On-line in-situ cleaning process for alternate tangential flow filter

Abstract

The present invention provides a method for in-line in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter in a filtration module in communication with a bioreactor. The method comprises the steps that diluted NaOH passes through an ATF filter, the diluted NaOH is 0.1-0.5 N NaOH, the volume of the NaOH is three to five times of the retention volume of the filter, and the flow velocity of the NaOH is 80-200 ml / L; flushing the filter with sterile water, wherein the volume of the flushing water is 10-20 times of the retention volume of the filter; and balancing the filter with the culture medium. The present invention also provides a system for in-line CIP of an alternating tangential flow (ATF) filter in a filtration module in communication with a bioreactor.

Description

TECHNICAL FIELD

[0001] The present invention relates to an online cleaning in place (CIP) process for alternating tangential flow (ATF) filters. In particular, the present invention relates to a method for cleaning in place (CIP) of alternating tangential flow (ATF) filters in a filtration module in communication with a bioreactor to mitigate protein retention in the bioreactor. In particular, the present invention provides an online CIP method for removing cell debris or clogging retained inside or on the surface of the filter, thereby facilitating prolonged reuse time of the ATF filter in the same batch. BACKGROUND

[0002] Cell culture technology has been widely used in the production of biological products, such as proteins, receptors, vaccines and antibodies, which have a wide range of applications, such as therapeutic drugs, scientific research and diagnosis. In order to address the challenges of low productivity, contamination, high cost, etc., various cell culture technologies have been developed.

[0003] Perfusion cell culture has attracted much attention due to its ability to maintain cells in exponential growth phase for a long time and achieve higher viable cell density. However, in the process of mammalian cell culture, due to the high density of the cell line used, a large amount of cell debris and cell waste will accumulate in the bioreactor, resulting in protein retention in the reactor, thereby reducing the yield of the protein. In order to solve these problems, researchers have developed an alternating tangential flow (ATF) system using hollow fiber filters for perfusion processes of prokaryotic and eukaryotic cell cultures to achieve efficient cell separation. ATF filters have been used to remove cell-free harvests.

[0004] In the ATF system, the cell retention device retains cells in the bioreactor; at the same time, the waste medium containing the product is collected. Then, the cells are pushed back into the bioreactor by alternating tangential flow. Therefore, the ATF system is considered an ideal separation system. However, there are also some disadvantages in using ATF filters; in particular, for single-use products, a new ATF filter needs to be replaced every time the filter is clogged, which will cause the production process to be interrupted and increase the production cost.

[0005] Although there are reports that the protein carryover is lower in alternating tangential flow (ATF) systems than in other tangential flow filtration (TFF) based perfusion systems, protein carryover still exists for most molecules. Protein carryover is often observed to varying degrees during perfusion processes of different CHO cell lines, such as glutamine synthetase (GS), dihydrofolate reductase (DHFR), and CHOK1, etc. Protein carryover in perfusion culture can be caused by a number of factors, including antifoam agents, cell debris of different particle sizes, media components, and protein related issues, which can act alone or in combination. The impact of the reciprocating flow of media during an ATF based process on cell health and protein quality is not fully understood; however, studies have found that reciprocating flow increases the shear stress experienced by the cells and protein product. Mammalian cells are particularly sensitive to shear or mechanical stress, and numerous studies have shown that high shear stress affects cell viability and growth. Therefore, the process flow must be carefully designed with operating parameters that avoid significantly perturbing the hydrodynamic conditions to prevent damage to the cells and protein production.

[0006] Some previous attempts include the method described in published PCT patent application WO2005097306A1, which discloses a cleaning membrane filter comprising hollow fibers and an internal skin. The method includes the steps of emptying the concentration chamber to release the liquid and suspended matter to be filtered therein; subsequently, a backflushing step is performed, in which liquid is caused to flow from the permeate chamber through the membrane into the concentration chamber to remove and release impurities deposited on the membrane, while circulating a gas in the concentration chamber. According to the present invention, in at least one backflushing phase, a pulse of backflushing liquid and / or gas is generated by the control means (5, 5a; 7, 7a), which can cause unnecessary stress to the cells, protein product and filter membrane, thereby adversely affecting the cells and protein product and making the membrane weak, vulnerable and less efficient.

