EA044363B1 - AUTOMATED COMPREHENSIVE CONTINUOUS SYSTEM AND BIOPROCESS FOR THE PRODUCTION OF THERAPEUTIC PROTEIN - Google Patents

Description

Field of invention

The present invention generally relates to a system for a continuous bioprocess for the production of a therapeutic protein. In particular, the present invention relates to an automated, end-to-end continuous bioprocess and bioprocess system that enables the continuous production of therapeutic protein and is scalable from laboratory to industrial scale.

State of the art

The description of the prior art includes information that may be useful for understanding the present invention. This is not an admission that any information presented herein is prior art or relates to an invention currently claimed, or that any publication specifically or impliedly referenced constitutes prior art.

Determining the significance of a therapeutic protein has led to a new revolution in the biopharmaceutical industry. However, during the production of a therapeutic protein, charged isoforms commonly arise, which significantly interfere with the isolation and purification processes necessary to achieve high yield and product quality.

Typically, therapeutic proteins are produced by biopharmaceutical companies using batch processes in which unit operations are performed and completed before the process flow moves on to the next step. Recently, biopharmaceutical companies have been using continuous bioprocesses to produce therapeutic proteins. In a continuous bioprocess, the products involved in the process are moved to the next stage after the completion of each unit process. Continuous bioprocessing has attracted much interest due to its various advantages such as steady-state operation, small equipment size, high volumetric throughput, optimized process flow, fast cycle time and reduced capital costs.

The current state of the art has attempted to produce therapeutic protein using continuous bioprocess methods. However, such existing continuous bioprocesses are pseudo-continuous processes containing autonomous units, each of which performs its own function. In such systems, each unit operation has limited communication between individual system parameters and operates primarily in isolation. Additionally, current continuous processes primarily involve offline chromatographic analysis with limited or no feedback to the ongoing process. As biopharmaceutical companies have grown, there has been an unmet need to create automated, end-to-end bioprocesses and systems for therapeutic protein production that can be scaled from the laboratory to the manufacturing level.

Objectives of the present invention

The objective of the present invention is to create an automated complex continuous bioprocess system for the production of therapeutic protein.

Another object of the present invention is to provide an automated, integrated, continuous bioprocess for therapeutic protein production using a control system equipped with communication and programmable control to regulate all process parameters using a master controller.

Another object of the present invention is to provide an automated, integrated, continuous bioprocess for the production of therapeutic protein that can be carried out continuously and continuously.

The essence of the invention

The present invention in some aspects relates to an automated end-to-end bioprocess system for continuous production of a therapeutic protein using a control system equipped with communication and programmable control functionality to regulate all process parameters using a master controller.

In one aspect of the present invention, there is provided an automated end-to-end bioprocess system for therapeutic protein production, comprising:

a bioreactor (103) for culturing mammalian cells capable of producing a therapeutic protein in a culture medium, said bioreactor (103) being provided with an alternating tangential flow (ATF) filter (109) for collecting material containing protein secreted into the culture medium;

a first chromatography system (119) connected to the ATF filtration system (109) of the bioreactor (103) without an intermediate storage vessel, for purifying the collected recombinant therapeutic protein and obtaining a protein A eluate;

a virus inactivation system (126), including (128) a virus inactivation vessel connected to a first chromatography system (119) for collecting protein A eluate and inactivating viruses that may be present in the eluate, and wherein the virus inactivation vessel (128) is configured with the ability to automatically adjust the pH of the protein A eluate;

- 1 044363 collection vessel (136) connected to a virus inactivation vessel (128) through one or more filters for receiving virus-inactivated, neutralized and filtered protein A eluate, said one or more filters (224) and (226) configured to remove impurities in the form of sediments from the eluate of neutralized protein A obtained from the vessel for inactivating the virus;

a second chromatography system (137) connected to a collection vessel (136) to receive the filtered protein A eluate from the collection vessel (136) and obtain further purified protein.

In one aspect of the present invention, an automated end-to-end bioprocess system is provided, optionally further comprising an additional collection vessel (140) connected to a second chromatography system (137) for receiving and storing purified protein, which can be further purified using one or more filters (142), and obtaining further purified therapeutic protein.

In one aspect of the present invention, an automated integrated continuous bioprocess system is provided, further comprising one or more control systems selected from an external control and data acquisition (SCADA) system (110); proportional-integral-derivative (PID) control (not shown); programmable logic circuit (PLS, PLC) (112); industrial PC, distributed control system (DCS) (not shown); input/output modules (130) or input/output blocks (132) and functionally connected to individual systems, for example, chromatographic system 1 (119); virus inactivation systems (128); vessel (136) for collection; chromatographic system 2 (137); and a message relay system (not shown).

In one aspect of the present invention, an automated integrated continuous bioprocess system is provided, further comprising one or more of electromagnetic or pneumatic pinch valves (114) and optionally consisting of flow meters, air bubble sensors, pressure sensors and load compartments to provide a feedback loop to maintain fluid flow between various system components, preventing the formation of air bubbles and regulating fluid flow.

In one aspect of the present invention, an automated integrated continuous bioprocess system is provided, further comprising a transfer bag (116) connected to the bioreactor through one of the pinch valves (114).

In one aspect of the present invention, an automated, integrated, continuous bioprocess system is provided, comprising a virus inactivation vessel (128), one or more pH sensors coupled to a pH transmitter used to measure the pH of a Protein A eluate, and an automatic titrator including a PLC and pumps ( 122), in turn connected to containers (124) containing the acid and base used for titration, level sensors to check the liquid level in the vessel, and built-in turbidity sensors to measure turbidity in nephelometric units (NTU) in real time.

In one aspect of the present invention, an automated end-to-end bioprocess system is provided in which each of the filters between the virus inactivation vessel and the collection vessel is a filter with a pore size of 0.2-0.45 μm.

In one aspect of the present invention, an automated end-to-end bioprocess system is provided in which the virus inactivation vessel, collection vessel, and additional collection vessel are made of glass or stainless steel.

In one aspect of the present invention, an automated, integrated, continuous bioprocess system is provided, wherein a first chromatography system includes one or more affinity chromatography columns and a second chromatography system includes one or more multimodal anion exchange columns and one or more cation exchange columns.

In one aspect of the present invention, an automated end-to-end bioprocess system is provided, which system further includes a clean-in-place (CIP) system (120) for periodically cleaning the chromatography system inlet, virus inactivation vessel, collection vessel, and fluid flow tubing.

In one aspect of the present invention, an automated integrated continuous bioprocess system is provided, the system further comprising automated sampling of the resulting material from the bioreactor for cell counting and nutrient analysis, as well as automated sampling at various locations for on-the-fly chromatographic analysis during the continuous bioprocess .

The present invention in certain other aspects relates to a continuous bioprocess for the continuous production of a therapeutic protein that can be scaled from laboratory to industrial scale.

In one aspect of the present invention, an automated, integrated, continuous bioprocess is provided for the production of a therapeutic protein, the process being controlled by one or more control systems selected from an external control and data acquisition (SCADA) system (110), a proportional-integral-derivative (PID) control system, programmable logic circuit (PLC), industrial PC (IPC), distributed control system (DCS) and message relay system, and the said process includes the steps:

(a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) equipped with an alternating tangential flow (ATF) filter (109), and collecting the released harvest material containing the protein secreted into the culture medium;

(b) supplying the harvested cell culture material containing the therapeutic protein from the bioreactor (103) to the first chromatography system (119) to obtain a protein A eluate;

(c) supplying the protein A eluate from the first chromatography system (119) to the vessel (128) to inactivate the viruses and allowing the viruses to be inactivated and the protein eluate to be neutralized and the protein A eluate to be neutralized;

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

(e) feeding the filtered protein A eluate to a second chromatography system (137) to obtain purified protein.

