximum hydrodynamic shear stress is caused by the pump. Furthermore, according to one embodiment of the invention, it must be ensured that the maximum shear stress occurring in the closed-loop system is higher than the maximum shear stress occurring in the bioreactor. To better understand the parameter bypass flow rate (… V ̇ bypass [L / min], shear stress frequency ( freq. [1 / min] and shear stress exposure duration (dwell time / exposure duration) t res The relationship between [min] is given by the following equation: Equation 1 [1 / min] in V reactor The bioreactor's packing volume [L], and Equation 2 [min] in V pump This is the wet volume of the pump [L]. Combined equations 1 and 2 explain... freq. and t res The relationship between them, Equation 3 [1 / min] as well as Equation 4 [min].

[0120] Therefore, this invention is applicable to various process control strategies, including perfusion. It is worth noting that variations in shear stress parameters other than the maximum shear stress can also lead to meaningful results. Not only the maximum hydrodynamic stress, but also other shear stress parameters contribute to the performance of the culture process.

[0121] Methods for determining maximum hydrodynamic shear stress It is not possible to simply use a measuring device to measure hydrodynamic shear stress and then measure the level of shear stress that occurs. Therefore, in order to determine the shear stress sensitivity of living cells, methods known in the prior art but requiring further development to address this specific problem are used. These methods always determine the maximum shear stress, and thus this is also an element of the present invention.

[0122] In step (4), the shear stress sensitive device is characterized by using a calibration method or by using computer calculation and simulation.

[0123] The calibration method in step (4) is, for example, a known calibration method selected from a method, in which the value of a shear stress-dependent quantity is determined and correlated with a known maximum shear stress value. A shear stress-dependent (physical) quantity is a physical value or physical variable or physical parameter whose value or magnitude varies depending on the applied shear stress, and in this case, varies depending on the maximum applied shear stress.

[0124] In this invention, calibration is the process of assigning values ​​of shear stress-dependent (physical) quantities to the maximum shear stress value. As previously mentioned, directly measuring the maximum shear stress value is not possible. However, in order to implement the method according to the invention, it is necessary to determine the values ​​of shear stress-dependent (physical) quantities. The values ​​of shear stress-dependent (physical) quantities can, in principle, either be measured and determined in an experimental procedure by reproducing prior art processes, or these specified values ​​can be obtained directly from the prior art. The maximum shear stress value can be determined by computer calculation, or the maximum shear stress value can also be obtained directly from these indicated values ​​in the prior art.

[0125] This shear stress-dependent quantity is, for example, the aggregation size of shear-sensitive aggregates. These are, for example, shear-sensitive aggregates of polymethyl methacrylate (PMMA) nanoparticles (e.g., Villiger et al.). (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) When shear-sensitive aggregates of PMMA nanoparticles are subjected to defined shear stress conditions, the size of the PMMA nanoparticle aggregates varies with the shear stress; therefore, a specific aggregate size implies a specific shear stress value. The aggregate size and associated shear stress value are known from the prior art. This method is referred to herein as the PMMA method.

[0126] Other known shear stress-sensitive aggregates have been referred to as so-called Blauton polymer flocculation systems (see, for example, DE 202010018640 U1). (15) And Stintzing, A. et al.(16) The Blauton polymer flocculation system consists of a cationic polymer (Praestol BC 650) and clay minerals (Blauton). By adding the cationic polymer to the clay minerals, a flocculation reaction is initiated, producing flocs of a defined size. The measured rate of change in aggregate size is a measure of the shear stress present in the system.

[0127] Characterizing a shear-sensitive device is a process in which values ​​of shear stress-dependent (physical) quantities obtained from / in a shear stress device are assigned to a maximum shear stress value. The values ​​of the shear stress-dependent quantities are measured in the shear stress-sensitive device and converted to a maximum shear stress value using a selected calibration procedure. According to the calibration procedure, the maximum shear stress value is known when the shear stress-dependent quantity has a specific value. When certain (defined) conditions are set in the shear stress-sensitive device, the values ​​of the obtained shear stress-dependent quantities can be used to infer the maximum shear stress value reflecting those conditions.

[0128] The general method for determining the maximum shear stress value in the equipment under inspection can be performed as follows: Calibration method: For example, if the aggregate size of the shear-sensitive aggregate is chosen as the shear stress-dependent quantity, a known calibration method using a known calibration apparatus is selected, which utilizes the aggregate size and correlates it with a known maximum shear stress value. For instance, the known calibration apparatus described in the prior art is reconstructed, and the known calibration method using such an apparatus is re-performed as described in the prior art. That is, the selected known calibration method is re-performed, and the aggregate size of the aggregate is measured. Based on the measured aggregate size values, a calibration curve is generated, in which the measured aggregate size values ​​are plotted against a known maximum shear stress value taken from the prior art.

[0129] Characterization methods: In the characterization method, it is convenient to use the same shear stress dependence quantity as in the calibration method. Therefore, the shear-sensitive aggregates used in the calibration method are also used in the characterization method. Thus, the same known calibration method that has already been used is repeated, particularly using the same aggregates as in the calibration method, but using the equipment according to the invention instead of the calibration apparatus. The equipment used according to the invention is a shear-sensitive device.

[0130] In calibration and characterization methods, for example, the characteristic aggregate size is selected as the radius of gyration of the aggregate, and the aggregate is selected from polymethyl methacrylate nanoparticle aggregates or Blauton polymer flocculation systems.

[0131] According to step (4) of the invention, a shear stress sensitive device is used to characterize the maximum shear stress that occurs therein.

[0132] Computer calculations and simulations: Another method for determining the maximum hydrodynamic shear stress is through computer calculations and simulations. In this invention, this specifically relates to the characterization of the maximum hydrodynamic shear stress occurring in a pump. Numerical flow simulations, such as computational fluid dynamics (CFD), are widely used tools for characterizing process engineering equipment. (17) When used properly, numerical flow simulations can provide useful information inexpensively and quickly. These simulations involve solving mathematical equations describing the conservation of mass, momentum, and energy in fluids. The primary goal of numerical flow simulations is to obtain detailed information on the velocity, pressure, temperature, and other relevant properties of the fluid throughout the system. Furthermore, the use of numerical methods to characterize the fluid dynamics shear stress in pumps is widely documented in the literature. (18)(19)(20) To characterize the hydrodynamic shear stress in the pump, the following steps should be performed using commercially available software (such as, but not limited to, Ansys Fluent, STAR-CCM+, M-Star CFD, COMSOL Multiphysics, and OpenFOAM): 1. Create detailed 3D geometry including moving and static parts. 2. Select a numerical method for solving the governing equations. a. Finite volume method b. Lattice Boltzmann method c. etc... 3. Select a turbulence model a. Reynolds-averaged Navier-Stokes equations (RANS) b. Large Eddy Simulation (LES) c. Direct Numerical Simulation (DNS) 4. Conduct mesh / mesh studies to ensure that the numerical solution is independent of the mesh size and that discretization error is minimized. 5. Run shear stress characterization simulations for different operating parameters, for example: a. Flow rate b. Pump head rotation frequency c. Pressure drop d. Fluid properties (viscosity).

[0133] 6. Analyze the simulation results to characterize the hydrodynamic shear based on the operating parameters. This may include visualizing the flow field, calculating shear strain and strain rate, and identifying regions of maximum shear stress.

[0134] Fluid dynamic shear stress is determined through mathematical formulas and numerical methods. The simplest type of shear stress occurs in laminar pipe flow and can be described by the Hagen-Poiseuille equations. The radial velocity distribution of laminar pipe flow is as follows: Figure 7 As shown. Figure 7 Visualization of radial velocity distribution in a cylindrical laminar pipe flow. (21) . Figure 7 The legend is given separately at the end of the description.

[0135] In laminar pipe flow, the shear stress τ can be directly derived from the velocity distribution. Under the assumptions of steady-state, fully developed, incompressible, Newtonian fluid, and radially symmetric (2D) flow, the shear stress... It depends only on viscosity and radial velocity gradient.

[0136] T is the shear stress [Pa]. H viscosity [Pa s] du / dr radial velocity gradient [1 / s] For three-dimensional transient flows, shear stress can be calculated using the strain tensor S. The three-dimensional transient flow field can be determined experimentally using thomo-PIV, 4D PTV, or numerical methods.

[0137] (20) T is the shear stress [Pa]. Si,j is the strain tensor [1 / s]. H viscosity [Pa s] Exemplary calibration method exist Figure 8A and 8B The following section details exemplary calibration methods of the prior art and characterization methods (step 4) of shear stress-sensitive devices based on embodiments of the present invention.

[0138] The calibration method explained here is the so-called PMMA method, based on Villiger et al. (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) The publication. This prior art calibration procedure is schematically shown in... Figure 8A middle: like Figure 8A As shown in step a), polymethyl methacrylate (PMMA) nanoparticles with an average aggregate size of 60 nm ± 20%, particularly 60 nm ± 10%, are provided in an aqueous suspension, for example, in a 10 wt.% aqueous suspension. The nanoparticles are commercially available, or can be obtained as described by Šrom et al. (12) Page 2 2.1. Synthesis of primary nanoparticlesThe preparation described below. For example, the aggregate size can be measured using static light scattering (SLS), dynamic light scattering (DLS), focused beam reflectance measurement, or other measurement methods known in the art.

[0139] The aggregate size of PMMA agglomerates represents the (average) radius of gyration (Rg) as previously described. The (average) radius of gyration (also known as the scattering mass radius) is a known quantity used to characterize the spatial extent or size of irregularly shaped aggregates. Therefore, in the context of this invention, the "agglomeration size" of PMMA nanoparticle aggregates is always understood as the (average) radius of gyration (Rg).

[0140] from Figure 8A Step a) shows that at least 5 mL of monodisperse nanoparticle suspension was used. Larger amounts may also be used. This indication should be understood as an example only. The nanoparticle weight fraction of the suspension is 10% (w / w).

[0141] Then according to Figure 8A Step b) as Šrom et al. (12) Page 2 2.2. Preparation of aggregates and their breakage As described, polymethyl methacrylate (PMMA) nanoparticles were aggregated by mixing them with NaCl solution, resulting in a final NaCl concentration of 300 mM in the suspension. The system was stirred during the aggregation process. A 1% (w / w) PMMA nanoparticle aggregate suspension was obtained. If 5 mL of nanoparticles were used, approximately 50 mL of suspension would remain after adding NaCl solution.

[0142] Subsequently Figure 8A In step c), the suspension is diluted 200-fold with water, resulting in no re-aggregation of nanoparticles under these conditions; fragmentation becomes the sole mechanism controlling aggregate size. A 200-fold dilution from the initial 5 mL yields 10 L of diluted suspension. The steady-state aggregates are then transferred to the device to be inspected. In the prior art, calibration is performed in a single device connected to two syringe pumps. (14) The constriction nozzle continuously pumps the aggregate, such as Figure 8A Step d) is illustrated schematically. A given nozzle configuration is characterized in terms of shear stress using computational fluid dynamics (CFD) calculations. This means that for each nozzle or given conditions used, the maximum shear stress T... max The known value of [Pa] is calculated by computer. Specific prior art equipment, detailed measurement procedures, and theoretical calculations are available from the cited prior art.

[0143] exist Figure 8AIn step e), the fragmentation of the PMMA aggregate as a function of the maximum hydrodynamic shear stress in the device of step d) is schematically shown.

[0144] In step f), the aggregate size of the shear-sensitive polymethyl methacrylate nanoparticle aggregates obtained in step e) is measured, for example by static light scattering (SLS). Dynamic light scattering (DLS), focused beam reflectance measurement (FBRM), or another method may also be used for measurement. For example, a Mastersizer 2000 or 3000 (Malvern Instruments, UK) can be used as the SLS / DLS measuring instrument, which should produce the same measurement. For further details, refer to Villiger et al. (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) Aggregate size measurements, with particular reference to Šrom et al. (12) Page 2 and onwards 2.2. Preparation of aggregates and their breakage The following content.

[0145] exist Figure 8A In the middle, "-> Figure 9B "This indicates that the value obtained from the measurement performed in step f) is plotted on" Figure 9B The diagram shown is in step g).

[0146] Therefore, in step g), a calibration curve is generated, in which the maximum shear stress value, calculated by CFD (Computational Fluid Dynamics), in [Pa] is plotted against the measured aggregate size of PMMA agglomerates (in [µm] as radius of gyration (Rg)). The calibration curve of step g) is as follows: Figure 9B As shown. According to one embodiment, the prior art procedure was re-performed, i.e., the prior art equipment was rebuilt, and the value of PMMA agglomerates was measured in the rebuilt equipment, expressed as radius of gyration (Rg) in [µm]. Then, a calibration curve was generated using the maximum shear stress value [Pa] corresponding to the agglomeration size given in the prior art. The radius of gyration (Rg) value in [µm] measured in the rebuilt equipment and the calculated value of the maximum shear stress value Tmax (tau) in [Pa] taken from the prior art are listed in Table 1 below: Table 1:

[0147] The entire process and the equipment used were designed by Villiger et al. (11) Šrom et al. (12) Harshe & Lattuada (13)and Soos et al. (14) A detailed description is provided, and its publicly available content is incorporated herein by reference in its entirety.

[0148] Exemplary characterization methods The purpose of characterizing the shear stress-sensitive device (whose setup has been selected in step (2) of the method according to the invention) is to determine the maximum shear stress present in the shear stress-sensitive device. As determined experimentally, the source of the maximum shear stress in the shear stress-sensitive device of the present invention is the pump, as previously described. Therefore, the maximum shear stress, expressed as a function of the pump speed, is used to characterize the shear stress-sensitive device.

[0149] Characterizing a shear stress-sensitive device means quantifying the maximum shear stress that occurs. This is done using calibration methods known in the prior art.

[0150] As previously mentioned, if a shear sensitivity-dependent quantity is selected and used in the calibration method, then in particular, the same shear sensitivity-dependent quantity is used in the characterization of the shear stress-sensitive device. For example, if the aggregate size of a specific aggregate is used as the shear sensitivity-dependent quantity in the calibration method, then the same aggregate size is also used in the characterization. In particular, even the same batch of aggregates purchased or manufactured is used for both calibration and characterization methods. The aggregates are, for example, polymethyl methacrylate (PMMA) nanoparticle aggregates. In particular, the aggregate size is the radius of gyration.

[0151] The characterization of shear stress-sensitive devices can be performed according to one implementation scheme as follows: Using the same aggregates as in the calibration method, specifically polymethyl methacrylate (PMMA) nanoparticles, but instead of existing equipment, a shear stress-sensitive device is used to re-perform the calibration method known from the prior art. In the shear stress-sensitive device, the aggregates are continuously circulated through the device in a liquid medium (such as water) by a pump until the aggregate size no longer changes. The liquid medium is the same as that used in the calibration method. The pump speed is varied in each subsequent run, specifically by increasing the pump speed sequentially. After each run, the aggregate size, particularly the radius of gyration of the particle aggregates, is measured, and a curve is generated, plotting the measured aggregate size against the pump speed. Then, using the calibration curve based on the prior art, the measured aggregate size values ​​of the shear stress-sensitive device are converted to maximum shear stress values, so that a shear stress value can now be assigned to each pump speed of the shear stress-sensitive device. This represents the characterization of the shear stress-sensitive device.