[0007] Another method is described in published PCT application WO2005028085A1, which discloses a method and apparatus for backflushing a membrane filtration system, wherein the permeate remaining in the filtration system when the filtration process is stopped or paused is used to provide backflushing liquid for the membrane pores during the backflushing process. This system requires stopping the filtration system; this can delay the overall production of the product, reduce the process efficiency and increase the cost of the product.

[0008] Currently, clean-in-place (CIP) systems and processes have been used to clean bioreactors, filtration devices and related equipment without disassembly. However, most CIP processes require the use of multiple aggressive agents, such as detergents, disinfectants, alkalis, surfactants and acids, at high concentrations and temperatures, which can leave harmful residues inside the system and are therefore not suitable for in-line applications. Typically, strong alkaline solutions and acids tend to dissolve proteins, and thus such processes are suitable for protein production devices, including ATF filtration devices.

[0009] Therefore, there is still an unmet need to provide an in-line cleaning process that can be specifically used for ATF filtration systems, including disposable ATF filters, to effectively clean the filters by removing cell debris, residue and residuals inside and outside the filters, thereby avoiding the need to replace the filters each time the filters are clogged, avoiding the need to stop the production process, allowing the reuse of the filters, and more importantly, solving the problem of protein product retention or accumulation in the bioreactor.

[0010] Objectives of the Invention The present disclosure aims to provide an in-line clean-in-place (CIP) process for alternating tangential flow (ATF) filters.

[0011] Another objective of the present disclosure is to provide a method for in-line clean-in-place (CIP) of alternating tangential flow (ATF) filters, allowing their reuse.

[0012] Another objective of the present disclosure is to provide a method for in-line clean-in-place (CIP) of alternating tangential flow (ATF) filters to solve the problem of protein retention in bioreactors. SUMMARY

[0013] This summary is intended to introduce some concepts in a simplified form, which will be described in more detail in the detailed description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to help determine the scope of the claimed subject matter.

[0014] The general aspect of the present disclosure provides an in-line clean-in-place (CIP) process for alternating tangential flow (ATF) filters.

[0015] Certain aspects of the present disclosure provide an in-line clean-in-place (CIP) process for alternating tangential flow (ATF) filters, which can overcome one or more drawbacks present in the prior art.

[0016] The present invention provides an online CIP filter process that helps to clear the cell debris or clogging inside the filter and helps to extend the ATF filter life time for the same batch of product.

[0017] This simple online CIP is suitable for industrial scale production bioreactor using different types of ATF 2, 4, 6 and 10.

[0018] Another aspect of the present disclosure is to provide a method to make ATF hollow fiber filter reusable by implementing CIP.

[0019] Another aspect of the present disclosure is to provide an improved process to address the protein hold up problem.

[0020] One aspect of the present disclosure provides a method for online in-situ cleaning in place (CIP) of an alternating tangential flow (ATF) filter in a filtration module in communication with a bioreactor, the method comprising the steps of: i) passing diluted NaOH through the ATF filter, the diluted NaOH being 0.1 to 0.5 N NaOH, the volume of NaOH being three to five times the hold up volume of the filter, the flow rate of NaOH being 80-200 ml / L; ii) rinsing the filter with sterile water, the volume of water being ten to twenty times the hold up volume of the filter; iii) equilibrating the filter with media.

[0021] The present invention also provides a system for online CIP of an alternating tangential flow (ATF) filter in a filtration module in communication with a bioreactor.

[0022] The present invention overcomes the protein hold up problem and allows long time batch processing without frequent change of ATF hollow fiber filter.