In one aspect of the present invention, an automated, end-to-end, continuous bioprocess is provided, optionally further comprising storing the purified protein obtained from the second chromatography system (137) in an additional vessel (140). In one aspect of the present invention, an automated, integrated, continuous bioprocess is provided, optionally further comprising one or more steps of passing the purified protein through one or more filters (142) to further purify the therapeutic protein.

In one aspect of the present invention, an automated, end-to-end, continuous bioprocess is provided in which mammalian cells are cultured in a bioreactor (103) that is a perfusion bioreactor that uses alternating tangential flow (ATF) technology.

In one aspect of the present invention, an automated, integrated, continuous bioprocess is provided in which the collected clarified cell culture material is directly fed from the bioreactor (103) to a first chromatography system (119) using a pump of the first chromatography system.

In one aspect of the present invention, an automated, end-to-end, continuous bioprocess is provided wherein a first chromatography 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.

In one aspect of the present invention, an automated, integrated, continuous bioprocess is provided in which the pH of the protein A eluate during the virus inactivation step is controlled automatically by one or more of a proportional integral derivative (PID) controller, a programmable logic controller (PLC), or an industrial computer controller ( IPC).

In one aspect of the present invention, an automated end-to-end bioprocess is provided in which the second chromatography step is performed using one or more multimodal anion exchange columns and one or more cation exchange columns, respectively, to obtain a purified protein.

In one aspect of the present invention, an automated, end-to-end, continuous bioprocess is provided, which process includes automated on-line sampling and clean-in-place (CIP) (120) for periodically cleaning chromatography systems, tubing, and vessels to maintain continuity of operation. .

In one aspect of the present invention, an automated, end-to-end, continuous bioprocess is provided in which the therapeutic protein produced is selected from an antibody, antibody fragment, monoclonal antibody, enzyme, recombinant protein, engineered protein, immunogenic protein, protein fragment, peptide, immunoglobulin, or any combination thereof.

Various objects, features, aspects and advantages of the subject matter will become more apparent from the following detailed description of preferred embodiments.

Brief description of drawings

The accompanying drawings are provided for further understanding of the present invention and are included in the present disclosure and form part of the description thereof. The drawings illustrate exemplary embodiments of the present invention and, together with the description, serve to explain the principles of the present invention.

Fig. 1 illustrates a typical flow of operations for the preceding process.

Fig. 2 illustrates a typical sequence of operations for the subsequent process.

Fig. 3 is a diagram showing an overview of an automated end-to-end bioprocess system for therapeutic protein production.

Fig. 4 is a diagram showing an overview of a virus inactivation system as part of an automated end-to-end bioprocess system for therapeutic protein production.

Fig. 5 is a diagram showing an overview of a CIP chromatography system 1 as part of an automated integrated continuous bioprocess system for therapeutic protein production.

Fig. 6 is a graph showing the effect of lactose versus glucose on cell growth on various days.

Fig. 7 is a chromatogram of the chromatography system 1, showing the pH peak, elution peak and conductivity peak.

Fig. 8 is a chromatogram of the chromatography system 2, showing the elution peak, the eluate peak and the conductivity peak.

Detailed Description of the Invention

Below is a detailed description of embodiments of the present invention. The embodiments are sufficiently detailed to clearly describe the present invention. However, the amount of detail provided is not intended to limit the expected changes in implementation options; rather, it is intended to cover all modifications, equivalents and alternatives falling within the spirit of the present invention as defined by the appended claims.

All publications are incorporated herein by reference to the same extent as if each individual publication or patent application had been specifically and separately identified as being incorporated by reference. If the definition or use of a term in an included source is inconsistent with or inconsistent with the definition of that term given herein, the definition of that term given herein is used and the definition of that term in the source is not applied.

In the present invention, reference to one embodiment or embodiment means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearance of expressions in the same embodiment or in an embodiment at different places herein does not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

In some embodiments, it should be understood that numbers expressing amounts of ingredients, properties such as concentration, reaction conditions, etc., used to describe and include certain embodiments of the invention in the claims, in some cases, are modified by the term approximately. Accordingly, in some embodiments, the numerical parameters set forth in the written description and the accompanying claims are approximate and may vary depending on the desired properties to be obtained by the particular embodiment. In some embodiments, numerical parameters should be interpreted in light of the number of significant figures recorded and using normal rounding techniques. Although the numerical ranges and parameters defining the broad scope of some embodiments of the invention are approximate, the numerical values set forth in the specific examples are given as accurately as practicable. The numerical values presented in some embodiments of the invention may contain certain errors due to the presence of the standard deviation detected in the corresponding test measurements.

As used herein and throughout the claims, the singular forms include the plural forms unless the context clearly indicates otherwise. In addition, as used herein, the preposition in includes in and on, unless the context clearly indicates otherwise.

Unless the context clearly indicates otherwise, throughout the following description, the word contain and its forms, such as contains and containing, are to be construed in an open, inclusive sense, as including but not limited to.

The listing of ranges of values in this document is intended to serve as a shorthand way of individually referring to each specific value that falls within that range. Unless otherwise indicated herein, each individual value is included in the specification of the present invention as if it were separately stated herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any examples or phrases implying the use of an example (eg, such as) presented with respect to certain embodiments herein is intended to better illuminate the present invention and does not limit the spirit of the invention as defined by the claims. Nothing in the specification of the present invention should be interpreted as indicating any element not included in the claims that is essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention described in

- 4 044363 of this document should not be considered as limitations. Each group member may be mentioned or included in the claims individually or in any combination with other group members or other elements found herein. One or more members of the group may be included in or removed from the group for reasons of convenience and/or patentability. With any such inclusion or deletion, the description herein will be deemed to contain the group as modified, thereby satisfying the requirements of the written description.

The following description and embodiments described herein are provided to illustrate an example or examples of specific embodiments of the principles and aspects of the present invention. These examples are provided for the purpose of illustration and not limitation of these principles and the present invention.

In addition, it should be appreciated that the present invention can be implemented in a variety of ways, including as a system, method or device. In the present description, these embodiments, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the described processes can be changed within the spirit of the invention.

The headings and abstract of the present invention presented herein are intended solely for convenience and not as an interpretation of the spirit or meaning of the embodiments.

The following discussion presents many exemplary embodiments of the subject matter. Although each embodiment represents a single combination of elements of the present invention, the subject matter of the present invention is intended to include all possible combinations of the described elements. Thus, if one embodiment contains elements A, B, and C, and a second embodiment contains elements B and D, then the subject matter is considered to contain other remaining combinations of A, B, C, or D, even if they are not expressly described. way.

The various terms used in this document are shown below. If a term used in the claims is not defined below, it should be given the broadest definition accepted for that term by those skilled in the art as reflected in published publications and patents issued at the time the application is filed.

As used herein, the term continuous bioprocess refers to any process involving two or more sequential processing steps, where the output of the previous step (unit operation) is continuously transferred to the next step (unit operation) until the final chromatography step, without necessarily completing the previous processing step before the next processing step begins. In a continuous process, some portion of the target product always passes through the processing system. Ideally, a continuous process is controlled so that each step or unit operation within a continuous process is performed to the greatest extent possible simultaneously and at substantially the same productivity. This ensures maximum cycle time compression and minimum possible completion time.

The term continuous transfer refers to the flow of product moving from a previous unit operation to the next unit operation, and means that the connections or links between two unit operations are such that the previous unit operation transfers the flow of product (directly or through other components) to the second (next) unit operation operation, and that the next unit operation begins before the completion of the previous unit operation (i.e., two successive unit operations process product streams flowing in them simultaneously for at least part of the progress of the overall process that includes the two specified unit operations).