[0152] The following example, using the PMMA method known in the prior art, explains this characterization in more detail. Another known calibration method can also be used. This characterization, based on the PMMA method, is schematically illustrated below. Figure 8Bmiddle.

[0153] Figure 8B Show steps a) to f) and Figure 8A The process is performed in exactly the same way, but in step d1), the shear stress sensitive device of the present invention is used. Figure 8B An implementation scheme (abbreviated as SSD) that replaces Figure 8A The existing nozzle device in step d). Figure 8A In the prior art equipment of step d), the use of a nozzle results in maximum shear stress, while according to the present invention... Figure 8B In the shear stress sensitive device of step d1), this is generated by the maximum shear stress, expressed as the pump speed. Incidentally, according to the present invention, a nozzle is not used in the shear stress sensitive device.

[0154] To characterize the shear stress-sensitive device, polymethyl methacrylate (PMMA) nanoparticle aggregates were continuously circulated in the loop system (and bioreactor) by a pump, thereby generating maximum shear stress in the shear stress-sensitive device. Different pump speeds resulted in different maximum shear stresses. The PMMA nanoparticle aggregates were exposed to maximum shear stress in the loop, and—as explained for the PMMA method—their aggregate size changed accordingly (step e).

[0155] In the illustrated embodiment, polymethyl methacrylate (PMMA) nanoparticle aggregates are placed in an aqueous suspension within a shear stress-sensitive device. The pump speed is set to a desired value, and the PMMA nanoparticle aggregates are continuously recirculated through the shear stress-sensitive device until the aggregate size no longer changes further. This is also referred to as "steady state" in the prior art.

[0156] For example, a specific pump speed is set, and the nanoparticle aggregates are circulated in the loop system until the agglomerate size no longer changes. Then, samples are taken and the agglomerate size is measured in step f). Then, for example, the pump speed is set to a higher value than before, and the aggregates still present in the shear stress-sensitive device are circulated again until the aggregate size no longer changes; samples are taken again and the aggregate size is measured. This process is repeated multiple times until a curve can be generated from the measured values.

[0157] According to one implementation, characterization begins with a low pump speed, which is then increased periodically, resulting in a gradual decrease in aggregate size. This simplifies the process, as the pump speed is simply increased, and the aggregate size is then measured after reaching a steady state.

[0158] exist Figure 8B In step f), the aggregate size (expressed as radius of gyration (Rg) of the shear-sensitive polymethyl methacrylate nanoparticle aggregates obtained in step e) is measured—as already determined. Figure 8A Interpretations—for example, through static light scattering (SLS), dynamic light scattering (DLS), focused beam reflection measurements, or another method.

[0159] For each pump speed set in the shear-sensitive device, the specific aggregate size of the shear-sensitive polymethyl methacrylate nanoparticle aggregates obtained in step e) is obtained, expressed as the radius of gyration (Rg).

[0160] exist Figure 8B In the middle, "-> Figure 9A "This indicates that the value obtained from the measurement performed in step f) is plotted on" Figure 9A The diagram shown (step g1) is shown.

[0161] exist Figure 8B In the middle, "-> Figure 9A + Figure 9B -> Figure 9C "Indicates use" Figure 9B (Step g) obtains the value or curve function from Figure 9A The value obtained in step g1 is converted to Figure 9C The value in step g2). That is, the symbol "+" represents Figure 9A and Figure 9B The combination produces Figure 9C .

[0162] Therefore, in Figure 8B In step g1), the measured aggregate size of the PMMA agglomerates (in the form of radius of gyration (Rg) in µm) is plotted against the set pump speed in rpm. The graph for step g1) is shown below. Figure 9A As shown.

[0163] In step g), a calibration curve has been obtained using existing calibration methods (see...). Figure 9B ).

[0164] In step g2), by combining step g1) ( Figure 9A ) and step g) Figure 9B )get Figure 9C The chart. That is, using existing technology to calibrate the curve (step g) by correlating the maximum shear stress value with the aggregate size: Figure 9B The PMMA nanoparticle aggregate size will be measured in a shear stress-sensitive device at a set pump speed (step g1): Figure 9A Convert to the maximum shear stress value (step g2): Figure 9C Therefore, the characterization of shear stress-sensitive devices, through the roundabout approach of the aggregation size of shear-sensitive aggregates, is used to determine the shear stress value corresponding to the adjusted pump speed.

[0165] According to one embodiment of the invention, if aggregate size is used as a shear stress-dependent quantity in the calibration and characterization methods in step (4), it is convenient to use the same batch of aggregates for both calibration and characterization. It can be assumed that the behavior of the aggregates may always differ slightly between different batches. Therefore, it makes sense to use the same batch of aggregates for both methods.

[0166] To better understand the basis Figure 8A Calibration and according to steps a) to g) Figure 8B The characterization of steps a) to g2) is explained in detail below using an exemplary embodiment of the present invention: In this exemplary embodiment, the maximum shear stress, expressed as the pump speed, is used as the source of maximum shear stress to characterize the shear stress-sensitive device for the maximum shear stress value that occurs. In this example, a magnetically levitated centrifugal pump, specifically the PuraLev i30 SU pump from Levitronix, Switzerland, is used. Other powered pumps may also be used. In this embodiment, a magnetically levitated centrifugal pump is used... Figure 1B The device is configured as a shear stress-sensitive device, based on a continuous mode such as irrigation mode. It can also be used... Figure 1A The equipment was used to study batch or fed-batch culture modes.

[0167] As previously described, the maximum shear stress value occurring within the shear stress sensitivity device was measured (see...). Figure 8B In this example, the PMMA method is used as a known calibration method. The generated PMMA nanoparticle aggregates (as described previously) Figure 8B Steps a) through c) are transferred to Figure 1B In shear stress sensitive devices (SSDs) Figure 8B Step d1) is used to characterize the PMMA aggregate suspension for the maximum shear stress. The PMMA aggregate suspension is recirculated in the loop system, and after a certain number of cycles, when the aggregate size no longer changes, samples are taken. This process is then repeated several times, each time increasing the pump speed (steps d1) and e). The resulting PMMA aggregate size is expressed as the radius of gyration (Rg), measured, for example, by static light scattering, dynamic light scattering, or another method. Figure 8B Step f). The obtained aggregate size measurement (Rg [µm]) is then processed in... Figure 9A Plot the values ​​relative to the corresponding set pump speed [rpm].

[0168] Figure 9A The results of the characterization of the shear stress sensitive device are shown: in the example experiment, the aggregate size ((mean) radius of gyration) Rg [µm] measured in the shear stress sensitive device is plotted against the rotational speed [rpm] of the magnetically levitated centrifugal pump used. Figure 9A The x-axis and y-axis values ​​are plotted on a logarithmic scale. Two black dashed lines represent the 95% prediction interval of the fitted graph. Figure 9A As shown, the curve follows a power-law function, R 2 The value is 0.98. The radius of gyration of the aggregate decreases with increasing pump speed [rpm]. The fitted curve follows the function y = a * x b Where a = 3.42e+04, b = -1.33, R 2 = 0.99, adjusted R 2 =0.98. Figure 9A Already Figure 8B The diagram (g1) is shown in a smaller format for illustrative purposes.

[0169] Figure 9B Calibration curves generated using calibration methods known in the prior art are shown. In this experiment, the PMMA method was used: calibration curves were generated using the values ​​given in Table 1, obtained by reproducing the process described in the prior art using equipment replicated from the prior art. (See Villiger et al.) (11) Šrom et al. (12) Harshe & Lattuada (13) and Soos et al. (14) ). Figure 9B The values ​​of the x-axis and y-axis are plotted on a logarithmic scale. Figure 9B The fitted curve in the function y = a * x follows the function y = a * x b Where a = 24.69, b = -2, R 2 = 0.98, adjusted R 2 = 0.96. Figure 9B Already Figure 8A and 8B As mentioned in step g).

[0170] like Figure 9B The calibration curve shown is generated according to existing technology and is used to... Figure 9A The aggregate size Rg [µm] measured at a given pump speed, as shown, is converted to a hydrodynamic shear stress value, such as... Figure 9C As shown. Therefore, Figure 9C The following is given: Figure 9A The final shear stress value [Pa] corresponding to the pump speed was derived from the measured data. Figure 9C The values ​​of the x-axis and y-axis are also plotted on a logarithmic scale. Figure 9C The two dashed lines in the diagram represent the 95% prediction interval of the fitted model. Figure 9C The fitted curve in the function y = a * x follows the function y = a * x bWhere a = 6.412e-07, b = 2.21, R 2 = 0.99, adjusted R 2 = 0.99. That is, a model was fitted, and the model produced R². 2 Power-law correlation = 0.99. R 2 This is the coefficient of determination, describing how well the measured values ​​fit the model function. The coefficient here is close to 1, indicating that the function describes the measured data very well. From... Figure 9C As can be seen, the maximum hydrodynamic shear stress increases with increasing pump speed, reaching 2 Pa at 1000 rpm and as high as 147 Pa at 6000 rpm. These values ​​are also listed in Table 2 below.

[0171] Table 2: Maximum shear stress of magnetically levitated centrifugal pump (PuraLev i30 SU centrifugal pump) at different speeds, determined by PMMA method.

[0172] Therefore, the shear stress measurement method of the present invention successfully converts the equipment process parameters used in shear stress sensitive devices into the maximum shear stress value in [Pa].

[0173] The maximum shear stress in a closed-loop system should be higher than that in a bioreactor. To obtain meaningful results in the inventive method, it is essential to ensure that the maximum shear stress occurring in the closed-loop system is higher than that occurring in a bioreactor (a shear stress-sensitive device without a closed-loop system). This is particularly relevant when performing calibration and characterization of shear-sensitive devices. This is appropriate because the maximum shear stress is typically the largest contributor to the shear stress intensity.

[0174] However, according to one implementation, it may be convenient to ensure that the shear stress intensity occurring in the closed-loop system is higher than that occurring in the bioreactor (a shear stress-sensitive device without a closed-loop system). This can be used without performing calibration and characterization on the shear stress-sensitive device. However, in short, it can also be assumed in this case that the maximum shear stress occurring in the closed-loop system is higher than that occurring in the bioreactor (a shear stress-sensitive device without a closed-loop system).

[0175] In any case, the maximum shear stress is used to characterize shear stress-sensitive devices and bioreactors, respectively, particularly by using calibration methods or by using computer calculations and simulations. This can be done in detail as described above.

[0176] Influence of fluid dynamic shear stress during cultivation To determine the effect of maximum hydrodynamic shear stress during culture, cell culture was performed. For this purpose, as described above, the cell type to be used, the composition and type of liquid culture medium, the culture conditions (e.g., the type and amount of liquid culture medium added) and the culture mode (batch, fed-batch, or continuous such as perfusion) were selected according to step (2).

[0177] Then—as described above—in step (3) select one, two, or three shear stress parameters to be studied. The shear stress parameters are selected from the following group: - Maximum shear stress expressed as pump speed; - Duration of shear stress exposure, expressed as the residence time of cell cultures within the pump; and / or - Shear stress frequency, expressed as the number of times the liquid culture medium and cells pass through the loop system.

[0178] The shear stress intensity is composed of these three shear stress parameters.

[0179] After selecting the shear stress parameter to be evaluated, the shear stress sensitive device used is characterized in (step 4) by measuring the maximum shear stress value, for example, by using a known calibration method, where the maximum shear stress is expressed by the rotational speed of the pump used. This has been described in detail.

[0180] In step (5), the cell culture process is performed multiple times, i.e., one, two, three, or more culture runs are conducted in a shear stress-sensitive device. Each run takes place within the same selected time period. One, two, or three shear stress parameters are changed between each run, and one or more performance characteristics of the cells are measured during each culture run. A curve showing the change of each performance characteristic over time is generated for each run. The culture process in step (5) is performed as follows, for example: For example, only one culture run, or only two culture runs, can be performed, where, for example, only one shear stress parameter is changed each time. Conclusions about changes in cell behavior / stress parameters can then be drawn from only said run.

[0181] In another implementation, a first culture run is performed, spanning a first time period. A second culture run is then performed, spanning a second time period. Subsequently, a third culture run is performed, spanning a third time period. Optionally, more culture runs are performed, spanning more time periods. The same culture run is performed throughout all runs—the first, second, third, and optionally more—using the same starting cell product, the same liquid culture medium, the same culture conditions, and the same culture mode selected in step (2). The time periods in all culture runs are chosen to be of equal length.

[0182] During each culture run, the liquid culture medium and cells in the shear stress-sensitive apparatus are continuously circulated through the loop system and the bioreactor, and all parameters of the shear stress-sensitive apparatus and all conditions of the culture process remain the same throughout all culture runs. Continuous circulation is provided by a power pump.

[0183] The term "circulation" refers to the circulation of a shear stress-sensitive device, i.e., from the bioreactor into the external loop system, back into the bioreactor, and then through the external loop system again, and so on. The term "recirculation" has the same meaning as "circulation," but for clarity and ease of understanding, the term "recirculation" is used in connection with the characterization of shear stress-sensitive devices, while the term "circulation" is used in connection with the circulation of liquid culture medium and cell-containing liquid during the culture process or operation.

[0184] During each culture run, one, two, or three shear stress parameters can be changed between runs. For example, only one of the three shear stress parameters can be changed between runs, or two or all three shear stress parameters can be changed between runs.

[0185] If only one of the shear stress parameters selected in step (3) is changed, it is set to a predetermined value and kept constant in the first culture run, and then set to a higher value for each subsequent culture run, with the value increasing between each culture run, while the set value of the shear stress parameter remains constant during each culture run. Alternatively, the shear stress parameter selected in step (3) is set to a predetermined value and kept constant in the first culture run, and then set to a lower value for each subsequent culture run, with the value decreasing between each culture run, while the set value of the shear stress parameter remains constant during each culture run.

[0186] If you need to change two or three shear stress parameters between each culture run, you can increase or decrease these parameters independently between each run. This depends on what you are studying.

[0187] Therefore, in step (5) of the present invention, the selected cells are cultured multiple times in a shear stress sensitive device while the entire cell culture containing the liquid medium and cells is continuously cycled for a selected period of time, wherein, for example, only the selected stress parameter is changed in each culture run of the culture process, and all other parameters—as far as possible—including the stress parameters that are not changed and all other conditions in the shear stress sensitive device remain unchanged.

[0188] Therefore, according to step (5), one, two, or three of the aforementioned shear stress parameters are selected and evaluated for the operation of the culture process. The effect of this one, two, or three parameters on the selected cells is then examined for the culture process. Other unselected shear stress parameters remain constant during the culture process. All other culture conditions and parameters selected before the start of the cell culture process also remain constant during the culture process. Only when all conditions and parameters remain constant during the culture process can changes in one, two, or three shear stress parameters be examined and meaningful results be obtained. However, in this context, it should be noted that although bypass flow rate is not a shear stress parameter but another parameter, it has a special status because it is related to other shear stress parameters. Therefore, to change the shear stress parameters, all other parameters except bypass flow rate may remain constant, but bypass flow rate may be increased or decreased accordingly. This will be explained in detail later.