[0023] Other aspects of the present invention will be apparent from the description that follows, some of which will be apparent from the description and others will become apparent to those of ordinary skill in the art upon implementation of the invention. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : shows a schematic of an experimental system set up in accordance with the present invention, the system comprising a bioreactor connected to an ATF filtration module and an online CIP process implemented by the said apparatus.

[0025] Figure 2 : shows the growth curve of denosumab in a perfusion batch.

[0026] Figure 3 : shows the productivity curve of denosumab perfusion harvest using the online CIP technique of the present invention.

[0027] Figure 4 Growth curve of cetuximab perfusion batch is shown.

[0028] Figure 5 Productivity curve of cetuximab perfusion harvest using online CIP process of the present application is shown. The top graph shows the batch cumulative bioreactor titer due to ATF filter clogging and the bottom graph shows the harvest rate, with peak harvest titer of 0.7 g / L / day observed after multiple CIPs.

[0029] Figure 6 Growth curve of ravulizumab-I is shown. The cell density at inoculation was 5 million cells / mL and the peak cell density of 87.6 million cells / mL was observed on day 6. IN CIP was performed on day 10 and day 14, respectively.

[0030] Figure 7 Productivity curve of ravulizumab-I perfusion harvest titer with online CIP of the present application is shown.

[0031] Figure 8 Productivity curve of ravulizumab-I perfusion bioreactor with online CIP process of the present application is shown. The bioreactor titer of 1.3 g / L is due to batch-to-batch titer accumulation due to ATF filter clogging. Peak harvest titer of 0.6 g / L / day was observed after multiple CIPs. Figure 9 Growth curve of ravulizumab-II in perfusion batch is shown. The cell density at inoculation was 0.5 million cells / mL and the peak cell density of 805 million cells / mL was observed on day 14. IN CIP was performed on day 10.

[0032] Figure 10 Productivity curve of ravulizumab-II perfusion harvest using online CIP technology of the present application is shown.

[0033] Figure 11 Productivity curve of ravulizumab-II perfusion bioreactor with online CIP system of the present application is shown. The bioreactor titer of 5 g / L is due to ATF filter clogging and accumulation during batch production. Peak harvest titer of 0.6 g / L / day was recorded after multiple CIPs.

[0034] Figure 12 Growth curve of tocilizumab in perfusion batch is shown. The cell density at inoculation was 0.5 million cells / mL and the peak cell density of 90 million cells / mL was observed on day 10. IN CIP was performed on day 15. Figure 13 : Shows productivity profile for tocilizumab perfusion harvest using online CIP implemented in the present application.

[0035] Figure 14 : Shows productivity profile for tocilizumab perfusion bioreactor using online CIP process of the present application. Bioreactor titer of 1.1 g / L was due to ATF filter clogging and accumulation during batch production. Post CIP, peak harvest titer was recorded at 0.8 g / L / day.

[0036] Figure 15 : Shows growth profile of nivolumab in a perfusion batch. Cell density at inoculation was 0.5 million cells / mL, peak cell density was 80 million cells / mL, and IN CIP was performed on day 15.

[0037] Figure 16 : Shows productivity profile for nivolumab perfusion harvest using online CIP implemented in the present application.

[0038] Figure 17 : Shows productivity profile for nivolumab perfusion bioreactor using online CIP process of the present application. Bioreactor titer of 1.5 g / L was due to ATF filter clogging and accumulation of clog during batch production, CIP was performed on day 15.

[0039] Figure 18 : Shows growth profile of pembrolizumab in a perfusion batch. Cell density at inoculation was 0.5 million cells / mL, peak cell density of 70 million cells / mL was observed on day 17. IN CIP was performed on days 22, 25, and 26.

[0040] Figure 19 : Shows productivity profile for pembrolizumab perfusion harvest using online CIP technology of the present application.

