Automated, integrated continuous systems and bioprocesses for the production of therapeutic proteins

Abstract

The present invention relates to an automated integrated continuous bioprocess system and a bioprocess that can continuously produce therapeutic proteins in an uninterrupted manner and can be expanded from laboratory scale to production scale. The present invention provides an automated integrated continuous bioprocess system and a bioprocess for producing therapeutic proteins, wherein the system and process are controlled by one or more control systems selected from a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID), a programmable logic circuit (PLC), an industrial PC (IPC), a distributed control system (DCS), an information relay system, and automated UPLC / HPLC sampling for online testing, so as to operate the system and process and continuously produce therapeutic proteins in an uninterrupted manner.

Description

Technical Field

[0001] The present invention generally relates to a system for continuous bioprocessing of therapeutic proteins. In particular, the present invention relates to an automated integrated continuous bioprocessing system and bioprocess that can continuously produce therapeutic proteins and can be expanded from laboratory scale to production scale. Background Art

[0002] The background description includes information that may be helpful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is an admission made that any publication specifically or implicitly referenced is prior art.

[0003] The identification of therapeutic proteins of interest has revolutionized the biopharmaceutical industry. However, production of therapeutic proteins often results in the observation of charged isomers, which significantly interferes with the separation and purification processes required to achieve high yields and product quality.

[0004] Traditionally, biopharmaceutical companies have used batch processing to manufacture therapeutic proteins, where unit operations are performed and completed before the process flow moves to the next step. More recently, biopharmaceutical companies are adopting continuous bioprocessing to manufacture therapeutic proteins. In continuous bioprocessing, as each unit process is completed, the processed product is moved to the next step. Continuous bioprocessing has attracted widespread attention due to its various advantages, including steady-state operation, small equipment size, high-volume productivity, simplified process flow, low cycle times, and reduced capital costs.

[0005] Various approaches to manufacturing therapeutic proteins using continuous bioprocessing methods have been attempted in the prior art. However, such existing continuous bioprocesses are pseudo-continuous processes consisting of independent units, each performing its own function. In such systems, each unit operation has limited communication between various system parameters and operates mostly in isolation. Furthermore, current continuous processes primarily involve offline chromatographic analysis with limited or no feedback on the ongoing process. As biopharmaceutical companies continue to grow, there is an unmet need to provide automated, integrated bioprocesses and systems that can be scaled from laboratory scale to production scale to manufacture therapeutic proteins.

[0006] Purpose of the Invention

[0007] An object of the present invention is to provide an automated integrated continuous bioprocessing system for producing therapeutic proteins.

[0008] Another object of the present invention is to provide an automated integrated continuous bioprocess for producing therapeutic proteins using a control system with communication and programmable control capabilities to regulate the overall process parameters using a master controller.

[0009] Another object of the present invention is to provide an automated integrated continuous bioprocess for the production of therapeutic proteins, which process can be performed in a continuous and uninterrupted manner. Summary of the Invention

[0010] Some aspects of the present invention relate to an automated integrated continuous bioprocessing system for producing therapeutic proteins in an uninterrupted manner using a control system with communication and programmable control capabilities to regulate overall process parameters using a master controller.

[0011] In one aspect of the present invention, an automated integrated continuous bioprocessing system (100) for producing a therapeutic protein is provided, comprising:

[0012] A bioreactor (103) for culturing mammalian cells capable of producing a therapeutic protein in a culture medium, wherein the bioreactor (103) is capable of using an alternating tangential flow (ATF) filter (109) to collect a harvest comprising the protein secreted into the culture medium;

[0013] a first chromatography system (119) connected to the ATF filtration system (109) of the bioreactor (103) without any intermediate holding vessel to purify the harvested recombinant therapeutic protein and provide a Protein A eluate;

[0014] a virus inactivation system (126) comprising a virus inactivation container (128) connected to the first chromatography system (119) to collect the Protein A eluate and inactivate viruses that may be present in the eluate, and the virus inactivation container (128) is configured to automatically adjust the pH value of the Protein A eluate;

[0015] a collection vessel (136) connected to the virus inactivation vessel (128) via one or more filters to receive the virus inactivated, neutralized, and filtered Protein A eluate, wherein the one or more filters (224) and (226) are configured to remove impurities in the form of precipitates from the neutralized Protein A eluate received from the virus inactivation vessel;

[0016] A second chromatography system (137) is connected to the collection vessel (136) to receive the filtered Protein A eluate from the collection vessel (136) and provide further purified protein.

[0017] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, which optionally also includes an additional collection vessel (140) connected to a second chromatography system (137) to receive and store purified protein, which can optionally be purified using one or more filters (142) and provide further purified therapeutic protein.

[0018] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises one or more control systems selected from the group consisting of: a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID) (not shown), a programmable logic circuit (PLC) (112), an industrial PC, a distributed control system (DCS) (not shown), input-output modules or IO boxes (130) and (132) operably connected to various systems such as chromatography system 1 (119), a viral inactivation system (128), a collection container (136), a chromatography system 2 (137) and a message relay system (not shown).

[0019] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises one or more electromagnetic or pneumatic pinch valves (114), and optionally comprises a flow meter, a bubble sensor, a pressure sensor and a load cell for generating a feedback loop to maintain fluid flow between the various components of the system, avoid bubble formation and regulate liquid flow.

[0020] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, further comprising a surge bag (116) connected to the bioreactor via a valve (114).

[0021] In one aspect, the present invention provides an automated integrated continuous bioprocessing system comprising a viral inactivation vessel (128), one or more pH probes connected to a pH transmitter for measuring the pH of a protein A eluate, an automatic titrator comprising a PLC and a pump (122) connected in turn to a vessel (124) containing an acid and a base for titration, a level sensor for checking the level of fluid in the vessel, and an online turbidity measurement sensor for measuring real-time nephelometric turbidity units (NTU).

[0022] In one aspect, the present invention provides an automated integrated continuous bioprocessing system wherein each filter between the viral inactivation vessel and the collection vessel is a 0.2-0.45 micron filter.

[0023] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the virus inactivation container and the collection container are made of glass or stainless steel.

[0024] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the first chromatography system comprises one or more affinity chromatography columns, and the second chromatography system comprises one or more multimodal anion exchange columns and one or more cation exchange columns.

[0025] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises a cleaning in place (CIP) system (120) for periodically cleaning the chromatography system inlet, virus inactivation container, collection container, and liquid flow tubes.

[0026] In one aspect, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises automated harvest sampling from the bioreactor for cell counting and nutrient analysis, and automated sampling at various locations for online chromatographic analysis in the continuous bioprocess.

[0027] In certain other aspects, the present invention relates to continuous bioprocesses for producing therapeutic proteins in an uninterrupted manner that can be scaled up from laboratory scale to production scale.

[0028] In one aspect, the present invention provides an automated integrated continuous bioprocess for producing a therapeutic protein, wherein the process is controlled by one or more control systems selected from the group consisting of a Supervisory Control and Data Acquisition (SCADA) control system (110), a Proportional Integral Derivative (PID), a Programmable Logic Circuit (PLC), an Industrial PC (IPC), a Distributed Control System (DCS), and a message relay system, and the process comprises the following steps:

[0029] (a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) capable of using an alternating tangential flow (ATF) filter (109), and collecting a bleeding harvest comprising the protein secreted into the culture medium;

[0030] (b) supplying the cell culture harvest containing the therapeutic protein from the bioreactor (103) to a first chromatography system (119) to provide a Protein A eluate;

[0031] (c) supplying the protein A eluate from the first chromatography system (119) to a virus inactivation container (128) and inactivating viruses in the protein A eluate;

[0032] (d) passing the virus inactivated and neutralized Protein A eluate through one or more filters to remove impurities in the form of any precipitates formed during the virus inactivation and neutralization steps, and collecting the filtered Protein A eluate in a collection vessel (136); and

[0033] (e) The neutralized and filtered Protein A eluate is supplied to a second chromatography system (137) to provide purified protein.