[0189] Therefore, a "culture run" means a culture process conducted over a selected time period (e.g., several days) using selected cells, cell culture conditions, and parameters, while continuously circulating liquid culture medium and cell-containing liquid culture medium within a shear stress-sensitive device. It is convenient if all culture runs are performed within the same selected time period.

[0190] The term "continuous circulation" means the continuous movement of returning to the bioreactor through a loop system and repeating these processes. These two terms should be understood as synonyms and are interchangeable.

[0191] The time period selected in step (5) depends on many factors, such as the type of cells selected, batch size, etc. Those skilled in the art can readily determine this in each case. For example, the time period could be several days. Exemplarily, the time span selected for the time progression of one of the culture methods can be, in particular, at least one day or at most 30 days or even longer.

[0192] Therefore, the same culture process as the first culture run is repeated multiple times in subsequent culture runs, thereby continuously circulating the liquid culture medium and cell-containing culture liquid in the shear stress-sensitive device via a pump. In one embodiment, only the selected shear stress parameter is changed in each case. These parameters(s) can be increased or decreased. In another embodiment, the selected shear stress parameter and the bypass flow rate are changed. Needless to say, a new starting cell stock, a new liquid culture medium, etc., are used in each new culture run. The cells and liquid culture medium used in the first culture run cannot be reused. This would distort the analysis and conclusions drawn from the process.

[0193] Therefore, in the first culture run, the conditions selected in the shear stress-sensitive device are maintained, where one or more initial values ​​are set for the selected stress parameters. For example, only one shear stress parameter is changed, and the selected shear stress parameter is the maximum shear stress corresponding to the pump speed [rpm]. Then, for example, in the first culture run of the culture process, the speed is set to 2000 rpm and culture is performed, for example, for 5 days. In the second culture run, for example, the speed is set to 3000 rpm and culture is performed again for 5 days. Other conditions and parameters are the same as those selected for the first culture run. In the third culture run, the speed is then set, for example, to 4000 rpm, and all other conditions and parameters of the culture process are the same as the culture process of the first culture run. The culture process is again performed for 5 days as in the first culture run. In the fourth culture run, the speed is then set, for example, to 5000 rpm, and the culture process is performed again for 5 days, and in the fifth culture run, the speed is then set, for example, to 6000 rpm, and the culture process is performed again for 5 days.

[0194] The number of repetitions or runs of the culture process can be determined by those skilled in the art in each specific case. It may be appropriate to perform at least two, three, four, or five culture runs.

[0195] Therefore, while performing the culture process according to step (5) in one or more runs, samples are taken from the cell culture and its performance characteristics are measured in each run. Multiple samples can be taken during each culture run of the cell culture, typically at regular intervals, such as once a day over several days. For example, at least 5, 6, 7, or more samples are taken in each culture run. In each culture run, the same performance characteristics are then measured, particularly periodically, especially at the same time interval as the first culture run. If multiple performance characteristics are to be measured, these characteristics are typically measured in all culture runs. The values ​​of the performance characteristics can be measured as online or offline data. That is, the data are measured inside the shear stress sensitive device, or samples are taken and examined and measured outside the shear stress sensitive device. These characteristic parameters of cell culture are, for example, the growth and productivity of the cell culture, particularly the relative viable cell density, viability, glucose concentration, lactate concentration, LDH concentration, titer (product concentration), product quality, etc.

[0196] As an example, the relative viable cell density is chosen as a performance characteristic of the cell culture. For instance, a 10-day time period is selected, and the relative viable cell density in the sample is measured daily: In the first culture run, the culture process is then carried out for 10 days in a shear stress-sensitive device, where, for example, the pump speed is chosen as the shear stress parameter, and all other parameters and conditions are selected to be the same. For example, in the first culture run, the pump speed is set to 5000 rpm. During the 10 days, the relative viable cell density is measured periodically, for example, daily. In the second culture run, the culture process is then carried out again for 10 days in a shear stress-sensitive device, where the pump speed is set, for example, to 6000 rpm. During the 10 days, the relative viable cell density is again measured periodically, for example, daily. In the third culture run, the culture process is then carried out again for 10 days in a shear stress-sensitive device, where the pump speed is set, for example, to 7000 rpm. During the 10 days, the relative viable cell density is again measured periodically, for example, daily.

[0197] According to step (5), curves are generated for one or more performance characteristics of the cells measured during each run of the culture process. That is, the measured performance characteristic values ​​are plotted for each culture run over a selected time period. Each curve represents a set value of the shear stress parameter selected for that culture run, which, as previously mentioned, can be converted into shear stress intensity. The obtained data shows the performance of the selected cells under the main stress conditions. Therefore, a set of curves is obtained for all culture runs.

[0198] For example, the pump speed is chosen as the shear stress parameter, which should be 3000 rpm in the first culture run, 4000 rpm in the second culture run, and 6000 rpm in the third culture run. The product titer of the cell culture is selected as the performance characteristic, and three culture runs are performed, each lasting 5 days. Cell samples are taken and examined daily during each run. Therefore, for each culture run, five product titer values ​​are obtained for each of the three culture runs, thus generating three curves. Plotting the product titer value against the number of days on each curve records the time progression of the performance characteristic. The first curve comes from the first culture run, where the pump speed is set to 3000 rpm. Using the PMMA method, the shear stress value measured on the shear stress-sensitive device for this pump speed is 39 Pa (see Table 2). For the second culture run, a pump speed of 4000 rpm results in a measured shear stress value of 55 Pa, and for the third culture run, a pump speed of 6000 rpm results in a measured shear stress value of 147 Pa. In other words, the cells were subjected to a maximum shear stress of 39 Pa in the first culture run, 55 Pa in the second, and 147 Pa in the third. The three curves obtained are considered as a set of curves for all culture runs. Therefore, this set of curves represents the cell behavior based on performance characteristics (here: product titer) as the shear stress intensity increases in a shear stress-sensitive device.

[0199] use Figure 9C The model function obtained can also be used to calculate the above values. Therefore, the model function allows for the verification of the reasonableness of the measurement data through calculation: using the model function, the following results are obtained: 3000 rpm -> 32 Pa, 4000 rpm -> 60 Pa, 6000 rpm -> 146 Pa. As mentioned earlier, the coefficient of determination R... 2 = 0.99, describing the goodness of fit between the measured values ​​and the model function. Since the coefficient here is close to 1, the function describes the measured data very well. Therefore, the calculated data are relatively close to the measured values, thus validating the measurement results through the model function, which supports the reliability of the results.

[0200] It should be noted that in the above example, an alternative existing calibration method could be used instead of the PMMA method. Alternatively, computer calculations or simulations could be used to determine the shear stress value.

[0201] To assess the effect of maximum shear stress, a control must be present where the shear stress is low, negligible, or only negligible. Therefore, in step (6), a control culture process is performed on each performance characteristic in a shear stress-sensitive device using the same culture process as in step (5) (i.e., the same cells, the same liquid culture medium, the same culture mode, and the same shear stress-sensitive device). During the control culture, a lower maximum shear stress than in step (5) is used, and control curves are generated for each performance characteristic over time. The control culture process is performed, for example, in the same manner as the first culture run of the culture process in step (5). The same cells, the same liquid culture medium, the same culture mode, the same culture process conditions, and the same shear stress-sensitive device are used, and the duration is the same as in step (5).

[0202] In the control culture process to be selected, it is necessary to distinguish between batch and fed-batch processes and continuous processes (e.g., perfusion). For example, for batch or fed-batch processes, the control culture process is conducted in a way that the shear stress sensitive device has no loop system and no pump. That is, the shear stress provided by the loop system and pump is absent. Therefore, such a device used for the control culture process is not actually a shear sensitive device, but simply a simple bioreactor. All parameters in the device and all conditions of the culture process are kept the same throughout the control culture process, and the same cell culture performance characteristics as in step (5) are measured at selected time periods, and the measured cell culture performance characteristic values ​​are plotted on the selected time periods to obtain a control curve for each performance characteristic examined.

[0203] In continuous culture processes (e.g., perfusion), the shear stress parameter that causes maximum shear stress can be set to a lower value than that used in step (5), for example, to the smallest possible value. In the context of this invention, the shear stress parameter that contributes most to the shear stress intensity is the maximum shear stress expressed by the pump speed. In other words, the pump speed is set lower than in step (5), for example, as low as possible, to meet this requirement. Ideally, the control culture process is performed at the minimum shear stress level of the perfusion default operation.

[0204] The curve set from step (5) is compared with the control curve for a certain performance characteristic in step (6), resulting in the selection of the curve from the curve set in step (5) that is closest to the control curve (step (7)). Those skilled in the art will understand that this depends on the operating conditions under which the curves are measured. Therefore, in step (7), the curve that is closest to the control curve is selected, so that its operating conditions best match those of the control curve. The shear stress value measured at the selected curve represents the cell's shear stress sensitivity and represents the shear stress limit of the cultured cells. If curves and control curves have been generated for multiple performance characteristics of the cell culture, the shear stress limit is usually the same or nearly the same value for all selected performance characteristics. If the values ​​are not equal, an average value is taken.

[0205] For example, if the shear stress limit is determined to be 60 Pa, meaning that shear stress values ​​above 60 Pa cause a significant deterioration in cell culture performance, this can be correlated with the shear stress parameters used, such as the specific rotational speed [rpm] of the pump used. For example, this might be 4000 rpm for the pump used. That is, this reference value can be used for culture processes performed in the same manner on a larger scale, allowing one to know in advance the conditions suitable for the culture process.

[0206] Therefore, by converting the process parameters of the equipment used in shear stress-sensitive devices into shear stress values ​​in Pa, the shear stress sensitivity or shear stress limit of the cells can be determined or estimated. This shear stress limit of the cells allows for very precise assessment of cell behavior under culture conditions. This allows for both scale-up and scale-down of the culture process. Shear stress can be selectively adjusted, allowing for back-calculation from a larger system to a smaller system (scale-up) or from a smaller system to a larger system (scale-up). For example, large-scale systems could be larger pumps, larger bioreactors, or other equipment.

[0207] Alternatively, in step (7), instead of determining the cell's shear stress sensitivity or shear stress limit, the shear stress behavior of the cell can be analyzed using curves of one or more performance characteristics obtained, particularly by comparison with control curves. This is evident from the fact that each curve represents a defined set of operating conditions. The shear stress behavior of the cell directly affects the performance characteristics, and therefore the obtained curves directly reflect how the cell responds to a given shear stress intensity, and in each case, to the applied level of shear stress intensity.

[0208] In particular, this enables reliable prediction of how optimally the culture process should be conducted. Studying the sensitivity of cells to shear stress intensity at small scale allows for accurate prediction of potential changes in process performance and product quality during scale-up or downsizing, and / or thus enables clone selection based on shear stress at small or large scales during cell line development. This allows for selection at a very early stage of which clones are suitable for production purposes and what mechanical stress resistance they exhibit. This is a screening procedure, for example, for distinguishing between high-yield and low-yield clones.

[0209] Operating modes of shear stress sensitive equipment As previously mentioned, one, two, or three shear stress parameters can be changed simultaneously in a shear stress-sensitive device while keeping all other parameters as constant as possible. According to another embodiment, one, two, or three shear stress parameters can also be changed simultaneously in a shear stress-sensitive device while keeping all other culture parameters constant. Shear stress-sensitive devices and the numerous modifiable shear stress parameters provide great variability and flexibility for various process control strategies. Examples are provided below to illustrate this.

[0210] Shear stress-sensitive devices used for cell culture can be operated in different ways, i.e., in different modes. Each mode maintains a different constant shear stress parameter, demonstrating the wide applicability of shear stress-sensitive devices. By changing the various shear stress parameters, the shear stress intensity also changes.

[0211] For example, one or more shear stress parameters can be varied as follows: - By keeping the pump speed at the same value, the maximum shear stress (in terms of speed) is kept constant; - Increase or decrease the maximum shear stress (in terms of speed) by setting the pump speed higher or lower. - If the pump speed is increased or decreased by using a device that controls fluid resistance (e.g., by changing the length and / or diameter of a variable tube in a shear stress-sensitive device loop system), the duration of shear stress exposure is kept constant. - If the pump speed is kept constant by adding an additional pump with the same speed [rpm] in series in the loop system, the duration of shear stress exposure is increased; - If the pump speed is kept constant by using a device that controls fluid resistance (e.g., by changing the length and / or diameter of a variable tube in a shear stress-sensitive device loop system), the duration of shear stress exposure can be increased or decreased. - If the pump speed is increased or decreased by using a device that controls fluid resistance (e.g., by changing the length and / or diameter of a variable tube in a shear stress-sensitive device loop system), the shear stress frequency is kept constant. - If the pump speed is kept constant by adding one or more external loops to the bioreactor or by adjusting the working volume of the bioreactor, the shear stress frequency is increased; or - If the pump speed is kept constant by providing one or more additional pumps in parallel, and all pumps are running at the same speed, the shear stress frequency is increased.

[0212] An important parameter is the bypass flow rate, which, according to the present invention, is not a shear stress parameter. It is related to the shear stress parameter, so understanding how it affects the bypass flow rate is useful. For example, when the maximum shear stress, expressed as by the pump speed, increases (assuming constant fluid resistance), the bypass flow rate increases. To change the bypass flow rate or keep it constant, a device for controlling fluid resistance can be used. The following gives some correlations between bypass flow rate and other shear stress parameters, as well as with devices for controlling fluid resistance. For devices for controlling fluid resistance, a variable pipe with variable length and / or variable inner diameter is used as an exemplary implementation. Other devices for controlling fluid resistance are possible and are known to those skilled in the art.

[0213] Possible measures or parameters for changing bypass flow include the following: - Maintain a constant bypass flow rate by keeping the pump speed constant; - If, while increasing the pump speed, the bypass flow rate is kept constant by increasing the length of the variable pipe in the shear stress-sensitive equipment loop system and / or decreasing its diameter; - If the bypass flow rate is kept constant while the pump speed is reduced, the length of the variable tube in the shear stress sensitive equipment loop system is reduced and / or its diameter is increased; - Increase or decrease bypass flow by increasing or decreasing pump speed; - If the pump speed is constant, the bypass flow can be increased by reducing the length of the variable pipe in the shear stress sensitive equipment loop system and / or increasing its diameter; - If the pump speed is constant, the bypass flow can be reduced by increasing the length of the variable pipe in the shear stress sensitive equipment loop system and / or reducing its diameter.