As used herein, the term perfusion cell culture process refers to perfusion culture performed by continuously introducing fresh media into the bioreactor and continuously removing cell-free waste media while maintaining cells in the reactor; thus, higher cell densities can be obtained in perfusion cultures compared to continuous cultures because the cells are retained in the reactor by a cell retention device. The rate of perfusion depends on the needs of the cell line, the concentration of nutrients in the supplied medium, and the level of accumulation of toxic substances.

The term cell culture medium refers to all types of media used in the context of cell culture. Typically, the cell culture medium contains amino acids, at least one carbohydrate as an energy source, trace elements, vitamins, salts and possibly additional components (for example, to influence cell growth and/or productivity and/or product quality.

The term therapeutic protein means a recombinant protein that is sufficiently purified from contaminating proteins, lipids and nucleic acids present in the liquid culture medium, and biological contaminants (eg, viral and bacterial contaminants), or isolated from a host cell (eg, mammalian, yeast or bacteria) that can be included in a pharmaceutical product for the treatment or prevention of various diseases or disorders. Typical examples of therapeutic proteins include, but are not limited to, antibody, antibody fragment, monoclonal antibody, enzyme, engineered protein, immunogenic protein, protein fragment, and immunoglobulin.

The term antibody refers to a functional component of serum and is often referred to as a collection of molecules (antibodies or immunoglobulins, fragments, etc.) or as a molecule. The antibody molecule is capable of binding to or reacting with a specific antigenic determinant, which in turn may result in a specific immunological effect or mechanism.

The term monoclonal antibody refers to an antibody produced by a single cell clone or cell line and consisting of identical antibody molecules.

As used herein, the term retention time refers to the time during which half of the amount of solute is eluted from the chromatography system. It is determined by the length of the column and the rate of migration of the solute and can range from 1 to 30 minutes.

As used herein, the term eluate refers to the liquid eluted from a chromatography column or chromatography membrane and containing a detectable amount of the recombinant therapeutic protein.

The present invention uses various abbreviations, the explanation of which is given below:

ATF: alternating tangential flow;

CV: column volume;

CEX: cation exchange chromatography (CEX);

CIP: cleaning in place;

DO: dissolved oxygen;

DCS: distributed control system;

HPLC: high performance liquid chromatography;

PPK: industrial PC;

min: minutes;

mM: millimolar;

mAU: milliunits of optical density;

ml: milliliter;

mS/cm: millisiemens/centimeter;

mA: milliamps;

NTU: nephelometric turbidity unit;

NaOH: sodium hydroxide;

PLC: programmable logic circuit;

PCC: periodic counterflow;

PID: proportional-integral-derivative control;

RV: reactor volume;

TTL: transistor-transistor logic circuit;

UF: ultraviolet;

With HPLC: ultra-performance liquid chromatography;

V I: virus inactivation;

V: volts;

V CC: number of viable cells.

The present invention, in some embodiments, relates to an automated end-to-end bioprocess system for continuous production of a therapeutic protein, controlled by a control system equipped with communication and programmable control functions to regulate all process parameters using a master controller.

The present invention will be more specifically described in the following embodiments with reference to a comprehensive automated system for continuous production of a therapeutic protein.

In fig. 3 illustrates a representative automated end-to-end system for continuous production of a therapeutic protein in accordance with an embodiment of the present invention described below herein:

The automated, complete system (100) for continuous production of therapeutic protein includes:

bioreactor (103) for cultivating mammalian cells capable of producing a therapeutic protein in a culture medium, wherein the bioreactor (103) is equipped with a stirrer (102) with attached blades (105), an inlet (104) for supply and is equipped with a perfusion filter (109) with a variable tangential flow (ATF) to collect harvest material containing protein secreted into the culture medium in the bioreactor;

a first chromatography system (119) connected to an ATF filtration system of a bioreactor (103) without an intermediate storage vessel for purifying a recombinant therapeutic protein produced by cultured mammalian cells from the culture medium and obtaining

- 6 044363 protein A eluate;

a virus inactivation system (126), including a virus inactivation vessel (128) connected to a first chromatography system (119) for collecting the Protein A eluate and inactivating viruses that may be present in the Protein A eluate, wherein the virus inactivation vessel (128) is configured with the ability to automatically adjust the pH of the protein A eluate;

a collection vessel (136) connected to a virus inactivation vessel (128) through one or more filters for receiving the neutralized Protein A eluate from the virus inactivation vessel (128) after the neutralized Protein A eluate has passed through one or more filters to remove impurities as precipitation from protein A eluate;

a second chromatography system (137) connected to a collection vessel (136) to receive the neutralized and filtered protein A eluate from the collection vessel (136) to obtain purified protein.

In one embodiment, the present invention provides an automated end-to-end bioprocess system, optionally further comprising an additional collection vessel (140) connected to a second chromatography system (137) for receiving and storing purified protein, which can be further purified using one or more filters (142), and obtain further purified therapeutic protein.

In one embodiment, the present invention provides an automated end-to-end bioprocess system, further comprising one or more control systems selected from an external control and data acquisition (SCADA) system (110); Proportional Integral Derivative (PID) control (not shown); programmable logic circuit (PLC), industrial PC (IPC), distributed control system (DCS) (not shown); input/output modules or input/output units (130) and (132) operatively coupled to individual systems, including chromatography system 1 (119); systems (126) for inactivating viruses; collection vessel (136); chromatographic system 2 (137); and message relay systems (not shown).

In one embodiment of the present invention, an automated integrated continuous bioprocess system is provided, further comprising one or more electromagnetic or pneumatic pinch valves (114) and optionally including flow meters, air bubble sensors, pressure sensors and cargo compartments to provide a feedback loop to maintain fluid flow between various system components, preventing the formation of air bubbles and regulating fluid flow.

In one embodiment of the present invention, an automated integrated continuous bioprocess system is provided, further comprising a transfer bag (116) connected to the bioreactor through one of the pinch valves (114).

In one embodiment, the present invention provides an automated end-to-end bioprocess system comprising a virus inactivation system (126), including a virus inactivation vessel (128), one or more pH sensors coupled to a pH transmitter used to measure the pH of a protein A eluate, and an automatic titrator including a PLC and pumps (122), in turn connected to containers containing the acid and base used for titration (124), optionally containing level sensors for checking the level of liquid in the vessel and built-in turbidity sensors for measuring turbidity in nephelometric units (NTU) in real time.

In one embodiment, the present invention provides an automated end-to-end bioprocess system in which each of the filters between the virus inactivation vessel and the collection vessel is a filter with a pore size of 0.2-0.45 μm.

In one embodiment, the present invention provides an automated end-to-end bioprocess system, wherein a first chromatography system includes one or more affinity chromatography columns and a second chromatography system includes one or more multimodal anion exchange columns and one or more cation exchange columns.

In one embodiment, the present invention provides an automated end-to-end bioprocess system in which the virus inactivation vessel, collection vessel, and additional collection vessel are made of glass or stainless steel.

In one embodiment, the present invention provides an automated, integrated, continuous bioprocess system in which fluid flow is maintained using tubing made from a suitable material, such as silicone and bioprene. In one embodiment, the connectors used are aseptic connectors to reduce bioburden on the system.

In one embodiment, the present invention provides an automated end-to-end bioprocess system, which system further includes a clean-in-place (CIP) system (120) for periodically cleaning the chromatography system, virus inactivation vessel, collection vessel, and tubing.

In one embodiment of the present invention, a system for automated

- 7 044363 complex continuous bioprocess, the system further including automated sampling of the resulting material from the bioreactor for cell counting and nutrient analysis, as well as automated sampling at various locations for chromatographic analysis during operation during the continuous bioprocess.

In one embodiment, the automated end-to-end bioprocess system optionally comprises ultra-performance liquid chromatography (UPLC) systems (108) and (138). In some embodiments, the first and second chromatography systems used in the present invention may be high pressure liquid chromatography (HPLC) systems. In yet another embodiment, the system provides the ability to integrate/analyze a system using an automated sampling system. The controller triggers the HPLC systems to run the analysis using a suitable one or more digital interfaces selected from, but not limited to, Open Platform Communications (OPC), Modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus and industrial Ethernet.