[0034] In one aspect, the present invention provides an automated integrated continuous bioprocess, which optionally further comprises storing the purified protein received from the second chromatography system (137) in an additional container (140). In one aspect, the present invention provides an automated integrated continuous bioprocess, which optionally further comprises one or more steps of passing the purified protein through one or more filters (142) to further purify the therapeutic protein.

[0035] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein mammalian cells are cultured in a bioreactor (103) that is a perfusion bioreactor capable of using alternating tangential flow (ATF) technology.

[0036] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein the fractionated cell culture harvest is fed from the bioreactor (103) directly into the first chromatography system (119) using a first chromatography system pump.

[0037] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein clarified cell culture harvest is fed directly from a bioreactor (103) to a first chromatography system (119) using a first chromatography system pump.

[0038] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein a first chromatography step is performed using one or more affinity chromatography columns to purify a cell culture harvest containing a therapeutic protein from a bioreactor and provide a Protein A eluate.

[0039] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein the pH of the Protein A eluate in the viral inactivation step is automatically adjusted by a proportional-integral-derivative (PID) controller, a programmable logic controller (PLC), or an industrial personal computer (IPC) controller.

[0040] In one aspect, the present invention provides an automated integrated continuous bioprocess wherein the second chromatography step is performed using one or more multimodal anion exchange columns and one or more cation exchange columns, respectively, to provide a purified protein.

[0041] In one aspect, the present invention provides an automated integrated continuous bioprocess that includes automated sampling for on-line testing and clean-in-place (CIP) using a system (120) for periodic cleaning of chromatography systems, tubing, and containers to maintain uninterrupted operation.

[0042] In one aspect, the present invention provides an automated integrated continuous bioprocess, wherein the therapeutic protein is selected from an antibody, an antibody fragment, a monoclonal antibody, an enzyme, a recombinant protein, an engineered protein, an immunogenic protein, a protein fragment, a peptide, an immunoglobulin, or any combination thereof.

[0043] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the disclosure and together with the description serve to explain the principles of the disclosure.

[0045] Figure 1 An exemplary process flow of an upstream process is shown.

[0046] Figure 2 An exemplary process flow for downstream processes is shown.

[0047] Figure 3 is a schematic diagram showing an overview of an automated, integrated, continuous bioprocessing system for manufacturing therapeutic proteins.

[0048] Figure 4 is a schematic diagram showing an overview of a viral inactivation system of an automated integrated continuous bioprocessing system for manufacturing therapeutic proteins.

[0049] Figure 5 is a schematic diagram showing an overview of the CIP system of chromatography 1 of an automated integrated continuous bioprocessing system for manufacturing therapeutic proteins.

[0050] Figure 6 is a graph showing the effects of lactose and glucose on cell growth at different days.

[0051] Figure 7 is the chromatogram of chromatography system 1, showing the pH peak, elution peak, and conductivity peak.

[0052] Figure 8 is the chromatogram of chromatography system 2, showing the elution peak, flow-through peak, and conductivity peak. DETAILED DESCRIPTION

[0053] The following is a detailed description of embodiments of the present disclosure. The embodiments are described in such detail as to clearly convey the present disclosure. However, the amount of detail provided is not intended to limit the intended variations of the embodiments; on the contrary, it is intended to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of the present disclosure as defined by the appended claims.

[0054] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0055] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0056] In some embodiments, the numerals for describing and claiming the amount of expression components, properties (such as concentration, reaction conditions, etc.) of certain embodiments of the present invention should be understood as being modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the specification and the appended claims are approximate values, which can be varied according to the desired characteristics sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted according to the number of significant figures reported and by applying common rounding techniques. Although the numerical range and parameters illustrating the broad scope of certain embodiments of the present invention are approximate values, the numerical value set forth in the specific embodiments is reported as accurately as possible. The numerical values ​​presented in some embodiments of the present invention may comprise certain errors inevitably caused by the standard deviation found in their respective test measurements.

[0057] As used in the description herein and in the claims that follow, the meanings of “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “among” and “on” unless the context clearly dictates otherwise.

[0058] Throughout the following specification, unless the context requires otherwise, the word "include" and variations such as "comprise" and "comprising" should be construed in an open and inclusive sense, ie, "including but not limited to."

[0059] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0060] Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein for certain embodiments is intended only to better illustrate the present invention and does not impose limitations on the scope of the claimed invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the implementation of the present invention.

[0061] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. For reasons of convenience and / or patentability, one or more members of a group may be included in a group or deleted from a group. When any such inclusion or deletion occurs, the specification is deemed to include the modified group to satisfy the writing requirement.

[0062] The following description and the embodiments described herein are provided by way of one or more examples of specific embodiments of the principles and aspects of the present invention. These examples are provided for the purpose of explaining these principles and the present invention, rather than for limitation.

[0063] It should also be understood that the present disclosure can be implemented in a variety of ways, including as a system, method, or device. In this specification, these embodiments or any other form that the present invention can adopt can be referred to as processes. Generally, the order of the steps of the disclosed processes can be changed within the scope of the present invention.

[0064] The titles and abstracts of the inventions provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0065] The following discussion provides a number of example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the inventive subject matter is also considered to include the remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0066] As used herein, various terms are as follows. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the relevant art have given it as reflected in printed publications and issued patents at the time of filing.

[0067] As used herein, the term "continuous bioprocess" refers to any process having two or more process steps in series, wherein the output from an upstream step (unit operation) is continuously transferred to a downstream step (unit operation) up to a final chromatography step, and wherein the upstream process step does not need to be run to completion before the next process step is started. In a continuous process, some portion of the target product is always moving through the process system. Ideally, the continuous process is adjusted so that, to the greatest extent possible, each step or unit operation of the continuous process runs simultaneously and at substantially the same production rate. In this way, cycle time is minimized and the shortest possible completion time is achieved.

[0068] The term "continuous transfer" refers to the transfer of a product stream from an upstream unit operation to a downstream unit operation, meaning that the connection or link between the two unit operations is such that the upstream unit operation transfers the product stream (directly or through other components) to the second (downstream) unit operation, and that the downstream unit operation begins before the upstream unit operation run is completed (i.e., the two continuous unit operations are simultaneously processing the product streams flowing into them for at least a portion of the overall operational run of which both unit operations form a part).

[0069] As used herein, the term "perfusion cell culture process" refers to perfusion culture, which is carried out by continuously supplying fresh culture medium to a bioreactor and continuously removing cell-free spent culture medium (while retaining the cells in the reactor); therefore, perfusion culture can achieve higher cell densities than continuous culture because the cells are retained in the reactor by a cell retention device. The perfusion rate depends on the requirements of the cell line, the concentration of nutrients in the feed, and the toxicity level.

[0070] The term "cell culture medium" refers to all kinds of culture media used in the context of culturing cells. Typically, a cell culture medium comprises amino acids, at least one carbohydrate as an energy source, trace elements, vitamins, salts and possibly additional ingredients (e.g., to influence cell growth and / or productivity and / or product quality).