[0214] Shear stress sensitive devices can be operated in a variety of ways. A summary of different exemplary operating modes of shear stress sensitive devices is provided below: For example, one of the following cases, namely cases 1 to 4, can be selected, and in each case, one or more shear stress parameters can be changed, thereby also changing the shear stress intensity: Scenario 1: Maintain a constant shear stress exposure duration and frequency throughout all culture runs, while increasing the maximum shear stress from one culture run to the next. or Case 2: Maintain the maximum shear stress and shear stress frequency constant throughout all culture runs, while increasing the duration of shear stress exposure from one culture run to the next. or Case 3: Keep the maximum shear stress and the duration of shear stress exposure constant throughout all culture runs, while increasing the frequency of shear stress from one culture run to the next. or Case 4: Maintain a constant maximum shear stress throughout all culture runs, while increasing the duration of shear stress exposure from one culture run to the next and decreasing the shear stress frequency.

[0215] Cases 1 through 3 are of particular interest because only one selected parameter is changed between each culture run, either increasing or decreasing between runs, while other shear stress parameters remain constant across all runs.

[0216] Case 4 is an example where two parameters are changed simultaneously (one parameter increases, the other decreases). Of course, there are many more possibilities for changing the shear stress parameters; for example, all three shear stress parameters could be changed simultaneously. The specific changes in shear stress parameters during the culture run depend on what is being studied.

[0217] Detailed explanation: Scenario 1: In Case 1, the maximum shear stress, expressed by the pump speed in the shear stress-sensitive device, is selected and studied as the shear stress parameter. The maximum shear stress in Case 1 increases from one culture run to the next, meaning each culture run is conducted at a higher pump speed than the previous run.

[0218] The maximum shear stress, as represented by the pump speed in a shear stress-sensitive device, is increased by setting the pump speed to a higher level.

[0219] Meanwhile, the duration and frequency of shear stress exposure were kept constant during each culture run.

[0220] One possibility for achieving this is to keep the bypass flow rate constant. If the bypass flow rate is kept constant, the duration of shear stress exposure and the frequency of shear stress are also equal (refer to Equations 1-4, which have already been explained in this regard). The bypass flow rate, representing the flow through the loop system, is kept constant, for example, by keeping the pump speed constant. However, if the pump speed is increased as in case 1, the bypass flow rate will also increase. Therefore, the bypass flow rate can be kept constant, for example, by using devices that control fluid resistance, particularly by increasing the length of the variable pipe in the shear stress-sensitive device loop system and / or decreasing its diameter, through the resulting pressure drop.

[0221] Therefore, in case 1, the effect of the maximum shear stress can be studied while other shear stress parameters remain constant.

[0222] Scenario 2: For example, by adding an additional pump with the same rotational speed [rpm] in series, the duration of shear stress exposure can be increased while keeping the pump speed constant.

[0223] By keeping the pump speed at the same value, the maximum shear stress is kept constant.

[0224] Maintaining the bypass flow rate, and therefore the shear stress frequency, can be done as described in Case 1.

[0225] Therefore, in Case 2, the effect of the duration of shear stress exposure can be studied while other shear stress parameters remain constant.

[0226] Scenario 3: For example, the shear stress frequency can be increased by providing a second or multiple additional loops, each with a pump having the same rotational speed. The shear stress frequency can also be controlled by adjusting the working volume of the bioreactor. There is always some flexibility regarding the operable volume of a bioreactor when operating it. As can be seen from Equations 1-4 explained below, the bioreactor volume has a corresponding effect on the shear stress frequency, and therefore it can be influenced accordingly.

[0227] Alternatively, the shear stress frequency can be increased by connecting two or more pumps operating at the same speed in parallel within the loop system. This allows for an increase in the total bypass flow, thereby increasing the frequency, while the bypass flow of each pump remains similar.

[0228] The maximum shear stress can be kept constant as described in Case 2.

[0229] Therefore, in case 3, the effect of the shear stress frequency can be studied while keeping other shear stress parameters constant.

[0230] Scenario 4: By using devices that control fluid resistance, particularly by increasing the pressure drop while keeping the pump speed constant (and thus the maximum shear stress constant), the length of variable piping in the loop system of shear stress-sensitive equipment can be increased and / or its diameter reduced, thereby reducing the pressure drop.

[0231] The reduced bypass flow rate leads to a decrease in shear stress frequency, while the duration of shear stress exposure increases.

[0232] Therefore, in Case 4, the effects of both shear stress exposure duration and shear stress frequency can be studied simultaneously, while the maximum shear stress remains constant. This is because the two shear stress parameters always behave in opposite ways: increasing the bypass flow rate increases the shear stress frequency and decreases the shear stress exposure duration, and vice versa. This allows us to determine which of the two parameters (shear stress exposure duration or shear stress frequency) has a greater impact.

[0233] An overview of the different exemplary operating modes of shear stress-sensitive devices according to conditions 1 to 4 is provided below. Figures 10A to 10E .

[0234] It is understood that cases 1 to 4 given are merely illustrative, and there are of course other modes of operating shear stress sensitive devices.

[0235] Explained below Figures 10A to 10E The figures exemplify how the settings of a shear stress-sensitive device can be changed from left to right to alter one or more shear stress parameters. These figures only show possible implementations and are not intended to limit the invention to these embodiments. Although bypass flow rate is not a shear stress parameter, it is also indicated in the figures for ease of understanding.

[0236] Figure 10A Case 1 is shown, illustrating three different settings of the shear stress sensitive device, with the pump speed increasing from left to right. Consequently, the maximum shear stress applied to the cell culture increases. As an exemplary device for controlling fluid resistance, a tube of variable length and / or variable diameter with two end reducers is shown, along with different winding configurations of the variable tube with two end reducers. The device for controlling fluid resistance causes a pressure drop, thereby maintaining a constant bypass flow rate, shear stress frequency, and duration of shear stress exposure of the cells (residence time within the pump). One or more valves can also be used to generate the pressure drop.

[0237] Figure 10AThe illustration shows that three different shear stress sensitivity settings can be used to adjust the stress parameters accordingly—as needed. Of course, more settings can be provided to achieve the desired variations in shear stress parameters. These three different settings are for illustrative purposes only. Each of the three settings should be considered separately from the others. The order of the three settings in Case 1 illustrates, for example, how to modify the shear stress sensitivity device if, to increase the pump speed while keeping other shear stress parameters constant. When culturing cells, for example from... Figure 10A Starting with the left-hand setting (Case 1), a low maximum shear stress is obtained in the form of a low pump speed. To increase the pump speed, for example, to a medium pump speed, then switch to... Figure 10A The intermediate equipment setup, in order to obtain high pump speed, switches to... Figure 10A The device settings on the right. Figure 10A Of all three settings, the other shear stress parameters are set to constant, i.e., modified by the shear stress device settings, as shown in the figure for constant shear stress frequency and constant shear stress exposure duration.

[0238] Figure 10B Case 2 is shown, where adding an additional pump with the same rotational speed [rpm] in series, instead of just one pump, can increase the shear stress exposure duration (residence time within the pump). This modification... Figure 10B The setup is shown from left to right. It should also be noted that when the pumps are connected in series, it is assumed that the cells are exposed to stress for a longer period due to the "instantaneous" series connection. In reality, the cells leave the pumps, thus leaving the site of shear stress, but this only happens for a very short time, with almost no volume between pumps 1 and 2; therefore, this assumption is acceptable for the method of the present invention. In the exemplary setup on the right side of the shear stress-sensitive device, two pumps are connected in series in the loop system. If pumps with the same rotational speed [rpm] are connected in series, the bypass flow rate will also increase. However, similar to case 1, a device for controlling fluid resistance is used in the loop system, which is introduced to cause a pressure drop, thereby reducing the bypass flow rate, as shown in the setup on the right side of the shear stress-sensitive device. Thus, the maximum applied shear stress, bypass flow rate, and shear stress frequency can be kept constant while studying the effect of the duration (length) of shear stress exposure.

[0239] Figure 10C Case 3A is shown, in which, from left to right, the maximum shear stress (rotation speed [rpm]), bypass flow rate, and shear stress exposure duration (cell residence time in the pump) are kept constant, while the shear stress frequency can be increased by a second loop or multiple additional loops. The shear stress frequency can also be controlled by adjusting the working volume of the bioreactor.

[0240] Figure 10DCase 3B is shown, in which, from left to right, the maximum shear stress (rotation speed [rpm]), bypass flow rate (for each pump), and shear stress exposure duration (the time the cell resides in the pump) remain constant, while the shear stress frequency can be increased by providing additional pumps in parallel and running them at the same rotational speed. As an example, two pumps are shown in parallel in the loop system. The bypass flow rate of the two pumps in total is approximately twice that of one pump, thus doubling the shear stress frequency. However, the bypass flow rate of each pump is the same, so the shear stress exposure duration remains constant.

[0241] Figure 10E Case 4 is shown, where multiple stress parameters are changed simultaneously. In the settings from left to right, by keeping the [rpm] constant, the maximum shear stress (pump speed [rpm]) remains constant, while the shear stress exposure duration (the residence time of the cells in the pump) increases. Conversely, in the settings from left to right, the bypass flow rate and shear stress frequency decrease due to pressure drops caused by devices controlling fluid resistance (e.g., through longer or smaller diameter pipes).

[0242] according to Figure 10E (Case 4) and Equations 1 to 2, bypass flow affects shear stress frequency ( freq. ) and shear stress exposure duration (dwell time / exposure duration) t res Bypass flow rate was positively correlated with shear stress frequency and negatively correlated with shear stress exposure duration. Since cultures with low bypass flow rates exhibited the lowest growth performance, while cultures with high bypass flow rates exhibited the highest performance, it can be concluded from the experiments that shear stress exposure duration has a stronger impact on performance than shear stress frequency. Overall, it was found that high shear stress exposure duration is weighted more heavily than low shear stress frequency. Figure 10B , 10C The parameters, along with the settings described in 10D (cases 2, 3A, and 3B), can also be studied independently.

[0243] although Figures 10A to 10E Only continuous modes such as perfusion mode are illustrated, but it should be noted that this setting is shown only as an example. However, the same applies to modes such as... Figure 1A The batch or replenishment batch settings are shown.

[0244] Embodiment of the present invention omitting step (4) (calibration and characterization method) One embodiment of the present invention relates to a method for determining the shear stress sensitivity of cells, wherein the characterization of the shear stress sensitivity device according to step (4) is omitted. In short, a culture process is selected, and a control process (reference) with a low or as low as possible shear stress level is selected. The control process is then reproduced in the shear stress sensitivity device by changing the shear stress parameter in the direction of the culture process, thereby deriving relevant information from the changes in the shear stress parameter. That is, only the relative shear stress levels are compared; the absolute values ​​need not be determined.

[0245] Therefore, the method according to this embodiment relates to a method for determining the shear stress sensitivity of cells in a shear stress-sensitive device during a culture process comprising a liquid culture medium, particularly for determining the effect of shear stress on cells, comprising the following steps: (1) A shear stress sensitive device is provided, comprising - Bioreactor, - A closed-loop system connected to and located outside the bioreactor; - A pump placed in a loop system, which is a power pump, and - A device for controlling fluid resistance in a control loop system; (2') Perform a culture process or use a culture process that has been performed, and provide one or more culture process curves in which one or more performance characteristics of the cells are plotted over selected time periods; (3) Select one, two, or three shear stress parameters to be studied from the following groups: - Maximum shear stress expressed as pump speed; - Duration of shear stress exposure, expressed as the residence time of cell cultures within the pump; and / or - Shear stress frequency, expressed as the number of times a cell culture containing liquid culture medium and cells passes through a loop system per unit time. Where shear stress intensity represents the combination of all three shear stress parameters; (4') Perform a control culture process or use a control culture process that has been performed, and provide one or more culture process curves of the control culture process, wherein one or more performance characteristics of the cells are plotted over a selected time period, wherein the control culture process is the same as the culture process in step (2'), but uses a lower shear stress intensity and uses the same cell performance characteristics and the same time period as in step (2'); (5') In a shear stress sensitive device, one, two, three or more control culture runs of the control culture process of step (4') are performed within the same selected time period. In each subsequent control culture run, one, two or three shear stress parameters are added or removed sequentially. Each subsequent control culture run is modified by changing the shear stress parameters in the direction of the selected culture process of step (2'). One or more performance characteristics of the cells are measured during each run of the control culture process, and a curve of the change over time is generated for each performance characteristic in each run. (6') Select the curve from the set of curves in step (5') that is closest to the performance characteristic for a certain performance characteristic; (7') By ​​comparing the obtained curves, determine how the shear stress intensity needs to be changed to switch from the control culture process to the culture process; This ensures that the shear stress intensity during the culture process is higher than that during the control culture process.

[0246] Therefore, according to this embodiment, the method includes or consists of steps (1), (2'), (3), (4'), (5'), (6'), and (7'). According to one embodiment, these steps are performed in the indicated order. According to another embodiment, no intermediate steps are performed between these steps.

[0247] According to this implementation method, step (1) is performed as described above.

[0248] Step (2) is replaced by step (2'), in which a culture process is performed or a previously performed culture process is used, and one or more culture process curves are provided, wherein one or more performance characteristics of the cells are plotted over selected time periods. Unlike step (2), not only are the cells, liquid culture medium, culture mode, and culture conditions for the culture process to be performed selected, but also the culture process itself that has been performed or will be performed in step (2') is selected. Then, either a culture process curve (where one or more performance characteristics of the cells are plotted over selected time periods) is selected from known data materials, or these curves are measured and generated during the culture process performed in step (2').

[0249] Step (3) is performed as described above. Step (4) is omitted, i.e., the calibration and characterization of the shear stress sensitive device are not performed, nor are they required.

[0250] According to this embodiment, step (4') can be performed in the same manner as step (6) already described, wherein a control culture process is performed. This control culture process is performed in the same manner as the culture process in step (2'), but using a lower shear stress intensity, for example, by using the lowest possible maximum shear stress through the use of the lowest possible pump speed. It can be performed as described in step (6). The control culture process can be performed in a shear stress-sensitive device, but this is not absolutely necessary, as it is only necessary to ensure that its shear stress intensity is lower than that of the selected culture process.

[0251] If a shear stress-sensitive device is used and the culture process is batch or fed-batch, for example, a lower maximum shear stress can be achieved using a modified shear stress-sensitive device (a loopless system without pumps). If a shear stress-sensitive device is used and the culture process is continuous, particularly perfusion, the shear stress-sensitive device should be used in a way that adjusts the shear stress intensity to be lower than during the culture process, especially as low as possible.

[0252] According to this embodiment, step (5') is performed in the same manner as step (5) already described, but the control culture process of step (4') is performed in a shear stress-sensitive device and modified by changing the shear stress parameter in the direction of the culture process of step (2'). Therefore, in the shear stress-sensitive device, one, two, three, or more control culture runs of step (4') are performed within the same selected time period as steps (2') and (4'), with one, two, or three shear stress parameters being added or removed sequentially in each subsequent control culture run. Each subsequent control culture run is modified by changing the shear stress parameter in the direction of the selected culture process of step (2'), and one or more cell performance characteristics are measured during each run of the control culture process. Based on the measurements, a curve is plotted for each performance characteristic and each run over time. Therefore, curves are again generated for the measured performance characteristics over time, but the shear stress parameter is changed during the run so that the curve obtained in the control run approximates the curve of the culture process.