[0041] Figure 20 : Shows productivity profile for pembrolizumab perfusion bioreactor titer using online CIP process of the present application. Bioreactor titer of 4.3 g / L was due to ATF filter clogging and accumulation of clog during batch production. Post multiple CIP cleanings, peak harvest titer was recorded at 2.5 g / L / day. DETAILED DESCRIPTION

[0042] The following is a detailed description of the embodiments of the present disclosure. The embodiments are sufficiently detailed to enable one of ordinary skill in the art to clearly understand the present disclosure. However, the details provided are not intended to limit the intended variations of the embodiments; rather, the purpose is to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure.

[0043] All publications cited herein are hereby incorporated by reference for the reason that the disclosure contained therein is considered to be part of the disclosure of this application. To the extent that any definition or usage of a term in an incorporated reference differs from the definition or usage of the same term in the disclosure herein, the definition or usage of the term in the disclosure herein shall prevail.

[0044] References in the specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" or "in an embodiments" in various places in the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0045] In some embodiments, to describe certain embodiments of the application, numbers have been used to quantify weight, percentages, ratios, and the like, and in some instances, these numbers should be understood to be modified by the word "about." Thus, in some embodiments, numerical parameters listed in the written description are approximations, and can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, these numerical parameters should apply and in other embodiments, should apply in light of the number of reported significant digits and normal rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, can contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0046] Various terms are used herein as follows. If a term is not defined herein, it should be interpreted as taking the broadest definition to be found in the published literature and issued patents as of the date of the application.

[0047] In the following description, the meaning of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In addition, in the following description, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0048] Unless the context requires otherwise, throughout the specification, the word "comprise," and variations of the word, such as "comprising" and "comprises," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0049] The recitation of ranges of values herein is merely intended to serve as a

[0050] Unless otherwise stated in this document, all methods described herein can be performed in any suitable order. Any examples, or exemplary language, provided herein, for instance with respect to certain embodiments, only serve to more effectively illustrate the application, and do not pose a limitation to the scope of the present application.

[0051] The grouping of alternative elements or embodiments disclosed herein should not be interpreted as limiting. Each group member can be referred to individually or in any combination with other group members or other elements in this document. For convenience and / or patentability, one or more members of a group can be added or removed as appropriate. When any such addition or removal occurs, the specification shall be construed to include only those group members which, at the time the specification is being interpreted, are included in the group.

[0052] The following description and embodiments therein serve only to illustrate particular embodiments of the principles and aspects of the present disclosure. These examples are provided for explanation and not limitation of the principles and the present disclosure.

[0053] It should also be understood that the present disclosure can be implemented in a number of manners, including as a system, method or apparatus. In this specification, any of these implementations or other forms the present disclosure can take are referred to as technologies. Typically, the order in which steps of disclosed technologies are carried out can be changed.

[0054] The title and summary of the application provided herein are merely for convenience and do not interpret the scope or meaning of the embodiments.

[0055] The following discussion provides several implementations of the inventive subject matter. Although each embodiment represents a single combination of the inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and another embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if such combinations are not explicitly disclosed.

[0056] The term "retention volume" as used herein refers to the volume retained in a filter or the volume used in a bioreactor.

[0057] The lag phase refers to the phase in the cell cycle where the cells do not divide. This is the period where the cells adapt to the culture conditions and prepare for cell division. In the present invention, the first 2-5 days are considered the lag phase.

[0058] The log phase, also known as the exponential phase, refers to the phase of the cell cycle where cells are actively proliferating and the cell density is increasing exponentially. Since the cell viability is the strongest at this time, it is recommended to assess the cell function at this phase. In the present invention, the first 5-12 days are considered as the log phase.

[0059] The stationary phase, or plateau phase, refers to the phase of the cell cycle where cell proliferation slows down due to growth limiting factors (e.g. depletion of essential nutrients and / or formation of inhibitory products) resulting in equal cell growth and death rates. In the present invention, the 12-30 days are considered as the stationary phase.

[0060] The decline phase refers to the phase of the cell cycle where cell death dominates and the number of viable cells decreases. In the present invention, the last 2-4 days of the growth cycle are considered as the decline phase.