[0071] The term "therapeutic protein" refers to a recombinant protein that has been sufficiently purified or separated from contaminating proteins, lipids, and nucleic acids present in liquid culture media or from host cells (e.g., mammalian, yeast, or bacterial host cells) and biological contaminants (e.g., viral and bacterial contaminants) and can be formulated into a pharmaceutical product for the treatment or prevention of various diseases or conditions. Representative examples of therapeutic proteins include, but are not limited to, antibodies, antibody fragments, monoclonal antibodies, enzymes, engineered proteins, immunogenic proteins, protein fragments, and immunoglobulins.

[0072] The term "antibody" refers to a functional component of serum and is generally referred to as a collection of molecules (antibodies or immunoglobulins, fragments, etc.) or molecules. Antibody molecules are able to bind or react with specific antigenic determinants, which in turn may lead to a specific immune effect or mechanism.

[0073] The term "monoclonal antibody" refers to an antibody that is produced by a single clone of a cell or cell line and is composed of identical antibody molecules.

[0074] As used herein, the term "retention time" refers to the time it takes for half the amount of solute to elute from a chromatography system. It is determined by the length of the column and the migration rate of the solute; it can range from 1 to 30 minutes.

[0075] As used herein, the term "eluate" refers to the fluid eluted from a chromatography column or chromatography membrane containing a detectable amount of a recombinant therapeutic protein.

[0076] This disclosure uses various abbreviations, the full forms of which are provided below:

[0077] ATF: Alternating Tangential Flow

[0078] CV: column volume

[0079] CEX: Cation exchange (CEX) chromatography

[0080] CIP: Cleaning in Place

[0081] DO: dissolved oxygen

[0082] DCS: Distributed Control System

[0083] HPLC: High Performance Liquid Chromatography

[0084] IPC: Industrial PC

[0085] min: minutes

[0086] mM: millimole

[0087] mAU: milliabsorptivity units

[0088] mL: milliliters

[0089] mS / cm: millisiemens / cm

[0090] mA: milliampere

[0091] NTU: Nephelometric Turbidity Unit

[0092] NaOH: sodium hydroxide

[0093] PLC: Programmable Logic Circuit

[0094] PCC: Periodic Countercurrent

[0095] PID: Proportional-Integral-Derivative

[0096] RV: reactor volume

[0097] TTL: Transistor-Transistor Logic

[0098] UV: Ultraviolet

[0099] UPLC: Ultra-Performance Liquid Chromatography

[0100] VI: Virus inactivation

[0101] V: Volt

[0102] VCC: Viable Cell Count

[0103] In some embodiments, the present invention relates to an automated integrated bioprocessing system for continuous production of therapeutic proteins in an uninterrupted manner, which is controlled by a control system having communication and programmable control capabilities to regulate overall process parameters using a master controller.

[0104] The present invention will now be described in more detail by way of the following embodiments with reference to an integrated automated system for continuous manufacturing of therapeutic proteins.

[0105] Figure 3 An exemplary automated integrated system for continuous production of therapeutic proteins in an uninterrupted manner according to one embodiment of the present invention is shown as follows:

[0106] An automated integrated system (100) for continuous production of therapeutic proteins comprises:

[0107] A bioreactor (103) for culturing mammalian cells capable of producing a therapeutic protein in a culture medium, the bioreactor (103) being equipped with an agitator (102) having blades (105) and a feed inlet (104), and being capable of utilizing an alternating tangential flow (ATF) filter (109) to collect a harvest comprising proteins secreted into the culture medium of the reactor;

[0108] a first chromatography system (119) connected to the ATF filtration system (109) of the bioreactor (103) without any intermediate holding vessel to purify the recombinant therapeutic protein from cultured mammalian cells and provide a Protein A eluate;

[0109] a virus inactivation system (126) comprising a virus inactivation container (128) connected to the first chromatography system (119) to collect the Protein A eluate and inactivate viruses that may be present in the Protein A eluate, and the virus inactivation container (128) is configured to automatically adjust the pH value of the Protein A eluate;

[0110] a collection container (136) connected to the virus inactivation container (128) through one or more filters to receive the neutralized Protein A eluate from the virus inactivation container (128) as the neutralized Protein A eluate passes through the one or more filters, thereby removing impurities in the form of precipitates from the Protein A eluate;

[0111] A second chromatography system (137) is connected to the collection vessel (136) to receive the neutralized and filtered Protein A eluate from the collection vessel (136) and provide further purified protein.

[0112] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, which optionally also includes an additional collection vessel (140) connected to a second chromatography system (137) to receive and store the purified protein, which may optionally use one or more filters (142) and provide further purified therapeutic protein.

[0113] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises one or more control systems selected from the group consisting of: a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID) (not shown), a programmable logic circuit (PLC) (112), an industrial PC (IPC), a distributed control system (DCS) (not shown), input-output modules or IO boxes (130) and (132) operably connected to a separate system including chromatography system 1 (119), a viral inactivation system (128), a collection container (136), chromatography system 2 (137), and a message relay system (not shown).

[0114] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises one or more electromagnetic or pneumatic pinch valves (114), and optionally comprises a flow meter, a bubble sensor, a pressure sensor, and a load cell for generating a feedback loop to maintain fluid flow between the various components of the system, avoid bubble formation, and regulate liquid flow.

[0115] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system further comprising a surge bag (116) connected to the bioreactor via one of the pinch valves (114).

[0116] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system comprising a virus inactivation vessel (126), one or more pH probes connected to a pH transmitter for measuring the pH of a protein A eluate, and an automatic titrator comprising a PLC and a pump (122) connected in sequence to a vessel (124) containing an acid and a base for titration, and optionally comprising a level sensor for checking the level of fluid in the vessel, and an online turbidity measurement sensor for measuring real-time nephelometric turbidity units (NTU).

[0117] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system wherein each filter between the viral inactivation vessel and the collection vessel is a 0.2-0.45 micron filter.

[0118] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the first chromatography system comprises one or more affinity chromatography columns, and the second chromatography system comprises one or more multimodal anion exchange columns and one or more cation exchange columns.

[0119] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the virus inactivation container and the collection container are made of glass or stainless steel.

[0120] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system in which tubing made of suitable materials (e.g., silicone and bioprene) is used to maintain fluid flow. In one embodiment, the connectors used are sterile connectors to reduce system bioburden.

[0121] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises a clean-in-place (CIP) system (120) for periodically cleaning the chromatography system, virus inactivation vessels, collection vessels, and tubes.

[0122] In one embodiment, the present invention provides an automated integrated continuous bioprocessing system, wherein the system further comprises automated harvest sampling from the bioreactor for cell counting and nutrient analysis, and automated sampling at various locations for online chromatographic analysis during continuous bioprocessing.

[0123] In one embodiment, the automated integrated continuous bioprocess system optionally includes an ultra-high performance liquid chromatography (UPLC) system (108) and (138). In some embodiments, the first chromatography system and the second chromatography system used in the present invention can be high pressure liquid chromatography (HPLC). In another embodiment, the system provides conditions for an integrated / analytical system using an automatic sampling system. The controller triggers the HPLC system to start the analytical run using one or more suitable digital interfaces, and the one or more digital interfaces are selected from but not limited to open platform communication (OPC), modbus TCP / IP, EtherCAT, Profibus, Profinet, profibus and industrial Ethernet interfaces.

[0124] In one embodiment, a controller regulates a multi-port electric rotary valve or flow selector valve to control liquid flow. The controller activates a precision pump and opens a valve in a bioreactor sampling port. Programmed amounts of fluid are collected and analyzed by an HPLC system. The HPLC system performs the analysis according to a pre-programmed procedure. The HPLC system transmits the analysis data to a SCADA system.