[0253] Therefore, according to another embodiment of the invention, there is a method in which the shear stress sensitive device is not calibrated and characterized, i.e., the maximum shear stress source is not determined, nor is the shear stress value appearing in the shear stress sensitive device quantified.

[0254] Therefore, the process involves systematically performing a control culture process by conducting one, two, three, or more control culture runs in a shear stress-sensitive device. In each subsequent control culture run, one, two, or three shear stress parameters are changed, and one or more cell performance characteristics are measured during each run, generating a curve for each performance characteristic over time. In this process, the shear stress parameters are changed in each of the one, two, three, or more control culture runs so that the obtained curve approximates the curve of the selected culture process (to obtain more information about that process).

[0255] In step (6') (replacing step (6) already described), the curve that best approximates the culture process curve for a certain performance characteristic is selected from the set of curves in step (5').

[0256] In step (7') (replacing step (7) already described), the shear stress intensity is then determined by comparing the obtained curves to determine how it needs to be changed from the control culture process to the culture process. By comparing the curves, approximate conditions simulating the selected culture process are obtained. In other words, the control culture process is thus calibrated to the selected culture process as a quasi-reference operation. That is, no absolute shear stress value is required, only variations in equipment settings attributable to these values, such as pump speed.

[0257] The culture process in step (2') can be arbitrarily selected and can be any type of culture process known to those skilled in the art.

[0258] Furthermore, it is ensured that the shear stress intensity occurring during the culture process is higher than that occurring during the control culture process. Typically, the culture process in step (2') has a higher shear stress intensity than the control culture process in step (4'), which is carried out at a lower shear stress intensity. Only in very rare special cases is this not the case or impossible. However, in such cases, this implementation cannot be carried out. Therefore, in this implementation, it is ensured that the shear stress intensity occurring during the culture process is higher than that occurring during the control culture process.

[0259] It goes without saying that, in order to obtain meaningful results, the selection conditions and parameters of the culture process in step (2') and the control culture process in step (4') should be as similar as possible. For example, the culture type, such as batch, fed-batch, or continuous (perfusion), should be the same; the type of organism to be cultured should be the same from a variety of organisms (e.g., eukaryotic cells); and the various performance characteristics that change over time should be the same, etc.

[0260] This implementation can also be used to study one or more modifications to the culture process compared to a control culture process. For example, this implementation can be used as a scale-down model: the culture process is then carried out on a large scale, while a control culture process is carried out on a smaller scale. The small-scale control culture process is then modified until it operates similarly to the large-scale culture process, thereby obtaining information about the shear stress level.

[0261] For example, this implementation can also be used as a scale-up model: the cultivation process is then carried out on a small scale, while a control cultivation process is carried out on a larger scale. The large-scale control cultivation process is then modified until it operates similarly to the small-scale cultivation process, thereby obtaining information about the shear stress level.

[0262] To better understand this embodiment of the invention, an exemplary embodiment is explained below by way of example. In this exemplary embodiment, the cultivation process is a production-scale cultivation process, while the control cultivation process is the same cultivation process, but carried out on a small scale.

[0263] Typically, processes with similar input parameters exhibit lower performance at production scale compared to small-scale or laboratory scale. (22),(23) For example, although processes are scaled up according to general principles of bioprocess engineering, the live cell density and titer in production-scale bioreactors are lower than at small scales. One parameter that typically does not remain constant during scale-up is hydrodynamic shear stress, which can explain performance differences between scales.

[0264] Therefore, simulating the shear stress of a bioreactor on a small scale allows for comparable process performance during scale-up.

[0265] exist Figure 11A and 11B Example curves of the culture process using CHO cell lines in a production-scale bioreactor (curve P) and a small-scale bioreactor (curve S) are shown, respectively, in terms of two performance characteristics, namely viable cell density (VCD)

[10] . 6 A plot of [cells / mL] and titer [g / L] versus time [h] shows that the small-scale bioreactor provides better performance than the production-scale bioreactor. To investigate shear stress as a possible cause of poor performance in the production-scale bioreactor, the method according to the invention was used. The process of the production-scale bioreactor was used as the culture process. The process of the small-scale bioreactor was used as the control culture process, which was identical to the selected culture process but modified only in scale.

[0266] To study the maximum hydrodynamic shear stress, a shear stress-sensitive device, for example, according to... Figure 10AOperate as described in Case 1. For this purpose, perform one, two, three or more runs of the culturing process identical to the control culturing process by changing one shear stress parameter, i.e., the maximum shear stress, between each culturing run. It should be noted, however, that the method is not limited to this exemplary embodiment, and it is understood that other variations of the shear stress parameter are possible.

[0267] In this exemplary embodiment, parallel control culturing runs can be performed under the same conditions but with an increased pump speed (Note: The same procedure was performed in Experiment 1 described in "Experimental Section I", but the shear stress sensitivity device was calibrated and characterized according to step (4); see Figures 13A-13F . The curves obtained with different pump speeds are shown in Figure 12A and 12B .

[0268] In this example, adjust the pump speed such that rpm A < rpm B < rpm C. Finally, compare curves A, B, and C of the resulting performance characteristics (in this example: VCD and titer) with the production-scale curve P. If the curves of the performance characteristics exhibit similar profiles over time, it can be concluded that the maximum shear stress conditions achieved with the shear stress sensitivity device match the conditions of the production-scale bioreactor. In this example, curve B matches curve P.

[0269] Therefore, the shear stress sensitivity device allows the performance of, for example, a production-scale culturing process to be achieved by simulating the maximum shear stress conditions. The advantage of this method is that it is not necessary to determine the maximum shear stress itself, nor is it necessary to perform complex calibration or characterization of the shear stress sensitivity device. By systematically increasing the shear stress intensity using a shear stress parameter (e.g., by the pump speed, as shown in Figure 10A Case 1), the shear stress conditions can be matched to those of, for example, a production-scale bioreactor simply by comparing the profiles or curves of the performance characteristics over time.

[0270] This way of simulating stress conditions is thus very widely applicable and is not limited to the conditions given in this exemplary embodiment.

[0271] The present invention also relates to a process for culturing cells in a liquid medium, wherein the shear stress sensitivity measured according to the method of the present invention is applied to the same culturing process, but at a scale larger or smaller than the scale at which the shear stress sensitivity was determined.

[0272] The present invention also relates to a process for culturing eukaryotic or prokaryotic cells in a liquid medium in a bioreactor, and performing in this culturing process the method according to the present invention for determining the cell shear stress sensitivity in a shear stress sensitivity device during the culturing process.

[0273] This invention also relates to a process for producing a recombinant protein, the process comprising the following steps: Step I) Culture eukaryotic or prokaryotic cells expressing the recombinant protein in liquid culture medium in a bioreactor; Step II) Harvest the recombinant protein; Step III) Purify the recombinant protein; In step (I), the method according to the invention for determining the shear stress sensitivity of cells in a shear stress sensitivity device during culture is performed.

[0274] The present invention also relates to the use of a device comprising... - Bioreactor, - A closed-loop system connected to and located outside the bioreactor; - A pump placed in a loop system, which is a power pump, and - A device for controlling fluid resistance in a control loop system; As a shear stress sensitivity measuring device or shear stress sensitivity instrument This device has no nozzle. Used to culture cells in liquid culture medium and to set a specific shear stress during the culture process.

[0275] The determined shear stress set during the cultivation process can be the maximum shear stress value obtained by changing the shear stress parameters in the device. The maximum shear stress value is preferably determined based on a shear stress-dependent physical quantity, particularly the aggregation size of the shear-sensitive aggregate. Preferably, the maximum shear stress value is determined based on the shear stress-dependent physical quantity as follows: the shear stress-dependent physical quantity is exposed to determined, preset conditions, whereby the physical quantity varies according to the magnitude of the maximum shear stress. Determined, preset conditions refer to conditions under which the maximum shear stress values ​​are known from existing calibration procedures. The shear stress-dependent physical quantity is exposed to unknown conditions in the device, whereby the physical quantity varies according to the magnitude of the maximum shear stress. Unknown conditions in the device refer to conditions where the maximum shear stress value in the device is unknown, but can be determined through existing calibration procedures, thereby determining the maximum shear stress value appearing in the device (characterization procedure). Then, the magnitude of the maximum shear stress value in the device can be inferred from the physical quantity obtained under the determined, preset conditions. The same magnitude of the physical quantity implies the existence of the same maximum shear stress value.

[0276] According to another embodiment, the determined shear stress can be a relative shear stress level derived from two runs of the culture process with different shear stress levels, one run serving as a control run, and the control run being repeated and modified by changing the shear stress parameters until its operation is similar to the other run. In this case, the calibration and characterization of the equipment are omitted; this procedure has also been explained in detail.

[0277] It is understood that the explanation of the method of the present invention should also apply to the use of the device, and vice versa.

[0278] Further experiments showed that shear stress sensitivity also applies to different cells, for example, different cell lines expressing completely different recombinant proteins. The results showed that each cell line had its own sensitivity to similar levels of shear stress. Any living organism grown in a suspension bioreactor can be studied for its shear stress sensitivity. In addition to the maximum hydrodynamic shear stress, this invention also allows for the independent study of parameters such as shear stress exposure duration and shear stress frequency. Figures 10A-10E These parameters have been shown to affect culture performance and are essentially relevant during the scaling up or scaling down of biological processes.

[0279] This invention has many different advantages: This invention provides, for the first time, a detailed examination and understanding of the effects of shear stress. Shear stress is measured directly during the culture process, where the total shear stress can be determined as a value of shear stress intensity by combining all shear stress parameters in the form of maximum shear stress, shear stress exposure duration, and shear stress frequency. This means that, for the first time, shear stress is fully defined and its components (maximum shear stress, shear stress exposure duration, and shear stress frequency) are described, where each parameter can be set individually and contributes to the total shear stress. Therefore, one, two, or three shear stress parameters can be changed simultaneously in a shear stress-sensitive device while keeping all other culture parameters constant. The shear stress-sensitive device and the modifiable different shear stress parameters provide great variability and flexibility for various process control strategies.

[0280] It has been found that the shear stress induced by aeration or agitation in conventional bioreactors, even if it contributes, is negligible to the total shear stress and therefore has a negligible impact on cell culture performance. In particular, shear stress never reaches levels >10 Pa, regardless of the conditions applied. A shear stress value of 10 Pa is generally too low in the context of biotechnology equipment, even for larger scales, especially when using recirculation pumps during perfusion. Therefore, agitation and aeration exist only as background shear stress and play almost no role in determining the shear stress sensitivity of living organisms. Thus, the actual shear stress parameter, rather than aeration or agitation, is the decisive factor in the culture system.

[0281] The method according to the invention is highly variable not only in terms of shear stress parameters but also in terms of the culture method under investigation. Any cell culture method, any cell, any liquid culture medium, any culture mode, and any culture conditions can be used with the method of the invention. The only limitation on the culture process is that it must be carried out in a liquid medium so that the cell culture can be circulated in a shear stress-sensitive device. In fact, any living organism grown in a suspension bioreactor can be examined for its sensitivity to shear stress. Experiments have shown that sensitivity to shear stress can also be applied to different cells, such as different cell lines expressing completely different recombinant proteins. It has also been found that each cell line has its own sensitivity to similar shear stress values.

[0282] The method of the present invention can also be carried out in various process operations (batch, feed-batch, perfusion) without affecting other parameters in the bioreactor.

[0283] Because the shear stress applied to the cells is quantified through calibration, the results are independent of the specific shear stress-sensitive device used (e.g., a separately inserted pump) and instead reflect the conditions of the corresponding complete culture system.

[0284] The method according to the invention also allows the device used in the shear-sensitive device to be reflected in the shear stress intensity. Each internal surface, tool, device, etc., which is part of the shear-sensitive device, interacts with the set process conditions to obtain the overall shear stress value acting on the cell. In other words, there is an interaction between all the individual features of the shear-sensitive device (e.g., the setup of the shear-sensitive device) and the set conditions, resulting in a synergistic overall effect that far exceeds isolated individual effects (e.g., measuring shear stress on the device wall or on the cell).

[0285] By converting the process conditions / equipment used in shear stress-sensitive devices into shear stress values ​​in Pa, the shear stress sensitivity or shear stress limit of cells can be determined or estimated. This allows for very precise assessment of cell behavior under culture conditions. This allows for both scale-up and scale-down of the culture process. Shear stress can be selectively adjusted, enabling backcomputation from a larger system to a smaller system (scale-up) or from a smaller system to a larger system (scale-up).

[0286] Cellular shear stress behavior directly affects performance characteristics; therefore, the curves obtained by the method of this invention directly reflect how cells respond to a given shear stress intensity and their response to the applied shear stress intensity level under each condition.

[0287] The shear stress-sensitive devices designed and used in the context of this invention have the advantage of approximating the culture methods of the target cell cultures. For example, the bioreactor used for culture can be configured in such a way that the shear stress sensitivity of the corresponding cells can be measured directly under the culture conditions.

[0288] Therefore, shear stress-sensitive devices can deeply analyze the relationship between cell behavior and hydrodynamic shear stress, thereby accurately inferring the applied shear stress level and providing a predictive basis for other culture processes at the same, larger, or smaller scales using similar or different hardware components. In particular, this enables reliable prediction of how to optimally conduct the culture process. Studying the sensitivity of cells to shear stress intensity at small scales can accurately predict potential changes in process performance and product quality during scale-up or downsizing, and / or thus enable clone selection based on shear stress at small or large scales during cell line development. This allows for the selection of which clones are suitable for production purposes and what shear stress resistance they exhibit at a very early stage. This is a screening procedure, for example, how to distinguish between high-yield and low-yield clones.

[0289] According to another embodiment of the invention, complex characterization procedures, including calibration of the shear stress-sensitive device, can be omitted. That is, only relative shear stress levels are compared, and absolute values ​​need not be determined. In short, a culture process is selected, and a control process (reference) with a low or as low as possible shear stress level is selected. The control process is then reproduced in the shear stress-sensitive device by changing the shear stress parameter in the direction of the culture process, thereby deriving relevant information from the changes in the shear stress parameter.

[0290] This embodiment of the invention can also be used, for example, to investigate shear stress as a possible cause of poor performance in production-scale bioreactors. For instance, this embodiment can be used as a scale-down model: the small-scale control culture process is then modified until its operation is similar to the large-scale culture process, thereby obtaining information about the shear stress level. This embodiment can also be used as a scale-up model: the large-scale control culture process is then modified until its operation is similar to the small-scale culture process, thereby obtaining information about the shear stress level.

[0291] Therefore, this invention provides surprising insights and knowledge about cell behavior and cell fluid dynamics shear stress, enabling accurate inference of the level of applied shear stress and aiding in the prediction of other culture processes of the same, larger or smaller scale.