In one embodiment, the controller controls the multiport electric rotary valve or flow selection valves to control fluid flow. The controller controls the startup of the precision pump and opens the bioreactor port valve for sampling. The programmed amount of liquid is collected and the HPLC system performs analysis. The HPLC system performs the analysis according to the set program. The HPLC system sends analysis data to the SCADA system.

In yet another implementation, a master controller consisting of an IPC with SCADA software manages data visualization, monitoring, and acts as a process parameter historian. The main controller communicates with the individual systems, for example, the bioreactor, the first chromatography system, the second chromatography system and the virus inactivation system using one or more of digital interfaces selected from OPC, modbus TCP/IP, EtherCAT, profinet, profibus or industrial Ethernet, but not limited to them.

In fig. 4 shows an overview of a virus inactivation system in accordance with an embodiment of the present invention. The virus inactivation system (200) contains a virus inactivation vessel (VI) (201), made of glass or stainless steel, for collecting the protein A eluate coming from the first chromatography system. Vessel VI (201) contains an overhead stirrer/magnetic stirrer (216) with multiple ports for adding and removing eluate. The virus inactivation system includes a pH sensor (220) coupled to a transmitter for measuring the pH of the protein A eluate obtained from the first chromatography system. The system further optionally includes cargo compartments (202), (206), (208), (210), (212) and (214) for storing various liquids, such as acid, base, NaOH, water and buffers. The system additionally contains filters (224) and (226) with a pore size of 0.2-0.45 microns to remove sediments that may have formed during the neutralization step. The system may further include flow sensors (250-1)-(250-3) for monitoring the flow of various liquids into the VI vessel (201) and the collection vessel (230). The system may also include level sensor(s) for monitoring the liquid level in vessel VI and load compartment sensors (248-1)-(248-6) for monitoring the liquid level in the respective load compartments. The control system has the ability to automatically switch the fluid path between the two filters if one of the filters becomes clogged during continuous operation, which may be based on the use of pressure sensors (2561) and (256-2) or a switch. The system can sound an alarm when the filters are clogged and switch for operator intervention. The system may further include a turbidity sensor (254) downstream of the inactivation vessel to measure turbidity in nephelometric units (NTU). If the threshold value is exceeded, the system will generate an alarm for operator intervention. An additional air bubble sensor (252) attached to the fluid path downstream of the virus inactivation vessel may switch the fluid path to prevent air bubbles from entering the chromatography system. An air bubble trap (not shown) prevents air bubbles from entering chromatography systems. The Virus Inactivation Vessel (VI) is specially designed to gently add water, eluate, acid, base and buffer(s) to avoid foaming and splashing of liquids. The system consists of a collection vessel (230), which is a vessel for collecting the eluate after inactivation of the viruses. The collection vessel (230) contains an overhead stirrer/magnetic stirrer (236) with multiple ports for adding and removing various liquids and eluate.

In one embodiment, the virus inactivation system includes various sensors for monitoring and regulating the virus inactivation system in a continuous and automatic manner without interruption.

In one embodiment, the virus inactivation system optionally includes level sensors or load cell sensors selected from sensors (248-1) for monitoring acid level, (248-2) for monitoring base level, (248-3) for monitoring NaOH level, ( 248-4) for monitoring water levels, (248-5) and (248-6) for monitoring buffer levels, but not limited to.

In one embodiment, the virus inactivation system optionally includes flow sensors for monitoring the flow of various liquids into the VI vessel (201) and the collection vessel (230), wherein the flow sensors are selected from sensors (250-1) for monitoring the flow of acid, (250-2) for monitoring the flow of base; and (250-3) for monitoring the flow of NaOH, water and buffers, but not limited to.

In one embodiment, the virus inactivation system is operably coupled to a host controller, such as a PID, PLC, or IPC, to measure and control pH in real time. The control unit is programmed with several parameters, such as set pH value, holding time, stirrer revolutions per minute, acid and alkali pump speed control, CIP cycle control. The virus inactivation system is controlled in a variety of ways, which are selected from various settings (238), main power supply (240), automatic or manual mode selection (242), alarm (244), calibration (258), and system shutdown ( 246), but are not limited to them. The controller provides signals to various actuators in the system, such as valves (222-1)-(222-10) and pumps (204-1)-(204-5), to regulate fluid flow and also control the addition of acid and base to protein A eluate to adjust the pH.

In one implementation, the controller receives and sends signals from chromatography systems, for example, analog signals, such as 0-10 V or 4-20 mA, or digital signals from TTL logic circuits or one or more of the more advanced interfaces selected from OPC, Modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus and industrial Ethernet, but not limited to. The signal from the first chromatography system starts the virus inactivation program. The transmitter measures the signal generated by the pH sensor (220) and transmits the values to the main controller. The system automatically adjusts the pH of the eluate based on set values in the controller, which activates the addition of acid (202) or base (206), followed by a dwell time for the virus inactivation step. Once the virus inactivation dwell time has been completed and the pH has been adjusted to set point 2, the system pumps the virus inactivation eluate from the virus inactivation vessel (201) to the collection vessel (230). Fluid paths are digitally controlled using normally closed solenoid/pneumatic pinch valves (222-1)-(222-3), which block and divert fluid flow as needed.

In one embodiment, after transferring the Protein A eluate from the virus inactivation vessel to the collection vessel, the controller sends signals to the second chromatography system, for example, analog signals such as 0-10 V or 4-20 mA, or digital signals from TTL or TTL logic circuits. one or more of the more advanced digital interfaces selected from, but not limited to, OPC, modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus and industrial Ethernet. The trigger mechanism initiates loading of the eluate from the collection vessel into a second chromatography system to further purify the Protein A eluate to produce purified protein.

In one embodiment, the controller initiates a CIP cycle in the vessel to inactivate viruses. In vessel VI, during the CIP cycle, the controller signals the pump (204-4) to start and opens the valve (222-5) to allow NaOH to flow from the load compartment (208). The flow of NaOH into vessel VI is monitored by a flow sensor (250-3) and controlled by a pump (204-4). After a predetermined time adjusted by the holding time setting means (218) for holding NaOH in vessel VI, the controller signals the waste pump (204-3) to remove NaOH from vessel VI and opens the waste valve (222-4) to remove waste (228). Emptying vessel VI of NaOH triggers the air bubble sensor (252), which sends a signal to the controller and stops the waste pump (252) (204-3) and its valve (222-4), alternatively, if there is no air bubble sensor, the controller it can also be programmed to run the pump for a set time until the vessel is emptied. The controller then signals the pump (204-4) to start and opens the valve (222-6), allowing water to flow from the load compartment (210) and regulating the flow of water through the valve (222-6) controlled by the sensor (250-3) and driven by a pump (204-4), into vessel VI (201). After the preset time for holding water in Vessel VI, the controller signals the waste pump (204-3) to remove water from Vessel VI and open the waste water valve (222-4). Emptying vessel VI of water triggers the air 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 air bubble sensor is not present the controller can also be programmed to start the pump for a set time until the tank is empty. The controller then signals the pump to start and opens the valve (222-7), initiating the flow of buffer from the load compartment (212). The signal to empty vessel VI from the buffer is triggered by the first chromatographic system when protein A is ready to be loaded into vessel VI. The controller receives this signal, turns on the waste pump to remove buffer from vessel VI and opens the waste valve (222-4). Emptying vessel VI of the buffer triggers the air 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 air bubble sensor is not present the controller can also be programmed to start the pump for a set time until the tank is empty.