[0125] In another embodiment, a master controller comprising an IPC with SCADA software controls data visualization, monitoring, and a historical record of process parameters. The master controller is connected to each system (e.g., the bioreactor, the first chromatography system, the second chromatography system, and the viral inactivation system) using one or more digital interfaces selected from, but not limited to, OPC, Modbus TCP / IP, EtherCAT, Profinet, Profibus, or Industrial Ethernet.

[0126] Figure 4An overview of a virus inactivation system according to an embodiment of the present invention is illustrated. The virus inactivation system (200) includes a virus inactivation (VI) container (201) made of glass or stainless steel to collect the protein A eluate from the first chromatography system. The VI container (201) includes an overhead stirrer / magnetic stirrer (216) with multiple ports for adding and removing eluate. The virus inactivation system includes a pH probe (220) connected to a transmitter for measuring the pH of the protein A eluate received from the first chromatography system. The system also optionally includes load cells (202), (206), (208), (210), (212) and (214) for accommodating various liquids (e.g., acid, alkali, NaOH, water and buffer). The system also includes 0.2-0.45 micron filters (224) and (226) to remove any precipitate that may form during the neutralization step. The system can optionally include flow sensors (250-1) to (250-3) to monitor the flow of various liquids to the VI container (201) and the collection container (230). The system can also include a liquid level sensor for checking the fluid level in the VI container, and a load tank sensor (248-1) to (248-6) for monitoring the liquid level in each load tank. If one filter in the control system is blocked during continuous operation, the fluid path can be automatically switched between the two filters. The system can be based on pressure sensors (256-1) and (256-2) or use a toggle switch. The system can issue an alarm for operator intervention when the filter is blocked and switched. The system can also optionally include a turbidity sensor (254) after the inactivation container to measure nephelometric turbidity units (NTU). When the threshold is exceeded, the system will trigger an alarm for operator intervention. An optional bubble sensor (252) connected in the fluid path after the virus inactivation container can trigger the switching of the fluid path to prevent bubbles from entering the chromatography system. A bubble trap (not shown) prevents bubbles from entering the chromatography system. The virus inactivation (VI) vessel is specifically designed to allow for gentle addition of water, eluent, acid, base, and buffer, thereby avoiding foaming and splashing of the liquid. The system consists of a collection vessel (230), which is a container for collecting the eluent after virus inactivation. The collection vessel (230) contains an overhead stirrer / magnetic stirrer (236) with multiple ports for adding and removing various liquids and eluents.

[0127] In one embodiment, the viral inactivation system includes various sensors to continuously monitor and adjust the viral inactivation system in a continuous and automated manner.

[0128] In one embodiment, the virus inactivation system optionally includes a liquid level or load cell sensor selected from but not limited to (248-1) for monitoring the liquid level of acid, (248-2) for monitoring the liquid level of alkali, (248-3) for monitoring the liquid level of NaOH, (248-4) for monitoring the liquid level of water, (248-5) and (248-6) for monitoring the liquid level of buffer.

[0129] In one embodiment, the virus inactivation system optionally includes flow sensors to monitor the flow of various liquids to the VI container (201) and the collection container (230), the flow sensors being selected from but not limited to (250-1) for monitoring the flow of acid, (250-2) for monitoring the flow of alkali, and (250-3) for monitoring the flow of NaOH, water, and buffer.

[0130] In one embodiment, the virus inactivation system is operably connected to a main controller, such as a PID, PLC, or IPC, for real-time measurement and control of pH. The control unit can be programmed for multiple parameters, such as pH set point, hold time, agitator rpm, acid and base pump rate control, CIP cycle control. The virus inactivation system is controlled by various devices selected from, but not limited to, for various settings (238), for main power (240), for automatic or manual mode selection (242), for alarms (244), for calibration (258), and for shutting down the system (246). The controller sends signals to various actuators in the system, such as (valves (222-1) to (222-10) and pumps (204-1) to (204-5)) to regulate liquid flow and control the addition of acid and base to the protein A eluent for pH regulation.

[0131] In one embodiment, the controller receives and sends signals from the chromatography system, such as analog signals (e.g., 0-10V or 4-20mA) or digital signals from one or more interfaces of TTL logic or higher, the one or more interfaces being selected from, but not limited to, OPC, modbus TCP / IP, EtherCAT, Profibus, Profinet, profibus, and industrial Ethernet. The signal from the first chromatography system triggers the virus inactivation procedure. The transmitter measures the signal generated by the pH probe (220) and transmits the value to the main controller. The system automatically adjusts the pH of the eluent according to the set point in the controller, which activates the addition of acid (202) or base (206), followed by the hold time of the virus inactivation step. After the virus inactivation hold time is completed and the pH value of set point 2 is adjusted, the system pumps the eluent after virus inactivation from the virus inactivation container (201) into the collection container (230). The fluid path is digitally controlled using normally closed solenoid valves / pneumatic pinch valves (222-1) to (222-3), which can block and divert fluid flow as needed.

[0132] In one embodiment, after the Protein A eluate is transferred from the viral inactivation vessel to the collection vessel, the controller sends a signal, such as an analog signal (e.g., 0-10V or 4-20mA) or a digital signal from TTL logic or one or more higher-level digital interfaces selected from, but not limited to, OPC, Modbus TCP / IP, EtherCAT, Profibus, Profinet, Profibus, and Industrial Ethernet, to the second chromatography system. A trigger initiates loading of the eluate from the collection vessel into the second chromatography system to further purify the Protein A eluate and provide purified protein.

[0133] In one embodiment, the controller initiates a CIP cycle in the viral inactivation container. In the VI container during the CIP cycle, the controller sends a signal to the pump (204-4) to start and open the valve (222-5) to allow NaOH to flow out of the load tank (208). The flow rate of NaOH to the VI container is monitored by the flow sensor (250-3) and controlled by the pump (204-4). After the set time adjusted by the holding time setting device (218) to keep the NaOH in the VI container has passed, the controller sends a signal to the waste pump (204-3) to remove the NaOH from the VI container and trigger the opening of the waste valve (222-4) for the waste outlet (228). The VI container emptying of NaOH triggers the bubble sensor (252), which sends a signal to the controller and stops the waste pump (204-3) and its valve (222-4), or, if the bubble sensor is not available, the controller can also be programmed to run the pump for a set time until the container is empty. The controller then signals the pump (204-4) to start and open valve (222-6) to allow water to flow from the load reservoir (210) and regulate the flow of water through valve (222-6), which is monitored by the water sensor (250-3) and controlled by the pump (204-4) to the VI container (201). After the set time for holding water in the VI container has passed, the controller signals the waste pump (204-3) to drain the water from the VI container and trigger the opening of the waste valve (222-4). Emptying the water from the VI container triggers the bubble sensor (252), which sends a signal to the controller and stops the waste pump (204-3) and its valve (222-4). Alternatively, if the bubble sensor is not available, the controller can also be programmed to run the pump for a set time until the container is empty. The controller then signals the pump to start and open valve (222-7) to start the flow of buffer from the load reservoir (212). This signal is used to drain the buffer from the VI container and is triggered by the first chromatography system once the Protein A eluate is ready to be loaded into the VI container. The controller receives this signal and triggers the waste pump to remove the buffer from the VI container and triggers the opening of the waste valve (222-4). The VI container draining of buffer triggers the bubble sensor (252), which sends a signal to the controller and stops the waste pump (204-3) and its valve (222-4). Alternatively, if the bubble sensor is not available, the controller can also be programmed to run the pump for a set time until the container is empty.