[0292] Experimental Section I Experiment 1 Effects of fluid dynamics shear stress during fed-batch culture according to Figure 1A The shear stress-sensitive apparatus setup shown was used to study a defined shear stress level in a fed-batch culture. In Experiment 1, the effect of hydrodynamic shear stress on CHO fed-batch cultures was investigated. The cell line used was CHO cells producing recombinant proteins (cell line A). The shear stress parameter selected and studied was the maximum shear stress (expressed as pump speed).

[0293] The same fed-batch process setup (the same shear stress-sensitive apparatus with identical culture conditions) was applied for all culture runs and tests. All parameters and conditions were kept constant throughout all culture runs. The glucose addition threshold was <3 g / L. Cells consumed glucose, and the consumption was recorded accordingly. Detailed information on the culture process can be found in Experimental Section II.

[0294] Only the selected shear stress parameter varied between different culture runs; that is, the pump speed remained constant during each culture run but increased in subsequent culture runs. The pump was a magnetically levitated centrifugal pump (in Experiment 1: PuraLev i30SU, Levitronix, Switzerland). As previously described, the cell culture was continuously circulated in a shear stress-sensitive device.

[0295] The measured performance characteristics of cell cultures over time are as follows: Figures 13A to 13F As shown. That is, Figures 13A to 13F Offline data for fed-batch CHO cell cultures exposed to different levels of hydrodynamic shear stress are presented. Offline data were obtained by sampling within a shear stress-sensitive device and measuring outside the device. See Experimental Section II for details.

[0296] Figures 13A to 13F The control group represents the standard feeding batch setting, with no bypass loop and no pump, so it is assumed that the shear stress value is negligible.

[0297] Figure 14 A and 14B show online data for the shear stress settings, i.e., values ​​measured within the shear stress apparatus itself during the cultivation process. Specifically, Figure 14 A and 14B show online data for the varying rotational speed [rpm] of the magnetically levitated centrifugal pump as previously described. Figure 14 A) and the constant bypass flow rate [L / min] measured online during fed-batch culture ( Figure 14 B). The corresponding settings for shear stress-sensitive devices are as follows: Figure 1A As shown, and according to Figure 10A The scenario shown is Scenario 1, but without the hollow fiber filter module.

[0298] Increase the shear stress level by increasing the rotational speed [rpm] of the magnetic levitation centrifugal pump. Figure 14 A). As previously stated (see step (4) of the method according to the present invention), see Figure 8A and 8B ,as well as Figure 9A , 9B and 9C), through Figure 9C The equation given in the figure calculates the rotational speed [rpm] into a shear stress value (Pa). Online signal measurements are very frequent (every few seconds), therefore displayed as a line. To allow for symbol indication outside the line, every 800th data point is represented by a symbol. The shear stress value calculated using the equation given in Figure 9C is as follows (see Figure 9C). Figure 14 A): Table 3:

[0299] In detail, Figure 14 A describes online data for fed-batch cultures with elevated shear stress levels. To maintain a constant shear stress level during culture, a magnetically levitated centrifugal pump was operated at a set speed setpoint that remained constant during each culture run. Figure 14 A). To maintain a bypass flow rate of ≈0.5 L / min, the pipe length was adjusted according to the rotational speed, such as... Figure 10A And three exemplary settings of the shear stress-sensitive device (pump speed increasing from left to right) are shown. However, due to the increase in relative viable cell density (VCD) and the resulting increase in viscosity, the bypass flow rate tends to decrease slightly during culture. Figure 14B). The feed batch process is characterized by an increase in volume over time. Therefore, the shear stress frequency varies with time (Equation 1). Since the feed batch process (and all its culture runs) remains the same, the change in fill volume over time is also the same, and the observed effect can be entirely attributed to the different levels of hydrodynamic shear stress.

[0300] The performance characteristics of cell culture operation over time are as follows: Figures 13A to 13F As shown. Therefore, six culture runs were performed, namely in Figure 1A In a shear stress-sensitive apparatus, under identical conditions (feed-batch) and using the same parameters, six culture cycles were performed on selected cells (here: CHO cells): one control run and five test runs. Each run was performed over a 14-day period. For each run, the time progression of the culture's performance characteristics was measured as offline data.

[0301] In Experiment 1, the characteristic parameter was the relative viable cell density (VCD). Figure 13A ), survival rate ( Figure 13B Lactate dehydrogenase (LDH) activity in the supernatant Figure 13C ), glucose concentration ( Figure 13D ), lactic acid concentration ( Figure 13E ) and relative titer (product concentration) Figure 13F ).

[0302] For each performance characteristic, its value was determined as offline data over time. For each run of the cultivation process, a curve was plotted for each performance characteristic over time, and the value of the performance characteristic was plotted against time and the corresponding shear stress value.

[0303] In addition, the control run used the same culture process, the same cells, the same liquid culture medium, the same culture mode, the same culture process conditions, and the same shear stress-sensitive device as before, but without the loop system and pump, and the control culture process was carried out within the same time period. Due to the lack of a pump and loop system, the shear stress-sensitive device in the control process in this example case is now simply a bioreactor. For the control run, control curves were also generated for the same performance characteristics as before, but the cell cultures were not subjected to shear stress due to the lack of a pump and loop system. Of course, in the control culture process, the same performance characteristics of the cell cultures were measured within the same selected time period as culture runs 1 to 5.

[0304] right Figures 13A to 13F The analysis yielded the following results: During the exponential growth phase, under conditions of increased shear stress, the relative viable cell density (VCD) decreases (see [reference]). Figure 13AFor the highest stress condition (174 Pa), the peak viable cell density (VCD) was lower, while the final viable cell density (VCD) on day 14 was comparable. The survival curves highlight that survival was lower under higher stress conditions until day 8, after which survival shifted to higher levels compared to lower stress conditions starting from day 9. Figure 13B This can be explained by the mechanism of survival determination via trypan blue staining. This method can detect dead cells but not lysed cells. Under higher shear stress rates, dead cells may be sheared at the end of the culture, meaning they cannot be measured by trypan blue staining (CEDEXHiRes, Roche Diagnostics), thus increasing survival. Lactate dehydrogenase (LDH) activity increases under higher stress conditions until day 10, followed by a transition (…). Figure 13C This shift may be because LDH activity is more strongly driven by total cell density (TCD) as the culture nears its end, while TCD is higher under lower shear stress conditions (TCD = VCD / survival rate). Figure 13D This indicates that glucose concentration increases under higher stress conditions. This is due to the negative impact of shear stress on growth. As growth slows, lactic acid accumulation decreases, and lactic acid levels decrease more rapidly under lower shear stress conditions after lactic acid metabolism shifts from production to consumption. Figure 13E ).at last, Figure 13F The relative titers shown also decreased under higher stress conditions (118, 137, and 174 Pa). Product quality measurements showed no significant differences (data not shown).

[0305] Based on these results, it can be concluded that this CHO cell line is resistant to shear stress levels up to ≈64 Pa during fed-batch culture. Overall, the curve at 64 Pa is closest to the control curve. Conditions ≥ 118 Pa showed reduced growth and titer. This indicates that the shear stress limit of this cell culture is approximately 64 Pa.

[0306] Experiment 2 Effects of fluid dynamic shear stress during perfusion culture According to Experiment 2, following Figure 1B The shear stress-sensitive apparatus setup shown was used to study a defined shear stress level in continuous mode during perfusion. In Experiment 2, the effect of hydrodynamic shear stress on CHO perfused cultures was investigated. The cell line used was CHO cells producing recombinant proteins (cell line A), the same cell line described for fed-batch culture. The glucose addition threshold was <3 g / L. The shear stress parameter selected and studied was the maximum shear stress (expressed as pump speed).

[0307] The same perfusion process setup (the same shear stress-sensitive apparatus with identical conditions) was applied for all culture runs and tests. All parameters and conditions were kept constant throughout all culture runs. Detailed information on the culture process can be found in Experimental Section II.

[0308] Only the selected shear stress parameter varied between different culture runs; that is, the pump speed remained constant during each culture run but increased in subsequent culture runs. The pump was a magnetically levitated centrifugal pump as described in Experiment 1. As previously mentioned, the cell culture was continuously circulated in the shear stress-sensitive device. The shear stress-sensitive device also included, as... Figure 1B The hollow fiber filter module shown.

[0309] The performance characteristics of cell cultures over time are as follows: Figures 15A to 15D As shown. Figures 15A to 15D Offline data of CHO cell perfused cultures subjected to different hydrodynamic shear stresses are presented. The same perfusion process settings were applied for all tested shear stress conditions.

[0310] Figures 15A to 15D The control represents a standard perfusion setup, where the pump, loop system, and hollow fiber module are still present, but the pump speed provides lower shear stress than during culture operation. Ideally, the control represents the minimum shear stress level for default perfusion operation. The bypass flow rate for feedback control is 0.5 L / min.

[0311] Figure 16 A and 16B show online data for the shear stress settings, i.e., values ​​measured within the shear stress-sensitive device itself during cultivation. These are the varying rotational speeds [rpm] of the magnetically levitated centrifugal pump (here: i30SU type pump). Figure 16 A) and the constant bypass flow rate [L / min] measured online during perfusion culture ( Figure 16 B). The corresponding settings for shear stress-sensitive devices are as follows: Figure 1B As shown, and according to Figure 10A The operation shown is Case 1, which is with the hollow fiber filter module shown.

[0312] Increase the shear stress level by increasing the rotational speed [rpm] of the magnetic levitation centrifugal pump. Figure 16 A). As previously stated (see step (4) of the method according to the present invention), see Figure 8A and 8B ,as well as Figure 9A , 9B and 9C), according to Figure 9CThe equation shown calculates the adjusted rotational speed [rpm] as a shear stress value (Pa). Online signal measurements are very frequent (every few seconds), therefore displayed as a line. To allow for symbol indication outside the line, every 350th data point is represented by a symbol. Figure 9C The shear stress values ​​calculated by the equations shown are as follows (see Figure 16 A): Table 4:

[0313] In detail, Figure 16 A and 16B show online data for shear stress settings during perfusion culture, with varying pump speed [rpm] and constant bypass flow. The speed of the magnetic levitation centrifugal pump (here: i30SU type pump) is as follows: Figure 16 As shown in Figure A, the bypass flow measured online is as follows: Figure 16 As shown in B.

[0314] Figure 16 A and 16B describe online data for perfusion culture with elevated shear stress levels. In addition to control conditions, elevated shear stress levels were achieved by increasing the rotational speed [rpm] while adjusting the tubing to maintain a bypass flow rate of ≈0.5 L / min. Figure 16 A and 16B). Complementary batch culture ( Figure 14 B) Similarly, due to the increase in relative viable cell density (VCD) and the resulting increase in viscosity, the bypass flow rate tends to decrease slightly during culture. Figure 16 B).

[0315] The performance characteristics of cell culture operation over time are as follows: Figures 15A to 15D As shown. Therefore, six culture runs, one control run, and five culture runs were performed, i.e., in Figure 1B In a shear stress-sensitive apparatus, under identical conditions (perfusion) and using the same parameters, five culture cycles were performed on selected cells (here: CHO cells). Each culture run lasted for a selected period of six days. For each culture run, the time progression of the selected culture performance characteristics was obtained as offline data. In Experiment 2, the performance characteristics were relative viable cell density (VCD) and viability (…). Figure 15A Lactate dehydrogenase (LDH) activity in the supernatant Figure 15B ), glucose concentration ( Figure 15C ) and lactic acid concentration ( Figure 15D Each item was measured as offline data over time. For each culture run, a curve was plotted for each performance characteristic over time, plotting the measured values ​​of the performance parameters against time and the corresponding shear stress values. This was achieved by increasing the rotational speed [rpm] of the magnetic levitation centrifugal pump (see...). Figure 16A) The conditions for increasing the level of shear stress have been achieved.

[0316] In addition, the control run used the same culture process, the same cells, the same liquid culture medium, the same culture mode, and the same culture conditions as before, and conducted the control culture process within the same time period, but the minimum possible shear stress level of the perfusion mode was set by the pump speed. For this control culture process, control curves were also generated for the same measured performance characteristics. Figures 15A to 15D As previously mentioned, standard perfusion settings were used, with the flow rate controlled at 0.5 L / min. Of course, during the control culture, the same performance characteristics of the cell cultures were measured within the same selected time periods as culture runs 1 through 5 in Experiment 2.

[0317] right Figures 15A to 15D The analysis yielded the following results: Under the highest stress conditions (174 Pa), the relative viable cell density (VCD) and survival rate decreased. Figure 15A Due to slower growth, glucose consumption decreases, lactic acid production decreases, while LDH activity increases. Figure 15B , 15C and 15D). Figures 15A-15D The offline samples shown also indicated that growth was slowed, glucose consumption and lactic acid production were reduced under 137 Pa conditions. Perfusion culture performance at shear stress levels up to 100 Pa was comparable to the control. The slightly increased LDH activity between day 1 and day 5 under 100 Pa conditions suggests that LDH activity in the supernatant is a sensitive indicator of increased stress levels. Figure 15B The sudden increase in LDH activity between day 5 and day 6 under control, 100 Pa, and 137 Pa conditions may be explained by improper sample processing or measurement error.

[0318] Based on the above data, it can be concluded that during the initial perfusion culture stage, growth performance is similar for cells with stress levels ≤100 Pa. In other words, the shear stress limit of this cell culture is approximately 100 Pa.

[0319] Experiment 3 Effects of fluid dynamic shear stress during perfusion culture In Experiment 3, the effect of constant and high (174 Pa) hydrodynamic shear stress with varying bypass flow rate was investigated in CHO perfused cultures. The same perfusion process setup was applied for all tested bypass flow rate conditions. [The text then abruptly shifts to a different topic:] ...using... Figure 1BThe shear stress sensitive device. The cell line used in this experiment is also CHO cells that produce recombinant proteins (cell line A). The glucose addition threshold is <3g / L. This is an implementation scheme that changes two shear stress parameters: the shear stress sensitive device is used according to situation 4 ( Figure 10E In Case 4, two shear stress parameters are changed between each culture run: the duration of shear stress exposure is increased, and the frequency of shear stress is decreased, while the maximum shear stress is kept constant.

[0320] The same perfusion process setup (the same shear stress-sensitive apparatus with identical conditions) was applied for all culture runs and tests. All parameters and conditions were kept constant throughout all culture runs. Detailed information about the culture process can be found in Experimental Section II.

[0321] As previously described, the cell culture is continuously circulated in a shear stress-sensitive device. The shear stress-sensitive device also includes, for example... Figure 1B The hollow fiber filter module shown.

[0322] The results of characteristic parameters of cell culture changes over time are as follows: Figures 17A to 17D As shown. Figures 17A to 17D Offline data of CHO cell perfused cultures subjected to different hydrodynamic shear stresses are presented. The same perfusion process settings were applied for all tested shear stress conditions.

[0323] No control culture process was performed because only individual correlations between different shear stress parameters were being investigated. Cultures with moderate bypass flow at the setpoint were used for orientation, as this experiment had already been conducted in this manner once (see [link]). Figures 15A to 15D And 16A and 16B).