Once the virus inactivation dwell time has been completed and the pH has been adjusted to a set point of 2, the system pumps the virus inactivation eluate from the virus inactivation vessel (201) to the collection vessel (230). Fluid paths are digitally controlled using normally closed solenoid/pneumatic pinch valves (222-1)-(222-3), which block and divert fluid flow as required.

In one embodiment, after inactivation of the viruses, Protein A is eluted from the VI vessel (201) through a digitally controlled solenoid/pneumatic pinch valve (222-1), followed by another solenoid/pneumatic pinch valve (222-2) and/or ( 222-3) digitally controlled passes through filters (224) and/or (226) with a pore size of 0.2-0.45 microns to remove sediments that may have formed during the virus inactivation step. Optionally, the fluid path pressure sensors (256-1) and (256-2) may be configured to automatically switch between the two filters if one of the filters becomes clogged during continuous operation. The system gives an alarm when the filters are clogged and switches for operator intervention. Optionally, there may be settings for a turbidity sensor (254) downstream of the inactivation vessel measuring NTU; When the threshold value is reached, the system will sound an alarm for operator intervention. Optionally, the air bubble sensor (252) may be configured downstream of the virus inactivation vessel to trigger the shutdown of fluid paths to prevent air bubbles from entering the chromatography systems, wherein the air bubble trap prevents air bubbles from entering the chromatography systems. When the virus-inactivated and neutralized protein is transferred from the VI vessel (201) after clarification through filters (224) and (226) to the collection vessel (230), the controller sends signals to the second chromatography system, for example, analog signals 0-10 V or 4- 20 mA or digital signals from TTL logic or one or more of the more advanced digital interfaces selected from, but not limited to, OPC, Modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus or industrial Ethernet. The trigger mechanism initiates loading of the protein eluate from the collection vessel into the second chromatography system through port (232).

In fig. 5 shows an overview of a clean in place (CIP) system for a first chromatography system in accordance with an embodiment of the present invention. The CIP system (300) for the chromatography system is operatively coupled to a controller that provides a signal to various actuators, such as valves and pumps, within the system to control fluid flow. The controller receives and sends signals from chromatography systems, such as analog signals such as 0-10 V or 4-20 mA, or digital signals from TTL logic circuits or one or more of the digital interfaces selected from OPC, Modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus and industrial Ethernet, but not limited to. A signal from the first chromatography system (314) triggers the sample inlet CIP program. The controller opens the bleed valve (306-2), directing fluid/material to the transfer bag (308) and activating the bleed pump (310). The controller opens the CIP NaOH valve (306-3), starts the pump (312) and simultaneously closes the valve (306-1). After a predetermined time has elapsed for NaOH flow through the first chromatography system, the controller signals the NaOH valve (306-3) to close and simultaneously opens the water valve (306-4). After a predetermined time for water flow through the first chromatography system has elapsed, the controller signals the water valve (306-4) to close and simultaneously opens the buffer valve (306-5). After a predetermined time for buffer flow through the first chromatography system, the controller signals the buffer valve (306-5) to close, and the bleed valve (306-2), pump (310) and valve (306-1) simultaneously open, causing in situ purification of the first chromatography system during the process is achieved.

Additional embodiments of the present invention relate to an automated, integrated biomanufacturing process that provides continuous production of a therapeutic protein, the process being controlled by one or more control systems selected from an external control and data acquisition (SCADA) proportional integral derivative (PID) system (110). ), programmable logic circuit (PLC), industrial PC (IPC), distributed control system (DCS) and message relay system, to start and continuously run the process.

In one embodiment, the present invention relates to an automated, end-to-end, continuous bioprocess for the continuous production of a therapeutic protein, scalable from laboratory to industrial scale according to the present invention and comprising the steps of:

(a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) equipped with an alternating tangential flow (ATF) filter (109), and collecting the released harvest material containing the protein secreted into the culture medium;

(b) supplying the harvested cell culture material containing the therapeutic protein from the bioreactor (103) to the first chromatography system (119) to obtain a protein A eluate;

(c) supplying the protein A eluate from the first chromatography system (119) to the virus inactivation vessel (128) and allowing viruses to be inactivated and the protein eluate to be neutralized that may be present in the protein A eluate;

(d) passing the virus-inactivated and neutralized protein A eluate through one or more

- 10 044363 more filters for removing impurities in the form of sediment formed during the stages of inactivation and neutralization of the virus, and collecting the filtered protein A eluate in a collection vessel (136) and;

(e) feeding the neutralized and filtered protein A eluate to a second chromatography system (137) to obtain further purified protein.

In one embodiment, the present invention provides an automated, integrated, continuous bioprocess in which mammalian cells are cultured in a bioreactor (103) that is a perfusion bioreactor equipped with variable tangential flow (ATF) means.

In one embodiment, the present invention provides an automated, integrated, continuous bioprocess in which collected clarified cell culture material is directly fed from a bioreactor (103) to a first chromatography system (119) using a first chromatography system pump.

In one embodiment, the present invention provides an automated, end-to-end, continuous bioprocess in which 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 to produce a Protein A eluate.

In one embodiment, the present invention provides an automated, integrated, continuous bioprocess in which the pH of the protein A eluate during the virus inactivation step is automatically controlled using a proportional integral derivative (PID) controller, a programmable logic controller (PLC), or an industrial personal computer (IPC) controller. .

In one embodiment, the present invention provides an automated, end-to-end, continuous bioprocess in which a second chromatography step is performed using one or more multimodal anion exchange columns and one or more cation exchange columns to obtain purified protein.

In one embodiment of the present invention, an automated end-to-end bioprocess is provided, optionally further including storing the purified protein obtained from the second chromatography system (137) in an additional collection vessel (140). In one embodiment, the present invention provides an automated, end-to-end, continuous bioprocess, optionally further comprising one or more steps of passing the purified protein through one or more filters (142) to further purify the therapeutic protein. The filter(s) can be selected from nanofilter, ultrafilter and diafilter.

In one embodiment of the present invention, an automated, end-to-end, continuous bioprocess is provided, the process including automated on-line sampling and cleaning in place (CIP) system (120) for periodically cleaning chromatography system(s) and their components, including chromatography columns, tubes and vessels to maintain continuity of operation.

In one embodiment, the bioprocess optionally includes automated HPLC sampling to verify process parameters during operation using an external control and data acquisition (SCADA) system, clean in place (CIP) system, and fluid flow path switching to maintain system continuity. In one embodiment, the bioprocess optionally further includes automated UPLC sampling to verify process parameters during operation using an external control and data acquisition (SCADA) system, a clean in place (CIP) system, and fluid flow path switching to maintain system continuity. .

In yet another embodiment, the complex continuous bioprocess of the present invention may be controlled by a master controller consisting of an IPC with SCADA software to control data visualization, monitoring, and acting as a process parameter historian. The master controller is capable of controlling individual systems, for example, the bioreactor, the first chromatography system, the second chromatography system and the virus inactivation system using one or more digital interfaces selected from OPC, modbus TCP/IP, EtherCAT, Profibus, Profinet, profibus or industrial Ethernet etc., but not limited to them.

In yet another embodiment, the automated bioprocess of the present invention provides a continuous transfer of product flow from the previous process to the next process.

In one embodiment, the previous process is performed in a bioreactor of an automated integrated continuous bioprocess system with control systems suitable for culturing mammalian cells. In one embodiment, the previous process is performed in a bioreactor using ATF technology, wherein the material containing the therapeutic protein produced is collected using an ATF HPLC system pump. A typical sequence of operations for the previous process is shown in FIG. 1.

- 11 044363

In one embodiment, the previous process for culturing mammalian cells in a bioreactor uses a perfusion cell culture process, and the duration of culturing the series of mammalian cells is selected from 3 to 16 days. In addition, the previous process can be performed by culturing the cells in another cell culture medium known to one skilled in the art to enhance the growth of mammalian cells in the bioreactor.