[0134] After completing the virus inactivation hold time and adjusting the pH value of set point 2, the system pumps the virus inactivated eluate from the virus inactivation container (201) into the collection container (230). The fluid path is digitally controlled by using normally closed solenoid valves / pneumatic pinch valves (222-1 to (222-3) that can block and divert fluid flow as needed.

[0135] In one embodiment, after viral inactivation, the protein A eluate from the VI container (201) passes through a digitally controlled solenoid valve / pneumatic pinch valve (222-1), then through another digitally controlled solenoid valve / pneumatic pinch valve (222-2) and / or (222-3), and is passed through a 0.2-0.45 micron filter (224) and / or (226) to remove any precipitate that may have formed during the viral inactivation step. Optionally, a device for pressure sensors (256-1) and (256-2) can be provided in the fluid path to allow automatic switching between the two filters if one of the filters becomes clogged during continuous operation. The system issues an alarm for operator intervention when the filter becomes clogged and switches. Optionally, a device for a turbidity sensor (254) can be provided in the container after inactivation to measure NTU, and when a threshold is exceeded, the system will trigger an alarm for operator intervention. Optionally, a bubble sensor (252) can be provided after the virus inactivation container, which triggers the closing of the fluid path to prevent bubbles from entering the chromatography system, and a bubble trap prevents bubbles from entering the chromatography system. After clarification by filters (224) and (226), the virus inactivation from the VI container (201) and the neutralization protein eluate are transferred to a collection container (230), after which a controller sends a signal to the second chromatography system, such as an analog signal (such as 0-10V or 4-20mA) or a digital signal from TTL logic or one or more digital interfaces of higher order, the digital interface being selected from, but not limited to, OPC, modbus TCP / IP, EtherCAT, Profibus, Profinet, profibus or industrial Ethernet. A trigger begins loading the protein eluate from the collection container into the second chromatography system via port (232).

[0136] Figure 5An overview of a clean-in-place (CIP) system for a first chromatography system according to an embodiment of the present invention is shown. The CIP system (300) for the chromatography system is operably connected to a controller that sends signals to various actuators (e.g., valves and pumps) in the system to regulate the flow of fluid. The controller receives and sends signals from the chromatography system, such as analog signals (e.g., 0-10V or 4-20mA) or digital signals from TTL logic or one or more higher-level digital interfaces selected from, but not limited to, OPC, modbus TCP / IP, EtherCAT, Profibus, Profinet, profibus, and industrial Ethernet. A signal from the first chromatography system (314) triggers a sample inlet CIP procedure. The controller opens the drain valve (306-2) to direct the fluid / harvest to the surge bag (308) and starts the drain pump (310). The controller opens the CIP NaOH valve (306-3), starts the pump (312), and simultaneously closes the valve (306-1). After a set time for NaOH to flow through the first chromatography system has passed, the controller sends a signal to the NaOH valve (306-3) to close it and simultaneously opens the water valve (306-4). After a set time for water to flow through the first chromatography system has passed, the controller sends a signal to the water valve (306-4) to close it and simultaneously opens the buffer valve (306-5). After a set time for buffer to flow through the first chromatography system has passed, the controller sends a signal to the buffer valve (306-5) to close it and simultaneously opens the drain valve (306-2), pump (310) and valve (306-1), thereby achieving in-situ cleaning of the first chromatography system during the process.

[0137] Further embodiments of the present disclosure relate to an automated integrated biomanufacturing process capable of continuously producing a therapeutic protein, wherein the process is controlled by one or more control systems selected from the group consisting of a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID), a programmable logic circuit (PLC), an industrial PC (IPC), a distributed control system (DCS), and a message relay system, thereby operating the process in an uninterrupted manner.

[0138] In one embodiment, the present invention relates to an automated integrated continuous bioprocess for producing therapeutic proteins in an uninterrupted manner, which is scalable from laboratory scale to production scale, comprising the following steps:

[0139] (a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) capable of using an alternating tangential flow (ATF) filter (109), and collecting an exudate harvest comprising the protein secreted into the culture medium;

[0140] (b) supplying the cell culture harvest containing the therapeutic protein from the bioreactor (103) to a first chromatography system (119) to provide a Protein A eluate;

[0141] (c) supplying the protein A eluate from the first chromatography system (119) to a virus inactivation vessel (128) and inactivating viruses that may be present in the protein A eluate;

[0142] (d) passing the virus inactivated and neutralized Protein A eluate through one or more filters to remove impurities in the form of any precipitates formed during the virus inactivation and neutralization steps, and collecting the filtered Protein A eluate in a collection vessel (136); and

[0143] (e) The neutralized and filtered Protein A eluate is supplied to a second chromatography system (137) to provide further purified protein.

[0144] In one embodiment, the present invention provides an automated integrated continuous bioprocess wherein mammalian cells are cultured in a bioreactor (103) that is a perfusion bioreactor capable of using alternating tangential flow (ATF) technology.

[0145] In one embodiment, the present invention provides an automated integrated continuous bioprocess wherein liquid culture medium is fed directly from the bioreactor (103) to the first chromatography system (119) using a first chromatography system pump.

[0146] In one embodiment, the present invention provides an automated integrated continuous bioprocess wherein a first chromatography step is performed using one or more affinity chromatography columns for purifying a cell culture harvest containing a therapeutic protein from a bioreactor and providing a Protein A eluate.

[0147] In one embodiment, the present invention provides an automated integrated continuous bioprocess wherein the pH of the Protein A eluate in the viral inactivation step is automatically adjusted using a proportional-integral-derivative (PID) controller, a programmable logic controller (PLC), or an industrial personal computer (IPC) controller.

[0148] In one embodiment, the present invention provides an automated integrated continuous bioprocess wherein one or more multimodal anion exchange columns and one or more cation exchange columns are used in the second chromatography step to provide a purified protein.

[0149] In one embodiment, the present invention provides an automated integrated continuous bioprocess, which optionally further comprises storing the purified protein in an additional collection container (140) received from a second chromatography system (137). In one embodiment, the present invention provides an automated integrated continuous bioprocess, which optionally further comprises one or more steps of passing the purified protein through one or more filters (142) to further purify the therapeutic protein. The filter can be selected from a nanofilter, an ultrafilter, and a diafilter.

[0150] In one embodiment, the present invention provides an automated integrated continuous bioprocess, wherein the process includes automated sampling for online testing and cleaning in place (CIP) system (120) for periodic cleaning of the chromatography system and its components (including chromatography columns, tubing, and containers) to maintain uninterrupted operation.

[0151] In one embodiment, the bioprocess optionally includes automated HPLC sampling for online testing of process parameters using a Supervisory Control and Data Acquisition (SCADA) control system, a Clean-in-Place (CIP) system, and switching of liquid flow paths to maintain uninterrupted system operation. In one embodiment, the bioprocess optionally further includes automated UPLC sampling for online testing of process parameters using a Supervisory Control and Data Acquisition (SCADA) control system, a Clean-in-Place (CIP) system, and switching of liquid flow paths to maintain uninterrupted system operation.

[0152] In one embodiment, the integrated continuous bioprocess of the present invention can be controlled by a master controller comprising an IPC with SCADA software for visualization, monitoring, and recording of control data as a historical record of process parameters. The master controller can control the individual systems (e.g., bioreactor, first chromatography system, second chromatography system, and viral inactivation system) using one or more digital interfaces selected from, but not limited to, OPC, Modbus TCP / IP, EtherCAT, Profinet, Profibus, or Industrial Ethernet.