[0061] ATF as used herein is an abbreviation for alternating tangential flow, which utilizes a hollow porous fiber membrane (pore size e.g. 0.1-5.0 microns or ultrafiltration membrane with a molecular weight cut-off of 750 kDa) to retain cells and other particulate matter. The ATF system includes a bioreactor, particularly a stirred tank reactor, for harvesting. The bioreactor is connected to the ATF via a pipe to maintain aseptic conditions. The ATF system utilizes a hollow porous fiber membrane and alternating tangential flow to perfuse the harvested cell culture into the bioreactor. In addition, the ATF system also includes a controller for controlling a diaphragm pump which performs the ATF utilizing the hollow porous fiber membrane.

[0062] The present invention aims to address various deficiencies in the art, such as protein retention in the bioreactor and process interruption due to replacement or cleaning of filters, among others.

[0063] In an embodiment, the present disclosure discloses a method for online in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter.

[0064] In an embodiment, the present disclosure discloses a method for online in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter using 0.1 to 0.5 N sodium hydroxide.

[0065] The present disclosure discloses a method for reusability of ATF hollow fiber by implementing CIP.

[0066] The present invention provides an improved process which addresses the challenge of protein retention in the bioreactor and related drawbacks.

[0067] In an embodiment, the present invention provides a method for online in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter in a filtration module in communication with a bioreactor, the method comprising the steps of: i) passing diluted NaOH through the ATF filter, the diluted NaOH being 0.1 to 0.5 N NaOH, the volume of NaOH being three to five times the retention volume of the filter, the flow rate of NaOH being 80-200 ml / L; ii) rinsing the filter with sterile water, the volume of rinsing water being ten to twenty times the retention volume of the filter; iii) equilibrating the filter with the culture medium.

[0068] In an embodiment of the disclosure, the process comprises monitoring the titer of the product in the bioreactor.

[0069] In an embodiment of the disclosure, the online CIP is initiated based on the titer value of the accumulated product in the bioreactor.

[0070] In an embodiment of the disclosure, the online CIP is performed one or more times based on the titer value of the accumulated product in a selected phase of the logarithmic phase, stationary phase and decline phase of the growth of the cells cultured in the bioreactor.

[0071] In an embodiment of the disclosure, the online CIP is performed based on the titer value of the accumulated product in the bioreactor being ≥ 20% in the lag phase, ≥ 40% in the logarithmic phase, ≥ 60% in the stationary phase, or < 40% and equal to 20% in the decline phase of the growth of the cells cultured in the bioreactor.

[0072] In an embodiment of the disclosure, the online CIP is repeated one or more times based on the titer value of the accumulated product in the bioreactor being 60% to 70%.

[0073] As Figure 1As shown, the experimental setup is used for in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter according to the present invention, wherein the system (100) includes: (a) an alternating tangential flow (ATF) filter module (101) and (b) a bioreactor (201), the ATF filter module (101) being in communication with the bioreactor (201). The bioreactor (201) includes a cell culture container (201a) housed within a housing (201b). The cell culture container contains cell culture medium (202), a stirring device (203), a port (204a) for injecting culture medium, a port (204b) for sample extraction, and a port (205) for connecting the bioreactor via a conduit (206) and a clamp (207) and communicating with the ATF filter module (101) via the port (106). The port (204a) or other ports (not shown) of the cell culture container allow the introduction of buffer solutions, air, and other desired input substances into the cell culture container (201a). The air / gas flow rate introduced into the cell culture container is controlled by a mass flow controller (MFC) (not shown) connected to the bioreactor. A conduit (206) allows waste culture medium containing cells and products to flow from the cell culture container (201a) of the bioreactor (201) to the ATF filtration module (101), and to return liquid culture medium containing isolated cells from the ATF filtration module (101) to the cell culture container (201a). The ATF filtration module includes a hollow fiber filter (102) and an inlet port (104) that allows diluted NaOH to be introduced from the container (103) and flow through the hollow fiber filter (102) located within the filtration module (101). The inlet port (104) also allows the introduction of sterile water to rinse the hollow fiber filter (102). An outlet port (105) allows the harvest containing the product to be collected to be removed from the ATF module (101) and collected into the container (107). Through pipe (206), port (106) allows NaOH and water to be discharged after flowing through filter (102) and collected in container (108).