In one embodiment, the following process includes the steps of purifying a therapeutic protein produced during a previous process in a continuous automated integrated bioprocess system, wherein the clarified cell culture liquid is fed to a first chromatography system containing one or more affinity columns to produce a protein A eluate, inactivation and neutralizing the protein A eluate in a virus inactivation system, and fed to a second chromatography system comprising one or more multimodal anion exchange chromatography columns and one or more cation exchange chromatography columns to obtain purified protein. A typical sequence of operations for the following process is shown in FIG. 2.

In various embodiments, during the preceding and subsequent processes and operation of the system of the present invention, cells selected from native, wild-type, mutated, or recombinant cells capable of producing the desired protein of interest may be used to produce the protein of interest; nutrient or culture media suitable for the cells used; various chemicals; reagents; resins used in chromatography steps; and any suitable materials. In some embodiments, the various buffers or buffer systems used contain chemicals suitable for use as a wash buffer, displacement buffer, equilibration buffer, elution buffer, or the like.

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

The therapeutic protein produced by the automated integrated continuous bioprocess in accordance with the present invention is selected from the group consisting of: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, al tumomab , amatuximab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab, tocilizumab, basiliximab, bestumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, biciromab, blinatumomab, canakinumab, certolizumab, tse tuximab, cixutumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab , efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab, imgatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab, nivolumab, obi nutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, secukinumab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, ustekinumab, vedolizumab, veltuzumab, zalutumumab, zatuximab, enzymes, proteins, immunogenic or antigenic proteins or protein fragments, alpha-glucosidase, laronidase, abatacept, galsulfase, alpha lutropin, antihemophilic factor , beta-agalsidase, interferon-beta Ia, darbepoetin-alpha, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, interferon-beta Ia, imiglucerase, dornase-alpha, epoetin-alpha, insulin or insulin analogues, mecasermin, factor VIII, factor VIIa , antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin-11, laronidase, idursulfase, galsulfase, alpha-1 proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, alpha-thyrotropin, acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A and hexosaminidase B.

Although various embodiments of the present invention have been described above, other and additional embodiments of the present invention can be developed without departing from the spirit thereof. The essence of the invention is determined by the following claims. The present invention is not limited to the embodiments, versions or examples described, which are included to enable one of ordinary skill in the art to make and use the invention in combination with information and knowledge available to one of ordinary skill in the art.

Examples

The present invention is further explained in the form of the following examples. At the same time, it should be understood that the following examples are purely illustrative and should not be construed as limiting the spirit of the invention.

Example 1.

Production and purification of cetixumab using an automated integrated continuous system and bioprocess.

Previous process - protein production

For the production of the protein of interest, the cell line used in the automated continuous process was the CHO-S cell line with catalog number 11619012, purchased from INVITROGEN, USA.

Before seeding mammalian cells, CHO calibration of dissolved oxygen (DO) was performed by bubbling air at a flow rate of 0.5 L/min. Once the DO value had stabilized, the DO sensor was calibrated to 100%.

For the continuous process, an ATF, filtration and perfusion system was used, which used alternating tangential flow through the filter cartridge for highly efficient filtration. This allowed cells to grow to densities of 80-100 million cells per milliliter with a viability of over 90%, resulting in extremely high protein yields.

The bioreactor was filled to the working volume with a culture medium, for example, ActiPro, and then seeded with mammalian cells, i.e. CHO cells, in an amount of 0.3-0.5x106 viable mammalian cells/ml. Cultivation was started for 3 days. Then the alternating tangential flow (ATF) system was started and within 5-10 minutes the reactor and ATF came to equilibrium. Alternating tangential flow through the filter cartridge was used for highly efficient filtration. This allowed cells to grow to densities of 80-100 million cells per milliliter with a viability of over 90%, resulting in extremely high protein yields. Cells were pumped into and out of the hollow fiber into the reactor at a recirculation rate of 1.5-6 L/min depending on cell concentration. The filtrate pump connected to the AKTA™ batch countercurrent chromatography (BCC) system was then started. Media used from days 3 to 16 for perfusion were PM-Basal media + 7a cell stimulation media (1 g/L) and cell stimulation media (1 g/L) + 8 g/L glucose and 8 mM glutamine.

Initial filtration rates were 0.25 reactor volume (RV) on days 3, 0.5 RV on days 4, 1 RV on days 5-7, 1.25 RV on days 8-9, and 1.5 RV on days 10-16 day. A check was made to ensure that the pump seal was secure enough to ensure that the filtrate side vacuum (transmembrane pressure) was maintained. This is because the ATF performs a reverse circulation in each cycle, which draws a small amount of liquid from the filtrate back through the filter into the reactor. Since the ATF system did not control the flow rate of the pump, the user manually controlled it by controlling the flow rate of the HPLC system pump.

Cell growth and cell viability were monitored daily in all cultures. The cell growth profile is presented in table. 1.

_________________________________Table 1

Culture age (days) VCC (million/ml) Viability (%) 0.0 0.58 99.0 1.0 0.89 99.2 2.0 1.8 99.7 h,o 2.7 98.5 4.0 4.8 98.1 5.0 I,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 and about 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

The data in the above table clearly shows that cell viability was maintained at an acceptable level (>90%) in all cultures under established culture conditions.

In addition, the effect of cell culture medium on cell growth was examined. Cell growth results using glucose and lactate as cell culture media are shown in FIG. 6.

Next process: purification of the produced protein.

A. Stage 1 chromatography was performed using a 4 co-affinity chromatography system.

- 13 044363 lonks with the parameters of the following process indicated in the table. 2(a).

Table 2(a)

Process stage Process parameters Affinity chromatography Affinity chromatography: - Speakers used: 4 speakers - Column diameter: 5 cm - Resin: Mab Select Sure LX - Layer height: 8.6 ± 0.2 cm. - Layer volume: ~ 168 ml - Residence time: ~4 min - Working flow: 42 ml/min - Equilibration buffer: 50 mM sodium phosphate buffer, pH 7.0 - Wash buffer 2: 50 mM sodium phosphate buffer + 1 MNaCl, pH 7.0 - Wash buffer 3: 50 mM sodium acetate buffer, pH 5.5 - Elution buffer: 200 mM acetic acid - Solution for regeneration and disinfection: 100 mM sodium hydroxide solution - Column storage solution: 20% ethanol or 2% benzyl alcohol. - Load factor: ~35 mg/ml - Elution: stepwise elution with elution buffer. - Collection after elution: for UV flow cell with with a path length of 2 mm, a fraction of the eluate from 50 milliunits of optical density on the rising side to three protein A column volumes (168 ml x 3) ml was collected.

The first chromatography system was connected to four columns with four different column positions in the AKTA PCC system, and ~3 CV of Milli Q water was passed through the columns to remove storage solution. Column Equilibration: The first container containing the chromatography system equilibration buffer was connected to the system and marked

Balancing. All columns were equilibrated by passing 3 CV buffer.

Loading: Line S1 of the first AKTA PCC chromatography system was connected to the ATF output and loading was started through it. For it, the Loading mark was checked and the command Automatic UV zeroing was given. Loading 1 cycle onto the first column took 219 minutes.

Post-Load Equilibration Buffer Wash: After loading for 219 min (1 cycle), the process was switched from the loading step to the wash step by directing flow to Chromatography Buffer-1 through another AKTA PCC system pump. The Post-Loading Equilibration Buffer Wash was checked and 3 CV of buffer was passed through the column.

When the process was switched from the loading step to the washing step in a given cycle, the loading step was simultaneously started in another column as another cycle.

Intermediate Wash 2: After the postload wash, 2 CV of Wash Buffer 2 was passed through the column.

Wash 3: After washing with Wash Buffer 2, 2 CV of Wash Buffer 2 was passed through the column.