[0153] In another embodiment, the automated bioprocess of the present invention provides for the continuous transfer of product streams from an upstream process to a downstream process.

[0154] In one embodiment, the upstream process is carried out in a bioreactor of an automated integrated continuous bioprocessing system with a control system suitable for mammalian cell culture. In one embodiment, the upstream process is carried out in a bioreactor capable of using ATF technology, wherein a HPLC system pump is used to collect the harvest containing the produced therapeutic protein from the ATF. An exemplary process flow of the upstream process is as follows Figure 1 shown.

[0155] In one embodiment, the upstream process for culturing mammalian cells in a bioreactor employs a perfusion cell culture process, and the batch duration of culturing the mammalian cells is 3 to 16 days. In addition, the upstream process can be performed by culturing the cells in various cell culture media known to those skilled in the art for promoting the growth of mammalian cells in bioreactors.

[0156] In one embodiment, the downstream process includes a purification step of a therapeutic protein produced during an upstream process in a continuous automated integrated bioprocess system, wherein clarified cell culture fluid is supplied to a first chromatography system comprising one or more affinity columns to provide a Protein A eluate, the Protein A eluate is inactivated and neutralized in a virus inactivation system, and supplied to a second chromatography system comprising one or more multimodal anion exchange chromatography columns and one or more cation exchange chromatography columns to provide a purified protein. An exemplary process flow of the downstream process is as follows: Figure 2 shown.

[0157] In various embodiments of the upstream and downstream processes and operations of the system for producing a protein of interest disclosed herein, cells selected from natural, wild-type, mutant, or genetically engineered cells capable of producing the desired protein of interest; nutrients or culture media suitable for the cells used; various chemicals; reagents; resins used in chromatography steps; and any suitable materials can be used. In some embodiments, the various buffers or buffer systems employed include chemicals suitable for use as wash buffers, chase buffers, equilibration buffers, elution buffers, and the like.

[0158] The automated integrated continuous system and bioprocess is capable of producing a therapeutic protein selected from the group consisting of an antibody, an antibody fragment, a monoclonal antibody, an enzyme, an engineered protein, an immunogenic protein, a protein fragment, an immunoglobulin, or any combination thereof.

[0159] The therapeutic protein prepared by the automated integrated continuous bioprocess according to the present invention is selected from the group consisting of panitumumab, omalizumab, abavotumab, abciximab, actosumab, adalimumab, adecamizumab, afelimomab, aftuzumab, aralizumab, aralizumab, alemtuzumab, alirocumab, atumomab, amelezumab, amelezumab, anrukinzumab, apolizumab, acitumomab, atenumab, tocilizumab, basiliximab, betumumab, belimumab, bevacizumab, bezolotoxumab, bevacizumab, bezlotoxumab, bevaciz ... Cetuximab, blinatumomab, canakinumab, certolizumab pegol, cetuximab, citrullumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, ifenguzumab, epratuzumab, ertuinomab, edarizumab, fentolimumab, golimumab, ibritumomab tiuxetan, igovomab, imaclizumab, infliximab, inomucosin, inotuzumab, labetuzumab, lerezumab, moxetumomab, natalizumab, nivolumab, obinutuzumab, ogavuzumab, palivizumab, panitumumab, panitumumab, Tocilizumab, ramucirumab, ranibizumab, rituximab, secukinumab, tocilizumab, tositumomab, trorocitumomab, tucotuzumab, trastuzumab, ustekinumab, vedolizumab, veltuzumab, zalutumumab, zatuximab, enzyme, protein, immunogenic or antigenic protein or protein fragment, alglucosidase alfa, laronidase, abatacept, galsulfase, luteinizing hormone alfa, antihemophilic factor, agalsidase-beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, interferon beta-1a, imiglucerase, Dornase alfa, epoetin alfa, insulin or an insulin analog, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-11, laronidase, idursuphase, galsulphase, alpha-1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alfa, acid beta-galactosidase, beta-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

[0160] Although various embodiments of the present disclosure have been described above, other and further embodiments of the present disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions, or examples, which, when combined with information and knowledge available to one of ordinary skill in the art, are included to enable one of ordinary skill in the art to make and use the invention.

[0161] Example

[0162] The present invention is further explained in the form of the following examples. However, it should be understood that the following examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0163] Example 1

[0164] Production and purification of cetuximab using an automated integrated continuous system and bioprocessing

[0165] Upstream Process - Protein Production

[0166] To produce the target protein, the cell line used in the automated continuous process was the CHO-S cell line with catalog number 11619012 purchased from INVITROGEN, USA.

[0167] Before inoculation of CHO mammalian cells, a dissolved oxygen (DO) calibration was performed by sparging with 0.5 LPM of air. Once the DO value was stable, the DO probe was calibrated to 100%.

[0168] For continuous processing, ATF is used, which is a filtration and perfusion system that uses alternating tangential flow through a filter cartridge to establish high-efficiency filtration. This allows cells to grow at densities as high as 80-100 million cells per milliliter with viability exceeding 90%, resulting in extremely high protein yields.

[0169] The bioreactor is filled to its working volume with culture medium such as ActiPro and then inoculated with mammalian cells (i.e., CHO cells) at a rate of 0.3-0.5×10 6 Live mammalian cells / mL were inoculated. Culture was continued for 3 days. Then the alternating tangential flow (ATF) system was started and within 5-10 minutes the reactor and ATF were in rapid equilibrium. Alternating tangential flow through the filter element was used to establish high efficiency filtration. This allowed the cells to grow at a density of 80-100 million cells per milliliter with a survival rate exceeding 90%, resulting in extremely high protein yields. Depending on the cell concentration, the cells were pumped into and out of the reactor from the hollow fiber at a recirculation rate of 1.5-6 L / min. Then the reaction with AKTA was started. TMPeriodic countercurrent chromatography (PCC) connected to the filtrate pump. From day 3 to day 16, the medium used for perfusion was PM-basal medium + cell Boost7a (1 g / L) and cell Boost (1 g / L) + 8 g / L glucose and 8 mM glutamine.

[0170] The filtration rate that begins is 0.25 reactor volume (RV) at the 3rd day, is 0.5RV at the 4th day, is 1RV at the 5th-7th day, is 1.25RV at the 8th-9th day, is 1.5RV at the 10th-16th day. Ensure that pump carries out enough tight sealing to ensure to keep the vacuum (it is transmembrane pressure) on filtrate side. This is because ATF applies backwash in each circulation, and it is pulled back through filter and enters reactor with a small amount of liquid from filtrate. Because ATF system does not control pump flow rate-it is manually controlled by the flow rate of user by controlling HPLC system pump.

[0171] Cell growth and cell viability were monitored daily throughout the culture process. Cell growth curves are provided in Table 1

[0172] Table 1:

[0173] Cultivation time (days) VCC (mill / mL) vitality(%) 0.0 0.58 99.0 1.0 0.89 99.2 2.0 1.8 99.7 3.0 2.7 98.5 4.0 4.8 98.1 5.0 11.4 99.2 6.0 29.6 99.0 7.0 47.8 98.8 8.0 59.5 98.2 9.0 68.0 98.2 10.0 78.0 97.5 11.0 85.0 98.1 12.0 88.0 96.8 13.0 92.0 95.2 14.0 100.0 95.0 15.0 94.3 93.2 16.0 92.3 90.0

[0174] The data in the above table clearly show that cell viability was maintained at acceptable values ​​(>90%) throughout the culture process under the established culture conditions.