[0074] Figure 1A process flow for online in-situ cleaning (CIP) of alternating tangential flow (ATF) filters in a filtration module (101) in communication with a bioreactor (201) is further depicted in accordance with the present application, the process flow comprising the steps of: flowing diluted NaOH from a container (103) through hollow fiber ATF filters (102) within the filtration module (101) via an inlet port (104), the diluted NaOH being 0.1 to 0.5 N NaOH, the volume of the NaOH solution being three to five times the hold-up volume of the filters (102), the flow rate of the NaOH solution being 80-200 ml / L; rinsing the filters (101) with sterile water via the inlet port (104), the volume of the rinse water being ten to twenty times the hold-up volume of the filters (102); equilibrating the filters with media via the inlet port (104).

[0075] Figure 1 In the figure, the double arrow indicates the flow of media containing cells and product from the bioreactor to the ATF filtration module, and the flow of separated cells and media from the ATF filtration module to the bioreactor. The single arrow indicates the flow of liquid from one component to another, as shown.

[0076] The method of the present application can be readily implemented using ATF modules of various capacities, for example:

[0077] The initiation or starting point of the online CIP depends on the stage of cell growth in the production culture system, which is divided into four stages: lag phase, log phase, stationary phase, and decline phase. In the lag phase, the online CIP is initiated when the titer in the bioreactor is in the range of 10-30%; while in the log growth phase, the online CIP is initiated when the titer in the bioreactor reaches 30% to 50%. In addition, in the stationary phase of cell growth, the online CIP is initiated when the difference in titer between the bioreactor and the harvest fluid is between 50% to 80%. In the decline phase, the online CIP is initiated when the titer in the bioreactor is 10% to 30%.

[0078] After CIP, the culture volume is lost by about 2% to 10%, depending on the size of the bioreactor, resulting in a slight decrease in cell number and dilution of the culture. However, contrary to the bioreactor productivity, the harvest productivity is surprisingly improved.

[0079] The method of the present disclosure solves the problems existing in the prior art. The present invention uses an online CIP process to clean partially or completely clogged hollow fiber pores, while solving the problem of protein retention. The online CIP process can remove various sizes of cell debris, blockages or deposition layers formed inside the hollow fiber filter by cell lysis, defoaming agents and other medium components, thereby prolonging the service life of the ATF filter.

[0080] In known cleaning methods, polyether sulfone (PES) membrane filter (ATF) is usually cleaned with a combination of strong acid and strong base. The present invention provides a method of online CIP using only very dilute 0.1 to 0.5N sodium hydroxide (NaOH), thereby avoiding the reduction in the strength of the membrane, allowing it to be used for a longer period of time and reused. In addition, the use of dilute NaOH also avoids damage or degradation of the protein product.

[0081] Although various embodiments of the present disclosure are described above, other embodiments can also be designed without departing from the basic scope of the present disclosure. The present invention is not limited to the described embodiments, versions or examples, which are intended to enable a person of ordinary skill in the art to make and use the invention in conjunction with the information and knowledge they possess.

[0082] Examples The present disclosure will be illustrated below by examples, which are intended to clarify the operation of the present disclosure, but not to limit the scope of the present disclosure. Although methods and materials similar or equivalent to those described herein can be used in practicing the disclosed methods and compositions, only exemplary methods, devices and materials are described herein. It should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as these methods and conditions can vary.