[0324] Figure 18 A and 18B show online data for the shear stress settings, i.e., values ​​measured within the shear stress apparatus itself during cultivation. These are the constant rotational speeds [rpm] of the magnetic levitation centrifugal pump (here: i30SU type pump). Figure 18 A) and the changed bypass flow rate [L / min] measured online during perfusion culture ( Figure 18 B). The corresponding settings for shear stress-sensitive devices are as follows: Figure 1B As shown, and according to Figure 10E The scenario shown is operation 4, which involves the hollow fiber filter module shown. Online signal measurements are very frequent (every few seconds), therefore displayed as lines. To allow for the use of symbols outside the lines, every 350th data point is represented by a symbol. Figure 18 A and 18B describe Figure 18 The actual rotational speed in A and Figure 18The bypass flow in B varies over time. The difference in bypass flow between low and high conditions is approximately 10 times. Figure 18 B).

[0325] As previously stated (see step (4) of the method according to the present invention), see Figure 8A and 8B ,as well as Figure 9A , 9B and 9C), according to Figure 9C The equation shown calculates the adjusted rotational speed [rpm] as a shear stress value (Pa). Using... Figure 9C The shear stress values ​​calculated by the equations shown are as follows (see Figure 18 A): Table 5:

[0326] The performance characteristics of cell culture operation over time are as follows: Figures 17A to 17D As shown. Therefore, in Experiment 3, the culture process was run three times, that is, in Figure 1B In a shear stress-sensitive apparatus, selected cells (here: CHO cells) were cultured three times under the same conditions (perfusion) and using the same parameters. Each culture run lasted for a selected period of 6 days. For each culture run, the time progression of the selected culture characteristic parameters was obtained as offline data. In Experiment 3, the performance characteristic was relative viable cell density (VCD). Figure 17A ) and survival rate ( Figure 17A Lactate dehydrogenase (LDH) activity in the supernatant Figure 17B ), glucose concentration ( Figure 17C ) and lactic acid concentration ( Figure 17D Each item was measured as offline data over time. For each culture run, a curve was plotted for each performance characteristic over time, and the values ​​of the corresponding performance characteristics were plotted against time and the corresponding shear stress values.

[0327] In detail, Figures 17A to 17D Offline data are presented for CHO cell perfusion cultures exposed to high levels of hydrodynamic stress (174 Pa) with variable bypass flow rates. Shear stress levels were kept constant by maintaining a constant rotational speed [rpm] of 6492 rpm while varying the bypass flow rate through the introduction of variable-length tubing.

[0328] The analysis of the charts yielded the following results: Although the maximum hydrodynamic shear stress remained constant at 174 Pa for all three culture runs, it can be observed that the culture with the highest bypass flow rate had the highest relative viable cell density (VCD), while the culture with the lowest bypass flow rate had the lowest relative viable cell density (VCD). Figure 17A This is consistent with metabolite concentrations, showing that high bypass flow cultures have the lowest glucose concentrations and the highest lactate concentrations, while low bypass flow cultures show the opposite. Figure 17C and 17D LDH activity is comparable ( Figure 17B ).

[0329] Figures 17A to 17D The results emphasize that growth performance is not solely attributable to maximum hydrodynamic stress. According to... Figure 10E (Case 4) and Equations 1 to 2, bypass flow affects shear stress frequency ( freq. ) and shear stress exposure duration (dwell time / exposure duration) t res Bypass flow rate is positively correlated with shear stress frequency and negatively correlated with shear stress exposure duration (residence time).

[0330] Since cultures with low bypass flow rates exhibited the lowest growth performance, while cultures with high bypass flow rates exhibited the highest performance, it can be concluded that this is related to the shear stress frequency ( freq. Compared to the duration of shear stress exposure (dwell time / exposure duration), t res The impact on performance is stronger.

[0331] Therefore, it can be deduced from Experiment 3 that two shear stress parameters can be changed simultaneously, thereby identifying the parameter with the greater impact.

[0332] Experiment 4 Effects of fluid dynamic shear stress during perfusion culture The effects of hydrodynamic shear stress on the culture of CHO cell lines (cell line B) expressing different recombinant proteins than those in Experiments 1 through 3 were investigated. Increasing the pump speed [rpm] to increase shear stress without controlling the bypass flow rate resulted in an increase in bypass flow rate with increasing speed [rpm]. However, the increased bypass flow rate also implied changes in shear stress frequency and shear stress exposure duration, as bypass flow rate is positively correlated with shear stress frequency and negatively correlated with shear stress exposure duration. Therefore, in Experiment 4, all three shear stress parameters were modified.

[0333] The same perfusion process setup (the same shear stress-sensitive device with identical conditions) was applied for all culture runs and tests under the same shear stress conditions. All parameters and conditions were kept constant throughout all culture runs. Only the selected shear stress parameters and bypass flow rates varied between each culture run. The pump was a magnetically levitated centrifugal pump (here: PuraLev i30 SU, Levitronix, Switzerland). As previously described, the cell culture was continuously circulated within the shear stress-sensitive device. The shear stress-sensitive device also includes, as... Figure 1B The hollow fiber filter module is shown. Detailed information about the cultivation process can be found in Experiment II.

[0334] The performance characteristics of cell cultures over time are as follows: Figures 19A to 19D As shown. Figures 19A to 19D Offline data of CHO cell perfused cultures subjected to different hydrodynamic stresses are presented. The same perfusion process settings were applied for all test shear stress conditions.

[0335] Figures 19A to 19D The control group represents the standard perfusion setting. The bypass flow rate for feedback control is 0.2 L / min.

[0336] Figure 20 A and 20B show online data for shear stress settings during perfusion culture, under varying rotational speed [rpm] and bypass flow rate. The rotational speed of the magnetic levitation centrifugal pump i30SU is as follows... Figure 20 As shown in Figure A, the bypass flow measured online is as follows: Figure 20 As shown in Figure B. The corresponding settings for the shear stress device are as follows. Figure 1B As shown. Increasing the rotational speed [rpm] to increase shear stress without controlling the bypass flow rate results in an increase in bypass flow rate with increasing rotational speed [rpm]. Online signal measurements are very frequent (every few seconds), therefore displayed as lines. To allow for symbol indication outside the lines, every 350th data point is represented by a symbol.

[0337] As previously stated (see step (4) of the method according to the present invention), see Figure 8A and 8B ,as well as Figure 9A , 9B and 9C), according to Figure 9C The equation shown calculates the adjusted rotational speed [rpm] as a shear stress value (Pa). Using... Figure 9C The shear stress values ​​calculated by the equations shown are as follows (see Figure 20 A): Table 6:

[0338] The analysis of the charts yielded the following results: Figures 19A to 19D Offline data for CHO cell perfusion cultures exposed to varying levels of hydrodynamic shear stress (with varying rotational speeds and bypass flow rates) are presented. In Experiment 4, performance characteristics were relative viable cell density (VCD) and viability (…). Figure 19A Lactate dehydrogenase (LDH) activity in the supernatant Figure 19B ), glucose concentration ( Figure 19C ) and lactic acid concentration ( Figure 19D ).

[0339] The same perfusion process settings were applied for all stress conditions tested. Relative viable cell density (VCD) showed decreased growth and survival rates compared to the control at maximum hydrodynamic shear stresses of 174 and 251 Pa, as shown in the figure. Figure 19A As shown. Cultures at 100 Pa showed comparable relative VCD, with slightly lower survival rates and lactate concentrations, and slightly higher glucose and LDH activity values ​​( ). Figures 19A to 19D These effects become more pronounced as the hydrodynamic shear stress levels increase to 174 and 251 Pa. Therefore, the shear stress limit in this experiment will be between 100 and 174 Pa.

[0340] Figure 20 A and 20B describe Figure 20 A medium speed and Figure 20 Online data for bypass flow rate in section B shows that the bypass flow rate increases with increasing rotational speed (rpm). This is because the experimental setup with increased hydrodynamic shear stresses (100, 174, and 251 Pa) is similar to the control setup. If no experimental adjustments are made to increase the pressure drop, the bypass flow rate is positively correlated with rotational speed [rpm] (see [link]). Figure 4A and 4B This setup requires no adjustments to the standard perfusion settings, thus representing the simplest experimental method for studying the effects of fluid dynamics shear stress. However, it should be noted again that the bypass flow rate is related to the shear stress frequency (…). freq. (Equation 1) and shear stress exposure duration (dwell time / exposure duration) t res (Equation 2) is relevant. Therefore, Figures 19A-19D The effect shown cannot be considered solely as the effect of the maximum hydrodynamic shear stress, because each shear stress parameter changes.

[0341] These data highlight that the setup for measuring shear stress sensitivity is also applicable to different CHO cell lines expressing entirely different recombinant proteins. It further demonstrates that each CHO cell line has its own sensitivity to similar levels of stress. However, the invention is not limited to CHO cells. Any living organism grown in a suspension bioreactor can be studied for its shear stress sensitivity. In addition to maximum hydrodynamic stress, the invention also allows for the independent study of shear stress parameters, such as the duration and frequency of shear stress exposure. Figures 10A to 10E These parameters have been shown to affect culture performance and are largely relevant during the scale-up of biological processes.

[0342] Experimental Part II 1. Installation of shear stress sensitive devices Experimental setups for fed-batch, perfusion, or flow-through cultures: A tank (e.g., a 3L tank) is connected to a tube (e.g., a 10mm x 6mm x 2mm silicone tube, 100mm long, with a connector such as a CPC coupler). This tube is then connected to the suction side of a pump (e.g., via an AseptiQuik S 3 / 8''). The pump is a magnetic levitation centrifugal pump (PuraLev i30 SU, Levitronix, Switzerland). On the discharge side, the pump is again connected (e.g., via an AseptiQuik S 3 / 8'') to a tube (e.g., a 10mm x 6mm x 2mm silicone tube, 100mm long). This tube is then connected to an additional variable-length tube via a CPC coupler. Thus, the length and diameter can be adjusted according to operating conditions. This tube is either directly connected to the bioreactor (feed-batch or batch mode) or to a hollow fiber filter module. A usable hollow fiber filter module is, for example, the Repligen S02-E65U-07. A flow meter (e.g., LFSC-i10X) is installed between the tank and the hollow fiber filter module or the variable tube. Pressure sensors can be used to determine the pressure drop across the hollow fiber filter module and the variable length tube.

[0343] 2. Bioreactor culture conditions Cell expansion Seed culture was primarily conducted in shake flasks, following standard protocols to ensure optimal cell growth (temperature = 36.5°C). At least one pre-stage culture (N-2) was performed prior to the N-1 stage, under controlled pH (6.7–7.2) and dissolved oxygen concentration (50%). Cultures were aliquoted approximately every 3 days.

[0344] Feed-in batch culture Fed-batch bioreactor cultures were conducted in a controlled 3L benchtop bioreactor using the CHO-K1 GS cell line (cell line A) and proprietary chemically defined basal and feed media. Concentrated feed media were continuously added via a feed pump. Feeding began on day 2 at a rate of 30 mL / L / day. The seeding cell density was <1.0 x 10⁻⁶ cells / day. 6 Cells / mL, with pure oxygen introduced via an immersion nebulizer to maintain dissolved oxygen concentration at 50%. The pH range for the standard process is 6.7–7.2, with constant power input (P / V). Temperature is maintained at 35.0°C. Glucose is added as needed once the measured glucose concentration is <3 g / L. The fed-batch process is performed over 14 days.

[0345] Perfusion culture Perfusion bioreactor culture was conducted in a controlled 3L benchtop bioreactor using CHO-K1 GS cell lines (cell lines A and B) and a proprietary, chemically defined basal medium. The perfusion system consisted of a centrifugal pump (PuraLev i30 SU, Levitronix, Switzerland), a clamp flow meter, a tangential flow filtration (TFF) membrane (hollow fiber filter module), and a scale for controlling reactor weight and perfusion rate. The perfusion rate was adjusted every 24 hours according to a preset rate. A chemically defined basal medium was used as the perfusion medium. The recirculation flow rate or centrifugal pump speed was adjusted according to experimental requirements. The seeding cell density was <2.0 x 10⁻⁶ cells / year. 6 Cells / mL. Dissolved oxygen concentration, pH range, and power input / volume ratio (P / V) were similar between the feed batch and perfusion processes. Temperature was maintained at 36.5°C. Glucose was added as needed once the measured glucose concentration was <3 g / L. The perfusion process lasted 5 to 6 days.

[0346] Offline sample analysis Bioreactor samples (feed-batch and perfusion) were analyzed to determine viable cell density (VCD), cell viability, glucose concentration, lactate concentration, and lactate dehydrogenase (LDH) activity. VCD and cell viability were analyzed using a Vi-CELL BLU (Beckman Coulter GmbH, USA) in combination with trypan blue and Accumax™ (Innovative Cell Technologies, USA). Glucose and lactate concentrations, as well as LDH activity, were determined using a Konelab Prime 60i (Thermo Fisher Scientific Inc., USA).

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Ozturk and Wei-Shou Hu; (25) Gaugler, L. et al.: “Mimicking CHO large‐scale effects in the single multicompartment bioreactor: A new approach to access scale‐up behavior“, Biotechnology and Bioengineering, (Received: 10 October 2023) 2024; 121:1243–1255; DOI: 10.1002 / bit.28647; (26) US 2007 / 034014 A1 and (27) US 2023 / 103671 A1. List of reference numerals 100 Shear-sensitive devices 110 Bioreactor 114 Bioreactor Outlet 115 Liquid Culture Medium 116 Bioreactor Inlet 118 Mixer 125a, 125b, 125d1, 125d2, 125d3 pipes 125C variable tube 130 pump 131 Pump casing 132 Pump Head 133 Impeller 134 Rotating Magnetic Ring 135 Motor / Bearing Winding 136 Motor / Bearing Stator 125C1, 125C2 reducing fittings 140 Flow Meter 150 Hollow Fiber Filter Module Arrows A, B, C, D, E legend: Figure 1A and 1B : Arrow A and Arrow B indicate the flow direction of cell culture in the shear-sensitive device loop. Arrow C: Permeate flow Figure 2 : Tmax is the maximum shear stress. VVM (Volume of air per minute per volume of culture medium) Figure 3A and 4A : ID tube inner diameter dP voltage drop Figure 3B and 4B : HF short hollow fiber filter module HF long hollow fiber filter module dP voltage drop Figure 5 : Rg Radius of gyration R 2 Coefficient of determination ID tube inner diameter HF short hollow fiber filter module HF long hollow fiber filter module Figure 6 : Arrow D enters the pump Arrow E indicates the pump is away. Figure 7 : u(r) Flow velocity [m / s] μ kinematic viscosity [Pa·s] Δp pressure difference [Pa / m] R is the pipe radius [m]. r Radial distance [m] Where 0 ≤ r ≤ R.