[0175] The effects of cell culture medium on cell growth were also investigated. The cell growth results using glucose and lactate as cell culture medium were as follows: Figure 6 shown.

[0176] Downstream processing: purification of produced proteins

[0177] A. Chromatography The first step was performed using an affinity chromatography system with 4 columns, and the following process parameters for downstream processing are provided in Table 2(a).

[0178] Table 2(a):

[0179]

[0180] The first chromatography system was connected to four columns at four different column positions of an AKTA PCC system and ~3 CV of Milli Q water was passed through the columns to remove the storage solution.

[0181] Column equilibration: Connect the first chromatography system equilibration buffer container to the system and set the label to "equilibrate". All chromatography columns are equilibrated with 3 CV of buffer.

[0182] Loading: The S1 line of the first chromatography system, AKTAPCC, was connected to the ATF outlet and loading was started through it. The setup flags for the "Load" and "Auto Zero UV" commands were given to it. The first cycle loading on the first column took 219 minutes.

[0183] Equilibration buffer wash after loading: After 219 minutes (1 cycle) of loading, the liquid was transferred to chromatography-1 buffer by different pumps of the AKTA PCC system, and the process was switched from the loading step to the washing step. The setting mark of "equilibration buffer wash after loading" was given, and 3CV of buffer was passed through the column.

[0184] In this cycle, when the process steps switch from the loading step to the washing step, the loading step is simultaneously started on another column as a different cycle.

[0185] Intermediate Wash 2: After the loading wash, pass 2 CVs of Wash 2 buffer through the column.

[0186] Wash 3: Wash 2 Buffer After washing, 2 CV of Wash 2 Buffer were passed through the column.

[0187] Elution: The desired protein was eluted using chromatography-1 elution buffer. The elution peak was collected from ↑50 mAU to 3 CV as the total elution volume.

[0188] Regeneration: After elution, the column was regenerated by passing 3 CVs of regeneration buffer.

[0189] MilliQ water wash: Pass 3 CVs of MilliQ water to remove the disinfection solution.

[0190] Equilibration buffer: Pass 3 CVs of EQB to re-equilibrate the column after regeneration.

[0191] Column storage: Pass 2 CV of Chromatography-1 storage solution for column storage (after all cycles are completed).

[0192] B. In an alternative process, an affinity chromatography system with two columns was used for downstream processing to reduce the resin utilization per batch. The process parameters followed are shown in Table 2(b):

[0193] Table 2(b):

[0194]

[0195] Thus, as a result of the first step of chromatography using affinity chromatography to capture the target protein using Mab Select Sure LX resin, the target protein binds to the resin and impurities are removed as they flow through. The chromatogram obtained in this step is shown in Figure 2. Figure 7As shown in the figure, the elution peak corresponding to the target protein eluted using a low pH buffer (pH–2.8) is shown, and the elution peaks of all cycles are consistent with the expected ones.

[0196] Low pH virus inactivation and neutralization

[0197] Perform viral inactivation and neutralization according to Table 3 using the following parameters:

[0198] Table 3:

[0199]

[0200] The pH of the Protein A eluate after mixing was maintained within the expected range of 3.5 ± 0.2 for each cycle (the control system ensured this range through a feedback mechanism). Taking the pH value into account, the protein solution was thoroughly mixed at room temperature (23 ± 2°C) for 45 ± 5 minutes, and the incubation start time, end time, incubation duration, and temperature were recorded.

[0201] After incubation for 45 ± 5 minutes, the pH of the protein solution was adjusted to 5.5 ± 0.2 with 2M Tris base solution by the control system based on a feedback mechanism. The conductivity after neutralization was expected to be about 6 mS / cm. After neutralization, the protein solution was passed through a peristaltic pump driven by the control system. The capsule was passed through a 0.2 μm filter and the filtrate was collected in a second collection vessel for further processing in multimodal and cation exchange chromatography steps.

[0202] Multimodal anion exchange chromatography and cation exchange chromatography

[0203] The protein was purified using a second chromatography system using multimodal anion exchange chromatography and cation exchange chromatography with the following parameters in Table 4:

[0204] Table 4:

[0205]

[0206] The first column as an anion exchange column of the second chromatography system was connected to the universal valve position 2 & 4 of the AKTA pure system, and the second column as a cation exchange column of the second chromatography system was connected to column position 1. 3 CV of MilliQ water was passed through the chromatography column to remove the storage solution.

[0207] High Salt Wash Buffer: Pass 2 CV of High Salt Wash Buffer through the anion exchange and cation exchange columns of the second chromatography system, ensuring that the pH and conductivity of the columns are within the range of the High Salt Wash Buffer.

[0208] Column equilibration: Both columns were equilibrated with 5 CV of equilibration buffer.

[0209] Loading and Buffer Tracking: The neutralized and filtered Protein A eluate sample collected in the collection vessel is loaded through the columns in series, where the target protein does not bind to the anion exchange column of the second chromatography system and is passed through as fluid, binding to the cation exchange column of the second chromatography system. After loading is complete, 100 mL of equilibration buffer is dispensed into the loading vessel and passed through the chromatography columns to ensure complete loading of the neutralized protein eluate sample.

[0210] Post-Loading Equilibration Buffer Wash: After the equilibration buffer chase, when the peak from the anion exchange column of the second chromatography system has stabilized, pass another 2 CVs of equilibration buffer to remove loosely bound and unbound proteins from the cation exchange column of the second chromatography system. The target protein bound to the cation exchange column of the second chromatography system is then eluted using the cation exchange chromatography elution buffer. For a 2 mm pathlength of the flow cell, the elution peaks from the ↑50 mAU peak to the ↓50 mAU peak are collected at UV at 280 nm. The eluted fractions from each cycle are stored at 2 to 8°C until further processing.

[0211] Regeneration: Pass 2 CV of anion exchange and cation exchange chromatography regeneration buffer through both columns.

[0212] MilliQ water wash: Pass 2 CV of MilliQ water to remove the regeneration solution.

[0213] Column Storage: Pass 2 CV of anion exchange and cation exchange storage solutions for column storage (at the end of all cycles).

[0214] Therefore, chromatography step 2 consists of two stages: a first stage of multimodal anion exchange chromatography using Capto Adhere Impres resin and a second stage of cation exchange chromatography using SP sepharose FF resin. After the multimodal anion exchange chromatography, the impurities bind to the column and the target protein passes through as the flow-through. The cation exchange chromatography column is connected in series with the anion exchange chromatography column, where the target protein binds to it. The protein is then eluted from the cation exchange chromatography column using a high salt buffer. The chromatogram obtained in this step is shown in Figure 2. Figure 8 As shown, the elution peak can be consistently observed in all cycles as expected.

[0215] Tangential flow filtration (ultrafiltration and diafiltration)

[0216] The eluates from the cation exchange chromatography were combined and further processed for tangential flow filtration to concentrate the cetuximab protein, which was buffer exchanged into formulation buffer without final excipients according to the parameters shown in Table 5 below:

[0217] Table 5:

[0218] TFF system Cogent μscale Processing box Pellicon Biomax 30 Box Area <![CDATA[0.1m 2 ]]> Box MOC PES Concentration of chromatography 3 eluate Up to 50 mg / mL Buffer exchange 8 diafiltration volumes pressure ≤1 bar Cutoff size / MWCO 30 kDa

[0219] The protein solution recovered from the TFF system was diluted to 5 mg / mL using formulation buffer containing the final excipient solution.