[0083] Example 1 In the production of various biomolecules, the process of online cleaning using alternating tangential flow (ATF) filters is processed To culture cells in perfusion mode, we used a Sartorius Biostat BDCUII bioreactor connected to an ATF module with online CIP options (Xcell® ATF). CHO-S cells were purchased from Catalant, USA. The inoculation concentration of the bioreactor was 0.5 to 7 x The initial production medium was ActiPro medium (producer: Cytiva) containing 6 mM glutamine. The medium was equilibrated for at least 12 hours before the start of the batch. The dissolved oxygen (DO) was controlled by a "cascade" mode of air and oxygen. The air flow was provided via a rotameter with a set point of 0.3 lpm and was cascaded with the oxygen flow. Oxygen was added via a MFC. The pH was controlled at 7.0 ± 0.1 by sparging CO2. (on / off - solenoid valve). Air, oxygen and CO2 were sparged using a ring sparger 。 Antifoam C (Sigma) was added manually as required to reduce foaming in the bioreactor. The perfusion was started on day 0 (0 hours) and the perfusion medium contained ActiPro and cell boosters 7a and 7b. The observed cell density peak ranged from 60 to 130 million cells / mL and depending on the VCC, the titers in the bioreactor and harvest could be observed from day 2 or day 3. The online CIP was started when the titer in the bioreactor exceeded 60-70%.

[0084] 3 to 5 times the retention volume (about 600 mL) of NaOH solution was passed through the ATF hollow fiber filter at a flow rate of 120-150 mL / L. Subsequently, a 10 times retention volume of autoclaved / filtrated water for injection (WFI) was used for rinsing. Thereafter, the ATF module was equilibrated with two times retention volume of base or perfusion medium and then restarted.

[0085] According to the present application, different biomolecules were produced using the online CIP process. Table 1 summarizes the different biomolecules produced.

[0086] Table 1: Summary of different molecule batch details:

[0087] The following table shows the impact of performing CIP on the titer values of the different biomolecules mentioned in Table 1: Table 2: Denosumab

[0088] Table 3: Cetuximab

[0089] Table 4: Ravulizumab-I

[0090] Table 5: Ravulizumab-II

[0091] Table 6: Tocilizumab

[0092] Table 7: Nivolumab

[0093] Table 8: Pembrolizumab

[0094] The above table shows that the harvest titer increased after CIP treatment, indicating the effectiveness of CIP.

[0095] The present application has been described in detail by specific implementation, but it should be understood that these implementations are only for illustration, and the present application is not necessarily limited to these implementations. Those skilled in the art should understand that modifications and variations of the present application can be easily seen from the present disclosure, and these modifications and variations will not deviate from the scope of the present application. Therefore, such modifications and variations of the disclosed implementations should be considered within the scope of the present application and its claims.

Claims

1. A method of performing online in-situ cleaning (CIP) of an alternating tangential flow (ATF) filter, the filter module being in communication with a bioreactor, the method comprising the steps of: i) passing diluted NaOH through the ATF filter, the diluted NaOH being 0.1 to 0.5 N NaOH, the volume of NaOH being three to five times the hold-up volume of the filter, the flow rate of the NaOH being 80-200 ml / L; ii) rinsing the filter with sterile water, the volume of water being ten to twenty times the hold-up volume of the filter; iii) equilibrating the filter with media.

2. The method of claim 1, wherein, The method comprises monitoring the titer of the product in the bioreactor.

3. The method according to claims 1 and 2, wherein, The online CIP is initiated based on the titer value of the accumulated product in the bioreactor.

4. The method of claim 1, wherein, The online CIP is performed one or more times based on the titer value of the accumulated product in a phase selected from the log phase, the stationary phase, and the decline phase of the growth of the cells cultured in the bioreactor.

5. The method of claim 4, wherein, The online CIP is performed based on the titer value of the accumulated product in the bioreactor being > 20% in the lag phase, > 40% in the log phase, > 60% in the stationary phase, or < 40% and equal to 20% in the decline phase of the growth of the cells cultured in the bioreactor.

6. The method of claim 1, wherein, The online CIP is repeated one or more times based on the titer value of the accumulated product in the bioreactor being 60% to 70%. The online CIP is repeated one or more times based on the titer value of the accumulated product in the bioreactor being 60% to 70%.

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