Elution: The desired protein was eluted with Chromatography Elution Buffer-1. The elution peak was collected from F50 milliunits of optical density to a total elution volume of 3 CV.

Regeneration: After elution, the column was regenerated by passing 3 CV of regeneration buffer.

Rinse with Milli Q water: run 3 CV of Milli Q water to remove the disinfectant solution.

Equilibration Buffer: Run 3 CV EQB to re-equilibrate the column after regeneration.

Column Storage: To store the column (after completion of all runs), 2 CV of Chromatography Storage Solution-1 was passed through.

B. In an alternative method, the following process was performed using a dual column affinity chromatography system to reduce resin usage per batch. The process parameters corresponded to those given in table. 2(b).

- 14 044363

Table 2(b)

Process stage Process parameters Affinity chromatography Affinity chromatography: - Speakers used: two - Column diameter: 5 cm - Resin: Mab Select Sure LX - Layer height: 4.2 ± 0.2 cm - Layer volume: ~84 ml - Residence time: ~2 min - Working flow: 42 ml/min - Equilibration buffer: 50 mM sodium phosphate buffer, pH 7.0 - Wash buffer 2: 50 mM sodium phosphate buffer + 1 M NaCl, pH 7.0 - Wash buffer 3: 50 mM sodium acetate buffer, pH 5.5 - Elution buffer: 200 mM acetic acid - Solution for regeneration and disinfection: 100 mM sodium hydroxide solution - Column storage solution: 20% ethanol or 2% benzyl alcohol. - Loading factor: ~35 mg/ml - Elution: stepwise elution with elution buffer. - Post-elution collection: For a UV flow cell path length of 2 mm, the eluate fraction from 50 mU absorbance on the rising side to three protein A column volumes (294 ml x 3) ml was collected.

Thus, as a result of 1 step of chromatography performed by affinity chromatography using Mab Select Sure LX resin capturing the protein of interest, said protein of interest was bound to the resin and impurities were removed as they passed through it. The chromatogram obtained at this stage is shown in Fig. 7, which shows the elution peak corresponding to the protein of interest eluted using a low pH buffer (pH 2.8), and the elution peak was systematically observed for all cycles, as expected. Low pH Virus Inactivation Treatment and Neutralization Virus inactivation and neutralization were performed using the following parameters listed in Table. 3.

Table 3

Virus inactivation at low pH Inactivation at low pH: - Inactivation pH: 3.5 ± 0.2 - Incubation time: 45 min ± 5 - Temperature: RT (23 ± 2) °C - Neutralization buffer: 2 M Tris base - Neutralization pH: 5.5 ± 0.2

The pH of the Protein A eluate in each cycle was maintained within the expected range of 3.5 ± 0.2 after mixing (the control system ensured this range through a feedback mechanism). After taking into account the pH, complete mixing of the protein solution was performed for 45 ± 5 min at room temperature (23 ± 2°C) and the start time, end time of incubation, incubation duration and temperature were recorded.

After 45 ± 5 min of incubation, the pH of the protein solution was adjusted to 5.5 ± 0.2 using 2 M Tris base solution using a feedback control system. The expected conductivity after neutralization was ~6 mS/cm. After neutralization, the protein solution was passed through a 0.2 μm filter in a Sartopore® capsule using a peristaltic pump driven by a control system, and the filtrate was collected in a second collection vessel for further processing in the multimodal and cation exchange chromatography step.

Multimodal anion exchange chromatography and cation exchange chromatography.

The protein was purified using a second chromatography system using multimodal anion exchange chromatography and cation exchange chromatography with the following parameters shown in table. 4.

- 15 044363

Table 4

Anion and cation exchange chromatography Anion exchange chromatography: - Column diameter: 2.6 cm - Resin: Capto adhere ImpRes - Layer height: ~ 15 ± 0.2 cm - Layer volume: ~80 ml - Residence time: ~4 min - Working flow: 20 ml/min - High salt wash buffer: 50 mM sodium phosphate buffer + 300 mM sodium chloride, pH 5.5 - Equilibration buffer: 50 mM sodium phosphate, pH 5.5 - Solution for regeneration and disinfection: 1 M sodium hydroxide + 2 M sodium chloride. - Column storage solution: 10 mM sodium hydroxide solution - Loading factor: ~35 mg/ml - Elution: flow (negative) mode - Peak collection: the leaking protein binds directly to a serially connected CEX column. Cation exchange chromatography: - Column diameter: 3.2 cm - Resin: SP Sepharose FF - Layer height: 20 cm ± 0.5 cm - Layer volume: ~160 ml - Residence time: ~8 min - Operating flow: 20 ml/min for loading and rinsing after loading and 32 ml/min during subsequent operation - Equilibration buffer: 50 mM sodium acetate buffer, pH 5.5 - Elution buffer: 50 mM sodium acetate buffer + 150 mM sodium chloride, pH 5.5 - Solution for regeneration and disinfection: 1000 mM sodium hydroxide + 2000 mM sodium chloride. - High salt wash buffer: 20 mM sodium acetate buffer + 300 mM sodium chloride, pH 5.5 - Column storage solution: 10 mM sodium hydroxide - Loading factor: ~25 mg/ml - Elution: stepwise elution - Post-elution collection: For a 2 mm path length UV flow cell, the eluate fraction from 50 milliU OD on the upstream side to 50 milliU OD on the downstream side was collected.

The first column, which was the anion exchange column of the second chromatography system, was connected to multiway valve positions 2 and 4 of the AKTA pure system, and the second column, which was the cation exchange column of the second chromatography system, was connected to column position 1. 3 CV of Milli Q water was passed through the columns to remove storage solution.

High Salt Wash Buffer: 2 CV of high salt wash buffer was passed through the anion and cation exchange columns of the second chromatography system and ensured that the pH and conductivity of the columns were within the range of the high salt wash buffer.

- 16 044363

Column equilibration: Both columns were equilibrated by passing 5 CV of equilibration buffer.

Loading and Buffer Displacement: A sample of the neutralized and filtered Protein A eluate collected in a collection vessel was loaded through columns connected in series in which the protein of interest did not bind to the anion exchange column of the second chromatography system and passed through it to bind to the cation exchange column of the second chromatographic system. Once loading was complete, 100 mL of equilibration buffer was added to the loading container and passed through the columns to ensure that the neutralized protein eluate sample was fully loaded.

Post-Load Equilibration Buffer Wash: After displacing the equilibration buffer, another 2 CV of equilibration buffer was passed to equilibrate the second chromatography system's anion exchange column peak flow and to remove loosely bound and unbound protein from the second chromatography system's cation exchange column. The protein of interest bound to the cation exchange column of the second chromatography system was then eluted using a cation exchange chromatography elution buffer. Peak elution was collected from T50 milliu OD to F50 milliu OD at 280 nm UV for a 2 mm path length flow cell. The eluate fraction of each cycle was stored at 2 to 8°C until further processing.

Regeneration: 2 CV anion exchange and cation exchange chromatography buffers were passed through both columns for regeneration.

Rinsing with Milli Q water: 2 CV of Milli Q water was passed through to remove the regeneration solution.

Column Storage: To store the columns (at the end of all runs), 2 CV of anion exchange and cation exchange storage solution were passed through.

Thus, stage 2 of chromatography consisted of two stages, the first of which was multimodal anion exchange chromatography, where Capto Adhere Impres resin was used, and the second was cation exchange chromatography, where SP-sepharose FF resin was used. After multimodal anion exchange chromatography, the impurities were bound to the column and the protein of interest passed through it. A cation exchange chromatography column was coupled to an anion exchange chromatography column in tandem and the protein of interest was bound to it. The protein was then eluted from the cation exchange chromatography column using high salt buffer. The chromatogram obtained at this stage is shown in Fig. 8, where the elution peak can be consistently observed for all cycles, as expected.