[0220] The resulting solution was sterile filtered through a 0.2 μm filter (MOC-polyethersulfone) into a PETG bottle. The filtrate thus obtained was the final protein product Cetuximab suitable for use as a drug substance.

[0221] The recovery data of exemplary representative batches are observed as follows according to Table 6:

[0222] Table 6:

[0223]

[0224] The above embodiments are illustrative only and should not be construed as limiting the scope of the present invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the present invention.

[0225] Advantages of the present invention

[0226] The present invention provides an automated bioprocess for continuous operation of a multi-day process without the need for repeated container / bag changes during the process.

[0227] The present invention provides an automated integrated continuous bioprocessing system that uses a controller to regulate and monitor the entire upstream and downstream processes using SCADA or DCS.

[0228] The present invention provides an automated integrated continuous bioprocessing system for producing therapeutic proteins, which includes a scalable and accurate real-time automatic pH adjustment system for viral inactivation

[0229] The present invention provides an automated bioprocess and system in which automated on-line sampling for HPLC analysis allows in-depth process analysis and feedback of process parameters.

[0230] The present invention provides an automated integrated continuous bioprocess for producing therapeutic proteins, the system including a pinch valve for trouble-free replacement of tubing after completion of each continuous batch, thereby facilitating easy operation.

Claims

1. An automated integrated continuous bioprocessing system for producing a therapeutic protein, comprising: a bioreactor (103) for culturing mammalian cells capable of producing a therapeutic protein in a culture medium, the bioreactor (103) using an alternating tangential flow (ATF) filtration system (109) to collect a harvest comprising the therapeutic protein secreted into the culture medium; a first chromatography system (119) comprising one or more affinity chromatography columns, connected to an alternating tangential flow (ATF) filtration system (109) of the bioreactor (103) without any intermediate holding vessels, to purify the harvest comprising the therapeutic protein and provide a Protein A eluate; a virus inactivation system (126) comprising a virus inactivation vessel (128) connected to the first chromatography system (119) to collect the Protein A eluate and inactivate viruses present in the eluate, wherein the virus inactivation vessel (128) is configured to automatically adjust the pH value of the Protein A eluate; a collection container (136) connected to the virus inactivation container (128) via a filter to receive the virus-inactivated, neutralized, and filtered Protein A eluate, wherein the filter is configured to remove impurities in the form of precipitates from the virus-inactivated and neutralized Protein A eluate received from the virus inactivation container; a second chromatography system (137) comprising one or more multimodal anion exchange columns and one or more cation exchange columns, connected to the collection vessel (136) to receive the virally inactivated, neutralized, and filtered Protein A eluate from the collection vessel (136) and provide further purified therapeutic protein; one or more solenoid or pneumatic pinch valves (114), flow sensors, bubble sensors, pressure sensors, and load cells for creating a feedback loop to maintain fluid flow between various components of the bioprocess system using tubing, prevent bubble formation, and regulate liquid flow; a clean-in-place (CIP) system (120) for periodic cleaning of the chromatography system, virus inactivation vessels, collection vessels, and tubing; One or more control systems selected from the group consisting of a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID) controller, a programmable logic controller (PLC), an industrial personal computer (IPC), a distributed control system (DCS), an input-output module operably connected to various systems including the first chromatography system (119), the viral inactivation system (126), the collection container (136), the second chromatography system (137), and a message relay system; The virus inactivation system (126) includes a virus inactivation container, a pH probe (220) connected to a transmitter for measuring the pH of the protein A eluate and an automatic titrator (122), a load cell sensor for monitoring the liquid level in each load cell, a level sensor for monitoring the liquid level in the virus inactivation container, a pressure sensor in the fluid path for automatically switching between two filters when one filter is clogged during continuous operation, an online turbidity measurement sensor (254) for measuring real-time turbidimetric turbidity units, and a bubble sensor (252) for preventing bubbles from entering the chromatography system.

2. The automated integrated continuous bioprocessing system according to claim 1, wherein the bioprocessing system further comprises a surge bag (116) connected to the bioreactor via a valve.

3. The automated integrated continuous bioprocessing system of claim 1, wherein each filter between the virus inactivation container and the collection container is a 0.2-0.45 micron filter.

4. The automated integrated continuous bioprocessing system according to claim 1, wherein the virus inactivation container and the collection container are made of glass or stainless steel.

5. The automated integrated continuous bioprocessing system of claim 1, wherein the cleaning-in-place (CIP) system (120) is used to clean a chromatography system, including cleaning components of the chromatography system, including chromatography columns, inlets, tubes, and containers.

6. The automated integrated continuous bioprocessing system of claim 1 , wherein the system further comprises automated harvest sampling from the bioreactor for cell counting and nutrient analysis, and automated sampling at various locations for online chromatography analysis in the continuous bioprocess.

7. An automated integrated continuous bioprocess for producing a therapeutic protein using the automated integrated continuous bioprocess system of claim 1, wherein the process is controlled by one or more control systems selected from the group consisting of a supervisory control and data acquisition (SCADA) control system (110), a proportional integral derivative (PID) controller, a programmable logic controller (PLC), an industrial personal computer (IPC), a distributed control system (DCS), an input-output module operably connected to various systems including a first chromatography system (119), a virus inactivation system (126), a collection container (136), and a second chromatography system (137), and a message relay system, and the process comprises the following steps: (a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) using an alternating tangential flow (ATF) filtration system (109), and collecting a harvest comprising the therapeutic protein secreted into the culture medium; (b) feeding the harvest containing the therapeutic protein from the bioreactor (103) to a first chromatography system (119) comprising one or more affinity chromatography columns to provide a Protein A eluate; (c) supplying the protein A eluate from the first chromatography system (119) to a virus inactivation container (128) and inactivating viruses in the protein A eluate, wherein the virus inactivation container (128) automatically adjusts the pH value of the protein A eluate; (d) passing the virus-inactivated and neutralized Protein A eluate through a filter to remove impurities in the form of any precipitates formed during the virus inactivation and neutralization steps, and collecting the filtered Protein A eluate in a collection vessel (136); and (e) supplying the virally inactivated, neutralized, and filtered Protein A eluate to a second chromatography system (137) comprising one or more multimodal anion exchange columns and one or more cation exchange columns to provide a purified therapeutic protein, and The process includes automated sampling for online testing and cleaning in place using a clean-in-place (CIP) system (120) for periodic cleaning of the chromatography system, virus inactivation vessels, collection vessels, and tubing to maintain uninterrupted operation.

8. The automated integrated continuous bioprocess according to claim 7, wherein the mammalian cells are cultured in a bioreactor (103) which is a perfusion bioreactor using alternating tangential flow (ATF) technology.

9. The automated integrated continuous bioprocess of claim 7, wherein the harvest is fed from the bioreactor (103) directly into the first chromatography system (119) using a first chromatography system pump.

10. The automated integrated continuous bioprocess of claim 7, wherein the pH of the Protein A eluate during viral inactivation is automatically adjusted using a proportional integral derivative (PID) controller, a programmable logic controller (PLC), or an industrial personal computer (IPC).

11. The automated integrated continuous bioprocess of claim 7, wherein the therapeutic protein is selected from the group consisting of an enzyme, a recombinant protein, an immunogenic protein, a peptide, an immunoglobulin, or any combination thereof.

12. The automated integrated continuous bioprocess according to claim 11, wherein the immunoglobulin is an antibody or an antibody fragment.

13. The automated integrated continuous bioprocess according to claim 12, wherein the antibody is a monoclonal antibody.

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