[0348] Figure 8A : NPs nanoparticles PMMA (polymethyl methacrylate) Rg Radius of gyration Tmax is the maximum shear stress. R 2 Coefficient of determination Figure 8B : NPs nanoparticles PMMA (polymethyl methacrylate) Rg Radius of gyration Tmax is the maximum shear stress. SSD Shear Stress Sensitive Device R 2 Coefficient of determination Figure 9A and 9B : Rg Radius of gyration R 2 Coefficient of determination Figures 10A to 10E : pump speed rpm freq. Shear stress frequency t res Shear stress exposure duration (residence time of cell culture in the pump) Figure 11A 11B, 12A and 12B: Curve P: Production-scale bioreactor Curve S: Small-scale bioreactor Curve A: Small-scale bioreactor / shear stress sensitive device: Rotation speed A Curve B: Small-scale bioreactor / shear stress sensitive device: Rotation speed B Curve C: Small-scale bioreactor / shear stress sensitive equipment: Rotation speed C Figure 13A , 15A, 17A, 19A Relative live cell density relative to VCD Figure 13C 15B, 17B, 19B: LDH lactate dehydrogenase

Claims

1. A method for determining the shear stress sensitivity of cells in a shear stress sensitivity device (100) during a culture process containing a liquid culture medium (115), comprising the following steps: (1) A shear stress sensitive device (100) is provided, comprising: - Bioreactor (110), - A closed-loop system connected to and located outside the bioreactor (110); - A pump (130) placed in the loop system, said pump (130) being a power pump, and - A device for controlling fluid resistance in a control loop system; (2) Select the cells, liquid culture medium (115), culture mode and culture conditions to be used in the culture process to be carried out; (3) Select one, two, or three shear stress parameters to be studied from the following groups: - Maximum shear stress expressed as pump speed; - Duration of shear stress exposure, expressed as the residence time of the cell culture within the pump (130); and / or - Shear stress frequency, expressed as the number of times a cell culture containing liquid culture medium (115) and cells passes through the loop system per unit time; (4) The shear stress sensitive device (100) is characterized by measuring the shear stress values ​​that appear under different maximum shear stresses by using calibration methods or by using computer calculation and simulation. (5) In a shear stress sensitive device, the same culture process in step (2) is run once, twice, three or more times within the same selected time period. In each subsequent culture run, one, two or three shear stress parameters are added or removed sequentially. During each culture run, one or more performance characteristics of the cells are measured, and a curve of the change over time is generated for each performance characteristic in each run. (6) In the shear stress sensitive device (100), for each performance characteristic, a control culture process is performed using the same culture process as in step (5), wherein a lower maximum shear stress than in step (5) is used, and a control curve is generated for each performance characteristic over time. (7) Select the performance characteristic curve that is closest to the control curve of the performance characteristic in step (6) from the curve set of step (5), the curve representing the shear stress sensitivity of the cell and constituting the shear stress limit of the cell. This ensures that the maximum shear stress occurring in the closed-loop system is higher than the maximum shear stress occurring in the bioreactor (110).

2. The method according to claim 1, Its features are, The culture mode in step (2) is selected from either batch or fed-batch culture. or The culture mode in step (2) is selected from the continuous culture mode, especially perfusion, in which a cell retention device is additionally provided in the shear stress sensitive device (100), the cell retention device being particularly selected from the hollow fiber filter module (150).

3. The method according to claim 1 or 2, Its features are, Devices for controlling fluid resistance in the control loop system are selected from variable tubes (125c) with variable length and / or variable diameter, reducing fittings (125c1, 125c2) for the variable tubes, valves such as pinch valves, and variable winding of the variable tubes (125c).

4. The method according to any one of the preceding claims, Its features are, The calibration method in step (4) is selected from a method in which a shear stress dependence quantity is determined and correlated with the measured maximum shear stress value, in particular the shear stress dependence quantity is the aggregate size of the shear-sensitive aggregate.

5. The method according to claim 4, Its features are, The same shear stress dependence quantity is used in the calibration method and the characterization of the shear stress sensitive device, in particular the shear stress dependence quantity is the aggregate size, more particularly the radius of gyration of the particle aggregates, especially the aggregates are selected from polymethyl methacrylate nanoparticle aggregates or Blauton polymer flocculation systems.

6. The method according to any one of the preceding claims, Its features are, The calibration method in step (4) is as follows: - Select the aggregate size of shear-sensitive aggregates as the shear stress-dependent quantity; - Select a calibration method using a calibration device, the method utilizing the aggregate size and relating the aggregate size to the measured maximum shear stress value; - Reproduce the selected calibration method and measure the aggregate size; - Generate calibration curves, in which the measured aggregate size values ​​are plotted against the measured maximum shear stress value. Specifically, the aggregate size is selected as the radius of gyration of the particle aggregate, and the aggregate is selected from polymethyl methacrylate nanoparticle aggregates or Blauton polymer flocculation systems.

7. The method according to claim 6, Its features are, The characterization in step (4) is performed as follows: - Choose the same calibration method as used in claim 6; - Use a shear stress sensitive device (100) instead of a calibration device to reproduce the selected calibration method, especially using the same aggregate as in the calibration method; - The aggregate is continuously recirculated in a shear stress sensitive device (100) by a pump (130), and the pump speed is increased or decreased in each subsequent run until the aggregate size no longer changes, and the aggregate size is measured after each run; - Generate curves in which the measured aggregate size is plotted against the rotational speed of the pump (130), and - The measured aggregate size is converted into the maximum shear stress value using the calibration curve of the calibration method or the calibration curve of claim 6; Specifically, the aggregate size is selected as the radius of gyration of the particle aggregate, and the aggregate is selected from polymethyl methacrylate nanoparticle aggregates or Blauton polymer flocculation systems.

8. The method according to any one of the preceding claims, Its features are, The cultivation process in step (5) is as follows: The first cultivation process was conducted in the form of the first cultivation run within the first time period. The second cultivation process was carried out in the form of a second cultivation run during the second time period. The third cultivation process will be carried out in the form of a third cultivation operation during the third time period. The cultivation process can be carried out more times over more time periods and in more runs. All time periods are selected to be of equal length. Thus, in each culture run, the cell culture containing liquid culture medium (115) and cells is continuously circulated in the loop system of the shear stress sensitive device (100). Therefore, the selected shear stress parameter is set to a predetermined value in the first culture run and kept constant during the first culture run, and for each subsequent culture run, each selected shear stress parameter is set to a higher or lower value between different culture runs.

9. The method according to any one of the preceding claims, Its features are, The control culture process in step (6) is as follows: - If the culture mode is batch or fed-batch, it is carried out in the same shear stress sensitive device (100) used in step (5), but without a loop system and without a pump (130); - If the culture mode is continuous, especially perfusion, it is carried out in the same shear stress sensitive device (100) used in step (5), wherein the maximum shear stress is adjusted to be lower than that used in step (5), especially as low as possible.

10. The method according to any one of the preceding claims, Its features are, Select one, two, three, or more of the following features: - The power pump (130) is selected from centrifugal pumps, especially magnetic levitation centrifugal pumps; - The loop system connects the outlet (114) (particularly located at the bottom) of the bioreactor (110) to the inlet (116) (particularly located at the top) of the bioreactor (110); - The device for controlling fluid resistance is located downstream of the power pump (130) in the loop system; - The pump (130) is located downstream of the outlet (114) of the bioreactor (110) in the loop system; - The cell retention device is arranged between the device for controlling fluid resistance in the loop system and the bioreactor (110); - The flow meter (140) is placed in the loop system, preferably between the bioreactor (110) and the variable tube (125c), or between the bioreactor (110) and the hollow fiber filter module (150); - Install one or more pressure gauges in the loop system; - The bioreactor (110) includes a stirrer (118); - In step (5), the performance characteristics of the cell culture are measured periodically during each culture run; - In step (6), the performance characteristics of the control cell culture are measured periodically; - In step (7), the shear stress behavior of the cells is analyzed using curves of one or more performance characteristics obtained, especially compared with control curves or without control curves; - The power pump (130) used has a replaceable pump head (132) that can be replaced after all culture runs of the cell culture process; - In step (5) and / or step (6), the time period is at least 1 day, and in particular up to 30 days; - The characterization of the shear stress sensitive device in step (4) is carried out by reproducing the calibration method, in particular by reconstructing the device of the calibration method and thereby reproducing the calibration method with the reconstructed device; - If the calibration method in step (4) is selected from a method in which the shear stress-dependent quantity is determined and the aggregate size is selected as the shear stress-dependent quantity, then the same batch of aggregates is used for the calibration and characterization methods; - In step (5), only one, two, or three shear stress parameters are changed, while all other parameters of the shear stress sensitive device (100) remain constant; and - In step (5), only one, two, or three shear stress parameters and the bypass flow rate are changed, while all other parameters of the shear stress sensitive device (100) remain constant.

11. The method according to any one of the preceding claims, Its features are, One or more performance characteristics of the culture process or control culture process are selected from parameters that change over time during cell culture, particularly from live cell density, viability, glucose concentration, lactate concentration, LDH concentration, cell culture titer, growth, productivity, and product quality.

12. The method according to any one of the preceding claims, Its features are, For all runs of the cultivation process, when changing one, two, or three shear stress parameters in the shear stress-sensitive device (100), choose one of the following modifications according to cases 1 to 4: Case 1: In all culture runs, while keeping the duration and frequency of shear stress exposure constant, increase the maximum shear stress from one culture run to the next. or Scenario 2: In all culture runs, while keeping the maximum shear stress and shear stress frequency constant, increase the duration of shear stress exposure from one culture run to the next. or Case 3: In all culture runs, while keeping the maximum shear stress and the duration of shear stress exposure constant, increase the frequency of shear stress from one culture run to the next. or Case 4: While keeping the maximum shear stress constant, increase the duration of shear stress exposure from one culture run to the next, and decrease the shear stress frequency from one culture run to the next. In particular, one or more of the following conditions are selected to control one, two, or three shear stress parameters: - By keeping the pump (130) speed at the same value, the maximum shear stress is kept constant in terms of speed, or - Increase or decrease the maximum shear stress (in terms of speed) by setting the pump (130) speed higher or lower. - If the pump speed (130) is increased or decreased by using a device for controlling fluid resistance, in particular by changing the length and / or diameter of the variable pipe (125c) in the shear stress sensitive device (100) loop system, the duration of shear stress exposure is kept constant. - If the speed of pump (130) is kept constant by adding an additional pump with the same speed in series in the loop system, the duration of shear stress exposure is increased; - If the pump (130) speed is kept constant by using a device to control fluid resistance, in particular by changing the length and / or diameter of the variable tube (125c) in the shear stress sensitive device (100) loop system, the duration of stress exposure is increased or decreased. - By using a device for controlling fluid resistance, particularly by changing the length and / or diameter of the variable tube (125c) in the shear stress sensitive device (100) loop system, the shear stress frequency is kept constant; - If the pump (130) speed is kept constant by adding one or more external loops to the bioreactor (110) or by adjusting the working volume of the bioreactor (110), the shear stress frequency is increased; and / or - If the speed of pump (130) is kept constant by providing one or more additional pumps in parallel and all pumps are running at the same speed value, the shear stress frequency is increased.

13. The method according to any one of the preceding claims, Its features are, Step (2) is replaced by step (2'), which is as follows: Perform a culture process or use a culture process that has been performed, and provide one or more culture process curves of the culture process, wherein one or more performance characteristics of the cells are plotted over a selected time period; Step (4) is replaced by step (4'), which is as follows: Perform a control culture process or use a control culture process that has been performed, and provide one or more culture process curves of the control culture process, wherein one or more performance characteristics of the cells are plotted over a selected time period, wherein the control culture process is the same as the culture process in step (2'), but uses a lower shear stress intensity than the culture process, and uses the same cell performance characteristics and the same time period as in step (2'); Step (5) is replaced by step (5'), which is as follows: In a shear stress sensitive device, one, two, three or more control culture runs of the control culture process of step (4') are performed within the same selected time period, with one, two or three shear stress parameters being added or removed sequentially in each subsequent control culture run, wherein each subsequent control culture run is modified by changing the shear stress parameters in the direction of the selected culture process of step (2'), and one or more performance characteristics of the cells are measured during each run of the control culture process, and a curve of change over time is generated for each performance characteristic in each run; Step (6) is replaced by step (6'), which is as follows: Select the curve of the cultivation process that is closest to the performance characteristic from the curve set of step (5'); Step (7) is replaced by step (7'), which is as follows: by comparing the obtained curves, determine how the shear stress intensity needs to be changed to switch from the control culture process to the culture process; This ensures that the shear stress intensity during the culture process is higher than that during the control culture process.

14. The method according to claim 13, Its features are, Choose the culture process in step (2') to carry it out on a larger scale than the control culture process in step (4').

15. A method for culturing cells in a liquid culture medium, wherein the shear stress sensitivity of the cells measured in the method of any one of claims 1 to 12 is applied to the same culture process, but on a larger or smaller scale.

16. A method for culturing eukaryotic or prokaryotic cells in a liquid culture medium in a bioreactor (110) and performing a method according to any one of claims 1 to 14 for determining the shear stress sensitivity of the cells in a shear stress sensitivity device (100) during the culture process.

17. A method for producing recombinant proteins, the method comprising the following steps: Step I) Eukaryotic or prokaryotic cells expressing the recombinant protein are cultured in liquid culture medium in a bioreactor (110); Step II) Harvest the recombinant protein; Step III) Purify the recombinant protein; In step I), the method for determining the shear stress sensitivity of cells in a shear stress sensitivity device (100) during culture is performed according to any one of claims 1 to 14.

18. Use of an apparatus (100), said apparatus comprising: - Bioreactor (110), - A closed-loop system connected to and located outside the bioreactor (110); - A pump (130) placed in the loop system, said pump (130) being a power pump, and - A device for controlling fluid resistance in a control loop system; As a shear stress sensitive device (100), The device (100) described there is no nozzle. It is used to culture cells in a liquid culture medium (115) and to set a limited shear stress during the culture process.

19. The use of the device (100) according to claim 18, Its features are, - As a limiting shear stress, the maximum shear stress value obtained by changing the shear stress parameter in the device (100) is used, the maximum shear stress value being determined based on a physical quantity that is dependent on shear stress, in particular the aggregate size of the shear-sensitive aggregate; or - As a limiting shear stress, relative shear stress levels derived from two runs of the culture process are used, the two runs having different shear stress levels, one of which is used as a control run, and the control run is repeated and modified by changing the shear stress parameters until its run is similar to the other run.

20. The use of the device (100) according to claim 19, Its features are, The maximum shear stress value is determined based on a physical quantity that depends on shear stress, wherein: - Expose the physical quantity that is dependent on shear stress to defined preset conditions, whereby the physical quantity varies according to the magnitude of the maximum shear stress; - The physical quantity that depends on shear stress is exposed to unknown conditions in the device (100), whereby the physical quantity varies according to the magnitude of the maximum shear stress; - The magnitude of the maximum shear stress value in the device (100) can be inferred from the physical quantity obtained under the limited preset conditions, whereby the same magnitude of the physical quantity means that the same maximum shear stress value exists.

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