Tangential flow filtration (ultrafiltration and diafiltration). The cation exchange chromatography eluate was pooled and further processed for tangential flow filtration to concentrate and replace the cetuximab protein buffer with pharmaceutical formulation buffer without the final excipient according to the parameters shown in Table 1 below. 5.

Table 5

TFF system Cogent μ scale Cassette Pellicon Biomax 30 Cassette area 0.1 ^m2 MOS cassettes PES Concentration of eluate 3 after chromatography Up to 50 mg/ml Buffer replacement 8 diafiltration volumes Pressure < 1 bar Threshold size/MW CO 30 kDa

The protein solution recovered from the TFF system was diluted to a concentration of 5 mg/mL using pharmaceutical formulation buffer with the final excipient solution.

The resulting solution was filtered through a 0.2 μm pore size filter (MOS-polyethersulfone) under aseptic conditions into a PETG bottle. The filtrate thus obtained was the final cetuximab protein product suitable for use as a drug substance.

The convergence data for a typical series corresponded to the following parameters given in Table. 6.

Claims (10)

Table 6 Stage Total protein (mg) Convergence (%) Loading, chromatography 1 30240 82 Pooled eluate, chromatography 3 24816 Loading TFF 24816 97.9 TFF concentrate 24300 Medicinal substance 24300 100 General output 80.35% The above examples are purely illustrative and should not be construed as limiting the spirit of the invention. Various changes and modifications to the described embodiments will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit of the invention. Advantages of the present invention The present invention proposes an automated bioprocess for continuous operation for several days without repeated changes of vessels/packages during the process. The present invention provides an automated end-to-end bioprocess system that uses a controller to regulate and monitor the entire upstream and downstream process using SCADA or DCS. The present invention provides an automated end-to-end bioprocess system for therapeutic protein production, including a scalable and accurate automated real-time pH control system for virus inactivation. The present invention provides an automated bioprocess and system in which automated in-line sampling for HPLC analysis allows for in-depth process analysis as well as feedback on process parameters. The present invention provides an automated, end-to-end, continuous bioprocess for the production of therapeutic protein using a system containing pinch valves for seamless tube replacement after each completion of a continuous run, thereby simplifying operations. CLAIM 1. System (100) of an automated integrated continuous bioprocess for the production of therapeutic protein, containing: a bioreactor (103) for culturing mammalian cells capable of producing a therapeutic protein in a culture medium, said bioreactor (103) being provided with a variable tangential flow filter (109) for collecting harvest material containing protein secreted into the culture medium; a first chromatography system (119) including one or more affinity chromatography columns coupled to a bioreactor filtration system (109) provided with an alternating tangential flow (ATF) filter (109) of the bioreactor (103) without an intermediate storage vessel for purification of the collected recombinant therapeutic protein and obtaining protein A eluate; a virus inactivation system (126) comprising a virus inactivation vessel (128) connected to a first chromatography system (119) for collecting the protein A eluate and inactivating viruses that may be present in the eluate, and a virus inactivation vessel (128) configured with the ability to automatically regulate the pH of the protein A eluate; a collection vessel (136) connected to a virus inactivation vessel (128) through one or more filters for receiving virus-inactivated, neutralized and filtered protein A eluate, said one or more filters (224) and (226) being configured to removing impurities in the form of precipitates from the neutralized protein A eluate obtained from the virus inactivation vessel; a second chromatography system (137) including one or more multimodal anion exchange columns and one or more cation exchange columns connected to a collection vessel (136) for receiving the filtered protein A eluate from the collection vessel (136) and obtaining further purified protein; a clean-in-place (CIP) system (120) for periodically cleaning the chromatography system(s), virus inactivation vessel, collection vessel, and fluid flow tubing; one or more electromagnetic or pneumatic pinch valves (114), and optionally includes flow meters, air bubble sensors, pressure sensors, and a load compartment to provide a feedback loop for maintaining fluid flow between various system components, preventing the formation of air bubbles, and regulating fluid flow ; And - 18 044363 one or more control systems selected from the external control and data acquisition (SCADA) system (110); proportional-integral-derivative (PID) control; programmable logic circuit (PLC), industrial PC (IPC), distributed control system (DCS); input/output modules or input/output units (130) and (132) operatively coupled to individual systems, including chromatography system 1 (119); virus inactivation system (126); collection vessel (136); chromatographic system 2 (137); and message relay systems. 2. An automated integrated continuous bioprocess system according to claim 1, characterized in that said system additionally contains a transfer bag (116) connected to the bioreactor through a valve (114). 3. An automated integrated continuous bioprocess system according to claim 1, characterized in that the virus inactivation system (200) contains a vessel (201) for inactivating viruses (VI), a pH sensor (220) connected to a transmitter for measuring the pH of the protein A eluate and autotitrator (122), load compartment sensors (248-1)-(248-6) for monitoring the liquid level in the corresponding load compartments, level sensor for checking the liquid level in vessel VI, sensors (256-1) and (256-2 ) pressure in the fluid path to automatically switch between two filters if one of the filters becomes clogged during continuous operation, an integrated turbidity sensor (254) for real-time turbidity measurement in nephelometric turbidity units (NTU) and an air bubble sensor (252) to prevent air bubbles from entering the chromatography system. 4. The automated integrated continuous bioprocess system according to claim 1, characterized in that each of the filters between the virus inactivation vessel and the collection vessel is a filter with a pore size of 0.2-0.45 microns. 5. An automated integrated continuous bioprocess system according to claim 1, characterized in that the virus inactivation vessel and the collection vessel are made of glass or stainless steel. 6. The automated integrated continuous bioprocess system according to claim 1, characterized in that the clean-in-place (CIP) system (120) for periodically cleaning the chromatography system(s) includes cleaning their components, including the chromatography column(s). ), inlet, tubes and vessels. 7. The automated integrated continuous bioprocess system according to claim 1, characterized in that said system further includes automated sampling of collected material from the bioreactor for cell counting and nutrient analysis, as well as automated sampling at various locations for chromatographic analysis during operation. time of continuous bioprocess. 8. An automated, integrated, continuous bioprocess for producing a therapeutic protein in a system according to claims 1 to 7, wherein said process is controlled by one or more control systems selected from an external control and data acquisition system (110), proportional integral derivative (PID), programmable logic circuit (PLC), industrial PC (IPC), distributed control system (DCS) and message relay system, and the said process includes the steps: (a) culturing mammalian cells capable of producing a therapeutic protein in a liquid culture medium in a bioreactor (103) equipped with an alternating tangential flow (ATF) filter (109), and collecting the released harvest material containing the protein secreted into the culture medium; (b) supplying the harvested cell culture material containing the therapeutic protein from the bioreactor (103) to a first chromatography system (119) including one or more affinity chromatography columns to obtain a protein A eluate; (c) supplying the protein A eluate from the first chromatography system (119) to the virus inactivation vessel (128) and allowing viruses to be inactivated in the protein A eluate as well; (d) passing the virus-inactivated and neutralized Protein A eluate through one or more filters to remove impurities in the form of sediment formed during the virus inactivation and neutralization steps, and collecting the filtered Protein A eluate in a collection vessel (136); (e) supplying the neutralized and filtered Protein A eluate to a second chromatography system (137) including one or more multimodal anion exchange chromatography columns and one or more cation exchange chromatography columns to obtain purified protein; and wherein said process includes automatic sampling for analysis during operation and cleaning in place using a system (120) (CIP) to periodically clean the inlet of the chromatography system to maintain continuous operation. 9. The automated complex continuous bioprocess according to claim 8, characterized in that the mammalian cells are cultured in a bioreactor (103), which is a perfusion bioreactor equipped with means for implementing alternating tangential flow (ATF). 10. Automated complex continuous bioprocess according to claim 8, characterized in that the liquid culture medium is directly supplied from the bioreactor (103) to the first chromatographic -